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The ULK1-FBXW5-SEC23B nexus controls autophagy

  1. Yeon-Tae Jeong
  2. Daniele Simoneschi
  3. Sarah Keegan
  4. David Melville
  5. Natalia S Adler
  6. Anita Saraf
  7. Laurence Florens
  8. Michael P Washburn
  9. Claudio N Cavasotto
  10. David Fenyö
  11. Ana Maria Cuervo
  12. Mario Rossi  Is a corresponding author
  13. Michele Pagano  Is a corresponding author
  1. NYU School of Medicine, United States
  2. Howard Hughes Medical Institute, University of California, Berkeley, United States
  3. CONICET-Partner Institute of the Max Planck Society, Argentina
  4. Universidad Austral, Argentina
  5. The Stowers Institute for Medical Research, United States
  6. The University of Kansas Medical Center, United States
  7. Albert Einstein College of Medicine, United States
  8. Howard Hughes Medical Institute, New York University School of Medicine, United States
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Cite this article as: eLife 2018;7:e42253 doi: 10.7554/eLife.42253

Abstract

In response to nutrient deprivation, the cell mobilizes an extensive amount of membrane to form and grow the autophagosome, allowing the progression of autophagy. By providing membranes and stimulating LC3 lipidation, COPII (Coat Protein Complex II) promotes autophagosome biogenesis. Here, we show that the F-box protein FBXW5 targets SEC23B, a component of COPII, for proteasomal degradation and that this event limits the autophagic flux in the presence of nutrients. In response to starvation, ULK1 phosphorylates SEC23B on Serine 186, preventing the interaction of SEC23B with FBXW5 and, therefore, inhibiting SEC23B degradation. Phosphorylated and stabilized SEC23B associates with SEC24A and SEC24B, but not SEC24C and SEC24D, and they re-localize to the ER-Golgi intermediate compartment, promoting autophagic flux. We propose that, in the presence of nutrients, FBXW5 limits COPII-mediated autophagosome biogenesis. Inhibition of this event by ULK1 ensures efficient execution of the autophagic cascade in response to nutrient starvation.

https://doi.org/10.7554/eLife.42253.001

Introduction

Macro-autophagy (more commonly referred to as autophagy) is a highly conserved process present in all eukaryotes, which allows the degradation of proteins and organelles by lysosomes (Hurley and Young, 2017; Klionsky et al., 2016; Lamb et al., 2013). It is characterized by the formation of the double-membraned autophagosome that transports cytoplasmic cargos to lysosomes, where the autophagic cargo is subjected to degradation. In a simplistic way, autophagy can be classified into ‘basal’ and ‘induced.’ The former is used to maintain cellular homeostasis by promoting the turnover of cytoplasmic components, and the latter is part of the cellular response to stressors (e.g., to produce amino acids upon nutrient deprivation). Due to its role in many cellular processes, it is not surprising that deregulation of autophagy plays a role in many human diseases, such as neurodegenerative disorders, cancer, and infection (Jiang and Mizushima, 2014; Rybstein et al., 2018).

The UNC51-like kinase 1 [ULK1, a.k.a. autophagy-related (ATG) protein ATG1] is a master regulator of autophagy (Dankert et al., 2016; Hurley and Young, 2017; Lamb et al., 2013; Mizushima, 2010). Specifically, in response to nutrient starvation or mTOR inhibition, ULK1 is activated and, in turn, this leads to enhanced activity of the autophagy-specific class III phosphoinositide 3-kinase (PI3KC3) complex, which is comprised of VPS34 (a.k.a. PIK3C3), p150 (a.k.a. VPS15 and PIK3R4), BECLIN-1 (a.k.a. ATG6), and ATG14 (Hurley and Young, 2017; Lamb et al., 2013). The activation of the PI3KC3 complex results in the production of PI(3)P (phosphatidylinositol 3-phosphate), which is necessary for the recruitment of downstream effectors and the subsequent nucleation of the autophagosome. Next, two consecutive ubiquitylation-like reactions catalyzed by certain ATG proteins mediate the attachment of phosphatidylethanolamine to LC3 family proteins (commonly referred to as LC3 lipidation), promoting the expansion and closure of the autophagosome (Mizushima et al., 2011). Originally, it was thought that the autophagosome only derives from mobile, cytoplasmic vesicles that are characterized by the transmembrane protein ATG9 and that are recruited to the ER. However, it has become clear that, in response to starvation, additional sources of membrane are necessary for the formation and growth of the autophagosome (Davis et al., 2017; Hurley and Young, 2017; Wang et al., 2014).

The coat protein complex II (COPII) is a multi-subunit protein complex essential for the transport of cellular cargos from the ER to the Golgi apparatus (Fromme et al., 2008; Zanetti et al., 2011). A key component of COPII is SEC23, whose importance in maintaining cellular homeostasis is highlighted by the fact that mutations in the two SEC23 paralogs (SEC23A and SEC23B) cause the human genetic diseases cranio-lenticulo-sutural dysplasia and congenital dyserythropoietic anemia type II, respectively (Boyadjiev et al., 2006; Lang et al., 2006; Schwarz et al., 2009). The other components of COPII are SEC13, SEC24, SEC31, and SAR1. COPII vesicles emerge from specialized domains of the ER called ER exit sites (ERES) (Zanetti et al., 2011). However, in response to starvation, when the secretory pathway is inhibited (Wang et al., 2014; Zacharogianni et al., 2014; Zacharogianni et al., 2011) and there is an urgent need for membranes to form and grow autophagosomes, ERES enlarge and patch along the ER-Golgi intermediate compartment (ERGIC) to function in autophagosome biogenesis (Davis et al., 2017; Egan et al., 2015; Ge et al., 2017; Ge et al., 2014; Hurley and Young, 2017; Sanchez-Wandelmer et al., 2015). In mammals, disruption of ERES inhibits autophagosome biogenesis at an early stage (Stadel et al., 2015; Zoppino et al., 2010). Moreover, in response to nutrient starvation, the PI3KC3 complex, which is activated by ULK1, promotes the recruitment of COPII components to the ERGIC (Egan et al., 2015; Ge et al., 2013; Ge et al., 2014; Karanasios et al., 2016). Next, specialized COPII vesicles budding from the ERGIC act as precursors for LC3 lipidation, a critical step in autophagosome biogenesis (Egan et al., 2015; Ge et al., 2017; Ge et al., 2014). The functions of COPII in the autophagic pathway are conserved along evolution. In fact, in response to starvation, yeast COPII components physically interact with core elements required for autophagy, and COPII vesicles provide membrane sources for the growing autophagosome (Davis et al., 2016; Ishihara et al., 2001; Lemus et al., 2016; Reggiori et al., 2004; Tan et al., 2013). However, how the components of COPII vesicles are regulated in response to nutrient deprivation to allow their contribution to autophagosome biogenesis is largely unknown.

SKP1-CUL1-F-box protein (SCF) complexes form a family of multi-subunit ubiquitin ligases, which, in turn, is part of the super-family of Cullin-Ring Ligase (CRL) complexes (Petroski and Deshaies, 2005; Skaar et al., 2013; Skaar et al., 2014). In human, 69 F-box proteins act as the substrate receptor subunits of SCF ubiquitin ligases, allowing the regulation of hundreds of substrate proteins. Thus, SCFs control a multitude of cellular processes whose deregulation is implicated in many pathologies, including cancer, neurodegenerative disorders, metabolic diseases, etc. (Frescas and Pagano, 2008; Wang et al., 2014). We had previously shown that, by promoting the activation of the PI3K-AKT-mTOR signalling cascade, the F-box protein FBXL2 inhibits autophagy (Kuchay et al., 2013). A targeted siRNA screen to deplete in U2OS cells 184 substrate receptors of human CRL complexes identified FBXW5 as a top hit involved in limiting autophagy in the presence of nutrients (YTJ and MP, unpublished results). Thus, we studied the role of FBXW5 in regulating autophagy as described herein.

Results

FBXW5 binds free SEC23B to promote its ubiquitylation and proteasomal degradation

To identify SCFFBXW5 substrates, Streptag-FLAG (SF)-tagged FBXW5 was transiently expressed in HEK293T cells and affinity purified for analysis by Multidimensional Protein Identification Technology (MudPIT) (Florens and Washburn, 2006; Jeong et al., 2013). MudPIT revealed the presence of peptides corresponding to SKP1 and CUL1 (as expected), as well as 15 unique peptides derived from the COPII coat subunit SEC23B (http://www.stowers.org/research/publications/libpb-1118). To confirm the binding between SEC23B and FBXW5 and its specificity, we screened a panel of nine human F-box proteins. SF-tagged F-box proteins were expressed in HEK293T cells and affinity precipitated to evaluate their interaction with SEC23B. We found that FBXW5 was the only F-box protein capable of co-precipitating with endogenous SEC23B (Figure 1A).

Figure 1 with 1 supplement see all
SCFFBXW5 interacts with SEC23B and targets it for ubiquitylation and proteasome-mediated degradation.

(A) HEK293T cells were transfected with either an empty vector (EV) or the indicated Streptag-FLAG-tagged (SF) F-box proteins (FBPs). Twenty-four hours after transfection, cells were treated with MLN4924 for 4 hr before harvesting them for affinity-purification (AP) with Streptactin (ST) beads and immunoblotting as indicated. (WCE, whole cell extracts). The white asterisks indicate individual F-box proteins. (B) HEK293T cells were transfected with an EV, FLAG-HA-tagged FBXW5 (FH-FBXW5), or FH-FBXW5(ΔF) together with SF-tagged SEC23B. Twenty-four hours after transfection, cells were harvested for immunoblotting. (C) HEK293T cells were transfected with FH-FBXW5 and SF-SEC23B in combination with either an EV, MYC-tagged DN-CUL1, or MYC-tagged DN-CUL4 as indicated. Twenty-four hours after transfection, cells were either left untreated (UT) or treated with MG132 for 6 hr, and finally harvested for immunoblotting. (D) U-2OS cells were transfected with either a non-targeting siRNA oligo (NT) or two different FBXW5 siRNA oligos (individually). Seventy-two hours after siRNA transfection, cells were treated with cycloheximide (CHX) for the indicated times and harvested for immunoblotting. The asterisk indicates a nonspecific band. The graph shows the quantification of SEC23B levels from three independent experiments. Error bars indicate standard deviation. (E) HEK293T cells were transfected with an EV, HA-tagged FBXW5, or HA-tagged FBXW5(ΔF) together with MYC-tagged SEC23B and FLAG-TR-TUBE cDNA as indicated. WCLs were immunoprecipitated (IP) with anti-FLAG resin and immunoblotted as indicated. The line on the right marks a ladder of bands corresponding to poly-ubiquitylated SEC23B. (F) HEK293T cells were transfected with either an EV or FH-FBXW5. Twenty-four hours after transfection, cells were either left untreated (UT) or treated with MLN4924 for 4 hr before harvesting them for immunoprecipitation (IP) with FLAG beads and immunoblotting as indicated. (G) HEK293T cells were transfected with SF-FBXW5 and increasing amounts of HA-tagged SEC24B as indicated. Twenty-four hours after transfection, cells were harvested for affinity-purification (AP) with Streptactin (ST) beads and immunoblotting as indicated. (H) WCEs from HEK293T cells were incubated with either unconjugated beads or beads coupled to a SEC23B peptide (a.a. 180–194, CEGISKSYVFRGTKD). Beads were washed with lysis buffer and bound proteins were eluted and subjected to SDS-PAGE and immunoblotting.

https://doi.org/10.7554/eLife.42253.002

Next, we investigated whether SEC23B is targeted for proteolysis by FBXW5. Expression of wild-type FBXW5 resulted in a reduction in the levels of both endogenous and exogenous SEC23B, as detected by immunoblotting or immunofluorescence microscopy (Figure 1B, Figure 1—figure supplement 1A–B, and Figure 1F). This reduction depended on the ability of FBXW5 to form an active SCF complex as demonstrated by the observation that the expression of FBXW5(ΔF), a mutant in which the F-box domain was deleted, did not affect SEC23B protein levels (Figure 1B). Moreover, either co-expression with dominant negative (DN)-CUL1 (a mutant of CUL1 lacking its C-terminus, which retains the binding to F-box proteins and SKP1, but not to the catalytic subunit RBX1) or addition of MLN4924 (a NEDD8 activating enzyme inhibitor used to inhibit the activity of SCFs) blocked the FBXW5-dependent degradation of SEC23B (Figure 1C and Figure 1F). In contrast, although it has been reported that FBXW5 can form a functional CRL4FBXW5 complex (Hu et al., 2008; Kim et al., 2013), co-expression of a CUL4 dominant negative mutant (DN-CUL4) did not interfere with the FBXW5-dependent degradation of SEC23B (Figure 1C). The observed reduction of SEC23B protein levels upon FBXW5 transfection was also rescued by the addition of the proteasome inhibitor MG132 (Figure 1C), indicating that the decrease in SEC23B levels was due to proteasome-mediated proteolysis.

To confirm that FBXW5 regulates the degradation of SEC23B, we used RNA interference to reduce FBXW5 expression. Depleting FBXW5 using three different siRNA oligos (each individually) induced an increase in both the steady state-levels and the stability of endogenous SEC23B in two different cell types (Figure 1D and Figure 1—figure supplement 1C). Moreover, expression of FBXW5, but not FBXW5(ΔF-box), promoted the in vivo ubiquitylation of SEC23B (Figure 1E). This was observed in HEK293T by expressing a FLAG-tagged, trypsin-resistant tandem ubiquitin-binding entity (TR-TUBE), which directly binds poly-ubiquitin chains and protects them from proteasome-mediated degradation (Dankert et al., 2016; Yoshida et al., 2015). After immunoprecipitation of FLAG-tagged TR-TUBE, high molecular weight ubiquitylated species of SEC23B were detected in lysates of cells expressing FBXW5, but not FBXW5(ΔF-box) (Figure 1E).

Altogether, these results indicate that FBXW5 controls the ubiquitin- and proteasome-mediated degradation of SEC23B. Intriguingly, SEC23A did not bind FBXW5, and its levels were not affected by FBXW5 silencing (Figure 1F and Figure 1—figure supplement 1D–E), indicating that SEC23B, but not SEC23A, is a substrate of SCFFBXW5.

Immunopurified FBXW5 does not co-precipitate other COPII subunits (Figure 1F and http://www.stowers.org/research/publications/libpb-1118), suggesting that the subpopulation of SEC23B interacting with FBXW5 is not integrated within COPII vesicles. SEC23B and SEC24 proteins form tight heterodimers (Fromme et al., 2008); therefore, we reasoned that FBXW5 and SEC24 proteins might compete for the binding to SEC23B. To examine this possibility, we co-transfected HEK293T cells with FBXW5 and increasing amounts of SEC24B and subjected the resulting lysates to affinity purification. In line with our hypothesis, increasing concentrations of SEC24B resulted in a marked decrease in the binding of endogenous SEC23B to FBXW5 (Figure 1G). Increasing amounts of SEC24B also induced an increase in the levels of endogenous SEC23B (Figure 1F), likely due to the ability of SEC24B to impair the interaction between SEC23B and FBXW5. These results support the hypothesis that SEC23B cannot simultaneously bind to FBXW5 and SEC24B, suggesting that these two proteins compete for the same binding region on SEC23B. In agreement with this model, using a panel of SEC23B deletion mutants, we found that the binding to FBXW5 is mediated by the TRUNK domain of SEC23B (Figure 1—figure supplement 1F–H), which has been shown to mediate the SEC23-SEC24 interaction (Mancias and Goldberg, 2008). In fact, SEC23B(100-767), a deletion mutant containing the TRUNK domain, was able to co-precipitate endogenous FBXW5, whereas SEC23B(400-767), a mutant missing the TRUNK domain, was not (Figure 1—figure supplement 1F–H).

Next, we used an immobilized synthetic peptide containing the previously reported SEC24-interacting sequence of SEC23B (a.a. 180–194 in human, see Figure 1—figure supplement 1F and Figure 1—figure supplement 1H) (Mancias and Goldberg, 2008) and tested its ability to bind endogenous FBXW5 present in cellular extracts. While the immobilized SEC23B peptide efficiently bound FBXW5, it failed to pull down other F-box proteins (Figure 1H), suggesting that this 14-amino acid region is sufficient for the binding to FBXW5.

Taken together, these results indicate that FBXW5 and SEC24B associate with SEC23B in a mutually exclusive fashion and may compete for the same binding region in SEC23B.

ULK1 phosphorylates SEC23B on serine 186 that is present in the binding motif for FBXW5 and SEC24

The interactions between F-box proteins and their cognate substrates are often regulated by post-translational modifications (most often phosphorylation) (Skaar et al., 2013). Therefore, we looked for phosphorylation consensus sequences for known kinases within the FBXW5-binding region of SEC23B and found that Serine 186 (S186) is part of a highly conserved ULK1 phosphorylation motif (Egan et al., 2015) (Figure 2—figure supplement 1A–B). We first investigated whether S186 is phosphorylated in cells. To this end, we generated a phospho-specific antibody against a peptide (a.a. 180–194) containing phosphorylated Serine at position 186. This antibody specifically detected the phosphopeptide, but not the unphosphorylated peptide (Figure 2—figure supplement 1C). Moreover, it recognized both wild-type SEC23B and a SEC23B(S186D) mutant (which mimics Ser186 phosphorylation), but not the SEC23B(S186A) mutant (Figure 2—figure supplement 1D), providing evidence that SEC23B is phosphorylated in vivo on S186.

Next, to test whether Ser186 on SEC23B can serve as a phospho-acceptor for ULK1, we co-expressed FLAG-tagged, wild-type SEC23B together with either wild type ULK1 or a kinase-dead (KD) mutant. Only wild-type ULK1 induced the phosphorylation of both endogenous and exogenous SEC23B on S186, as detected with our phospho-specific antibody (Figure 2A and Figure 2—figure supplement 1E) (endogenous phosphorylated SEC23B was detected in whole cell extracts, and exogenous phosphorylated SEC23B was detected in both cell extracts and anti-FLAG immunoprecipitates). In contrast, in cells expressing SEC23B(S186A), only endogenous SEC23B was phosphorylated by ULK1(Figure 2A and Figure 2—figure supplement 1E). Addition of a specific ULK1 inhibitor, SBI-0206965, blocked the ULK1-dependent phosphorylation of endogenous SEC23B in a dose-dependent manner, similar to what observed for ATG13 (Figure 2B), a canonical substrate of ULK1. We further confirmed that SEC23B is an ULK1 substrate by using purified proteins in an in vitro phosphorylation assay (Figure 2C).

Figure 2 with 1 supplement see all
ULK1 phosphorylates SEC23B on Serine 186.

(A) HEK293T cells were transfected with either FLAG-tagged SEC23B or FLAG-tagged SEC23B(S186A) in combination with MYC-tagged ULK1 or MYC-tagged ULK1-KD as indicated. Twenty-four hours after transfection, cells were harvested for immunoprecipitation (IP) and immunoblotting. exo and endo indicate the exogenous and endogenous SEC23B, respectively. (B) HEK293T cells were transfected with the SF-ULK1 or SF- ULK1-KD as indicated. Twenty-four hours after transfection, cells were treated with various doses of SBI-0206965 (an ULK1 inhibitor) for 4 hr before harvesting them for immunoblotting. (C) In vitro kinase assays were performed using purified SEC23B (wild-type or the S186A mutant) and ULK1 (wild-type or a kinase-dead mutant) as substrate and kinase, respectively. Purified SEC23B and ULK1 proteins were prepared by immunoprecipitation (followed by elution) from extracts of HEK293T cells transfected with each corresponding plasmid. (D) HEK293T cells were nutrient-starved with EBSS for the indicated times and harvested for immunoblotting. (E) HEK293T cells were nutrient-starved with EBSS for the indicated times (in the presence or absence of SBI-0206965) and harvested for immunoblotting at the indicated times. (F) HEK293T cells were recovered from nutrient-starvation (EBSS for 4 hr) for the indicated times and harvested for immunoblotting.

https://doi.org/10.7554/eLife.42253.005

Next, we studied how nutrient deprivation, a condition that activates ULK1 and autophagy, modulates the phosphorylation of SEC23B on S186. After substituting the growth medium, which contains 10% fetal bovine serum, with Earle's balanced salt solution (EBSS), we observed de-phosphorylation of ULK1 and phosphorylation of ATG13 and BECLIN-1, two known substrates of ULK1 (Figure 2D–E), indicating that mTORC1 activity is inhibited and ULK1 and autophagy are activated. Starvation also promoted the co-localization of SEC23B and ULK1 (Figure 2—figure supplement 1F). Moreover, EBSS induced a time-dependent increase in the phosphorylation of endogenous SEC23B similar to what observed for ATG13 and BECLIN-1, and this phosphorylation was inhibited by treating cells with SBI-0206965 (Figure 2D–E). Recovery from nutrient deprivation by addition of fetal bovine serum for three hours resulted in the de-phosphorylation of SEC23B and a decrease in its total levels (Figure 2F).

We concluded that, in response to starvation, ULK1 phosphorylates SEC23B on S186.

ULK1-dependent phosphorylation of SEC23B inhibits its interaction with and degradation via FBXW5

To investigate whether phosphorylation on S186 affects the SEC23B-FBXW5 interaction, we used a phosphorylated version of the synthetic peptide employed for the binding experiments. While the non-phosphorylated peptide efficiently bound FBXW5, as previously observed (Figure 1H), a corresponding peptide containing phosphorylated Ser186 displayed a strongly reduced ability to bind FBXW5 (Figure 3—figure supplement 1A). This indicates that Ser186 phosphorylation inhibits the interaction between these two proteins, in agreement with the fact that the binding of FBXW5 to the phospho-mimetic SEC23B(S186D) mutant is abolished, while FBXW5 binding to SEC23B(S186A) is increased (Figure 3A). Accordingly, endogenous SEC23B phosphorylated on Ser186 was not affinity purified with FBXW5 (Figure 3B). Similarly, upon nutrient deprivation (when SE23B becomes phosphorylated on Ser186) the interaction between FBXW5 and SEC23B decreased (Figure 3—figure supplement 1B).

Figure 3 with 1 supplement see all
ULK1-mediated phosphorylation of SEC23B on S186 prevents the FBXW5-dependent degradation of SEC23B.

(A) HEK293T cells were transfected with either EV, SF-SEC23B, or the indicated SF-SEC23B mutants. Twenty-four hours after transfection, cells were treated with MLN4924 for 4 hr before harvesting them for affinity-purification (AP) with Streptactin (ST) beads and immunoblotting. The asterisk indicates a nonspecific band. (B) HEK293T cells were transfected with SF-FBXW5 in combination with either MYC-tagged ULK1 or MYC-tagged ULK1-KD. Twenty-four hours after transfection, cells were treated with MLN4924 for 4 hr before harvesting them for affinity-purification (AP) with Streptactin (ST) beads and immunoblotting as indicated. (C) HEK293T cells were transfected with FH-FBXW5 in combination with SF-SEC23B or SF-SEC23B(S186D). Twenty-four hours after transfection, cells were harvested for immunoblotting. (D) U-2OS cells stably infected with viruses expressing either SEC23B or SEC23B(S186D) were treated with cycloheximide for the indicated times. The cells were then harvested for immunoblotting. (E) RPE1-hTERT cells were transfected with siRNAs against the indicated mRNAs. Sixty-eight hours after transfection, cells were nutrient-starved with EBSS for 4 hr and harvested for immunoblotting (left panel) and real-time PCR using ULK2 and GAPDH primers (right panel). The asterisk indicates the nonspecific band.

https://doi.org/10.7554/eLife.42253.008

Consistent with the observed impaired interaction with FBXW5, the levels of SEC23B(S186D) did not decrease in the presence of FBXW5 (Figure 3C) and it exhibited a longer half-life than wild-type SEC23B (Figure 3D). These results suggest that ULK1 phosphorylates Ser186 in SEC23B, inhibiting its interaction with and degradation via FBXW5. Concurring with this hypothesis, silencing of ULK1, but not ULK2, reduced the levels of SEC23B phosphorylated on Ser186 and induced a decrease in the levels of endogenous SEC23B in starved cells (Figure 3E). Moreover, co-silencing FBXW5 rescued the decrease in SEC23B levels induced by the depletion of ULK1 but did not alter the amount of phosphorylated SEC23B (Figure 3E), confirming that ULK1 and FBXW5 have an antagonistic effect on the control of SEC23B protein abundance. Finally, ULK1 silencing had no effects on the levels of SEC23A (Figure 3E), and SEC23A levels do not increase upon starvation (Figure 2D).

Significantly, although Ser186 is located at the interface between the SEC23 and SEC24 heterodimer binding region (Mancias and Goldberg, 2008) (Figure 1—figure supplement 1H) and its phosphorylation blocks the SEC23B-FBXW5 interaction (Figure 3A–B), endogenous phosphorylated SEC23B interacted with SEC24B (Figure 3—figure supplement 1C). Moreover, the phospho-mimetic SEC23B(S186D) mutant interacted with SEC24A, SEC24B, SEC13, SE16, and SEC31 (Figure 3—figure supplement 1D, see also Figure 6A). In addition, both wild-type SEC23B and phospho-mimetic SEC23B(S186D) efficiently bound to the small GTPase SAR1(H79G), a GTP-bound SAR1 mutant that is constitutively associated with COPII vesicles, but not with the cytosolic GDP-bound SAR1(T39N) mutant (Venditti et al., 2012) (Figure 3—figure supplement 1E).

Altogether, these results indicate that the ULK1-dependent phosphorylation of SEC23B blocks its FBXW5-dependent degradation but does not interfere with either the formation of the SEC23B-SEC24 heterodimer or its recruitment to the COPII vesicle coat by SAR1.

The FBXW5-mediated degradation of SEC23B limits the autophagic flux in the presence of nutrients

Since ULK1 plays an essential role in the induction of autophagy, and since COPII proteins, in addition to their role in secretion, are also required for the proper execution of the autophagic program (see Introduction), we investigated whether the ULK1-dependent regulation of the interaction between FBXW5 and SEC23B regulates the autophagic flux. Automated quantification of both endogenous LC3 (Figure 4A) and exogenous GFP-LC3 (Figure 4—figure supplement 1A) showed that the LC3 puncta area increased upon FBXW5 downregulation in cells grown in the presence of nutrients. Measurement of area of LC3-positive puncta was preferred to individual number of puncta per cell because clustering of the vesicular compartments in some images made delineation of individual vesicles inaccurate. The increase in LC3-positive puncta could in principle reflect either an increase in autophagic activity or an impairment in the lysosome-dependent degradation of lipidated LC3 (Klionsky et al., 2016). Compared to untreated cells, treatment of FBXW5-depleted cells with bafilomycin A1, a proton ATPase inhibitor that blocks the degradation of lipidated LC3 but not the formation of autophagosomes, significantly increased LC3 puncta (Figure 4A and Figure 4—figure supplement 1A), as well as the amount of lipidated LC3 (Figure 4—figure supplement 1B). These results suggest that depletion of FBXW5 increases autophagic flux during unstressed conditions (i.e., it increases basal autophagy). Consistent with this hypothesis, FBXW5 downregulation did not induce a further increase in the appearance of LC3 puncta upon induction of autophagy by nutrient starvation (Figure 4—figure supplement 1C), a condition under which the FBXW5-mediated degradation of SEC23B is inhibited by ULK1 (Figure 3). We also evaluated the turnover of p62, a cargo adaptor and substrate of autophagy (Galluzzi et al., 2018). Upon FBXW5 silencing, p62 degradation was activated in two different cell lines under nutrient-rich conditions (Figure 4B).

Figure 4 with 1 supplement see all
The FBXW5-mediated degradation of SEC23B limits the autophagic flux in the presence of nutrients.

(A) RPE1-hTERT cells were transfected with a non-targeting (NT) oligo or a FBXW5-targeting siRNA oligo. Forty-eight hours after transfection, cells were re-plated onto coverglass for immunofluorescence with an anti-LC3 antibody. Where indicated, cells were treated with Bafilomycin A1 (BafA1) for 4 hr before fixation. Images of endogenous LC3 puncta underwent automated processing with at least 300 cells counted per sample. Because in several images LC3·puncta were too close to be distinguished, we adopted LC3 puncta area as a criterion for our analysis. The data are presented as mean ±SD (right panel). Scale bar, 10 μm. (B) HeLa and RPE1-hTERT cells were transfected with the indicated siRNAs. Seventy-two hours after transfection, the cells were harvested for immunoblotting. (C) U-2OS cells stably expressing tandem fluorescent-tagged LC3 (pBabe-mCherry-EGFP-LC3) were transfected with a NT oligo or a FBXW5 siRNA oligo, alone or in combination with a SEC23B-targeting siRNA oligo as indicated. Forty-eight hours after transfection, cells were replated onto coverglass, followed by fixation twenty-four hours after replating. Images of mCherry-EGFP-LC3 puncta underwent automated processing with at least 100 cells counted per sample. The data are presented as mean ±SD (right panel). The yellow and red bars represent green +red double positive LC3 puncta (autophagosome) and red only positive LC3 puncta (autolysosome), respectively. Scale bar, 10 μm. (D) U-2OS cells were infected with lentiviruses expressing either wild-type SEC23B or SEC23B(S186D). Twenty-four hours after infection, cells were fixed for immunofluorescence. Images of endogenous LC3 puncta underwent automated processing with at least 300 cells counted per sample. The data are presented as mean ±SD (right panel). Scale bar, 10 μm. (E) U-2OS cells were infected with lentivirus expressing SEC23B(S186D). Where indicated, forty-eight hours after infection, cells were treated with BafA1 prior to harvest and immunoblotting. (F) U-2OS cells were infected with the increasing amounts of lentivirus expressing SEC23B. Forty-eight hours after infection, cells were harvested for immunoblotting. (G) RPE1-hTERT cells stably expressing GFP-tagged LC3 were transfected with a NT oligo or the indicated siRNA oligos. Forty-eight hours after transfection, cells were replated onto coverglass, followed by treatment with EBSS for 1 hr and fixation. Images of GFP-LC3 puncta underwent automated processing with at least 300 cells counted per sample. The data are presented as mean ±SD.

https://doi.org/10.7554/eLife.42253.011

Next, we used a tandem fluorescent-tagged LC3 construct (mCherry-GFP-LC3) that allows monitoring autophagosome maturation as a change from double-positive, green +red (i.e., yellow) fluorescent vesicles (autophagosomes), to single-positive, red fluorescent vesicles (autolysosomes) deprived of GFP fluorescence due to its quenching at low pH (Klionsky et al., 2016). Using this method, we demonstrated that the majority of LC3 puncta that accumulated upon FBXW5 depletion were autolysosomes and not autophagosomes (Figure 4C), further suggesting that increased levels of SEC23B promotes autophagic flux.

Importantly, co-depletion of SEC23B almost completely prevented the increase in autophagy mediated by FBXW5 silencing (Figure 4C and Figure 4—figure supplement 1B). Moreover, expression of SEC23B(S186D) significantly induced higher levels of LC3 puncta compared to wild-type SEC23B (Figure 4D) and activated p62 degradation under nutrient-rich conditions (Figure 4E). We also observed that high levels of wild-type SEC23B were able to induce p62 degradation (Figure 4F), suggesting that a threshold of SEC23B protein expression (and not the presence of the phospho-mimetic mutation per se) is a critical determinant to induce autophagy. In agreement with a role for SEC23B in promoting autophagy, SEC23B silencing inhibited autophagy in response to starvation (Figure 4G). Finally, although, expression of SEC23B(S186D) is sufficient to induce autophagy in the presence of nutrients, it was unable to rescue autophagy in ULK1 knockdown cells (Figure 4—figure supplement 1D), likely because ULK1 is necessary to phosphorylate additional pro-autophagic substrates (Hurley and Young, 2017) and promote the assembly of COPII complexes (Joo et al., 2016).

Ser186 in SEC23B is necessary for its localization to the ERGIC and an efficient autophagic response upon nutrient deprivation

The ERGIC compartment produces vesicles that are active for LC3 lipidation (Egan et al., 2015; Ge et al., 2013; Ge et al., 2014). We found that, whereas upon starvation wild-type SEC23B colocalized approximately twice more with the ERGIC membrane marker ERGIC53, the localization of SEC23B(S186A) did not change after nutrient deprivation (Figure 5A). Notably, SEC23B(S186D) colocalization with ERGIC53 was already high in the presence of nutrients. Accordingly, compared to wild-type SEC23B, SEC23B(S186D) co-distributed approximately twice more with ERGIC53 in fractionated membranes (Figure 5—figure supplement 1A) and ULK1 silencing inhibited SEC23B-ERGIC53 colocalization upon starvation (Figure 5B). Finally, evaluation of the secretory pathway by assaying secreted Gaussia Luciferase (Badr et al., 2007) indicated that overexpression of either SEC23B(S186D) or ULK1, but not wild-type SEC23B, SEC23B(S186A), or ULK1 knockdown, resulted in an inhibition of secretion in the presence of nutrients, similarly to what observed when cells were staved (Figure 5C–D).

Figure 5 with 1 supplement see all
Ser186 in SEC23B is necessary for its localization to the ERGIC and an efficient autophagic response upon nutrient deprivation.

(A) U-2OS cells were transfected with either FLAG-HA-tagged wild-type SEC23B, SEC23B(S186A), or SEC23B(S186D). Twenty-four hours after transfection, cells were fixed for immunofluorescence as indicated. Images were analysed by ImageJ with at least 100 cells counted per sample. Quantification of SEC23B overlapping with ERGIC53 was performed using the Pearson's correlation coefficient. The data are presented as mean ±SD (bottom panel). Scale bar, 10 μm. (B) U-2OS cells stably expressing FLAG-HA-tagged SEC23B were transfected with ULK1 siRNAs. Seventy-two hours after transfection, cells were fixed for immunofluorescence as indicated. Images were analysed by ImageJ with at least 100 cells counted per sample. Quantification of SEC23B overlapping with ERGIC53 was performed using the Pearson's correlation coefficient. The data are presented as mean ±SD (bottom panel). Scale bar, 10 μm. (C) HEK239T cells were transfected with a plasmid expressing Gaussia luciferase in combination with the indicated constructs. Twenty-four hours after transfection, cells were replated onto 96-well plates. After another forty-eight hours, fresh media was added to the cells, and four hours after, the culture media were collected to measure Gaussia luciferase activity. The data are presented as mean ±SD of the Gaussia luciferase activity of triplicate samples. Expression of FBXW5 was used as a positive control since it results in the downregulation of SEC23B and, therefore, it is expected to inhibit trafficking. (D) A375 cells stably expressing Gaussia luciferase were plated onto 96-well plates. After forty-eight hours, either fresh media or EBSS was added to the cells and four hours after, the culture media were collected to measure Gaussia luciferase activity. The data are presented as mean ±SD of the Gaussia luciferase activity of triplicate samples. (E) A375 parental cells or SEC23B(S186A)-A357 knock-in cells were starved with EBSS for the indicated times (±BafA1) and harvested for immunoblotting as indicated. (F) A375 parental cells or SEC23B(S186A)-A357 knock-in cells were starved with EBSS and fixed for immunofluorescence. Images of endogenous LC3 puncta underwent automated processing with at least 100 cells counted per sample. The data are presented as mean ±SD (right panel). Scale bar, 10 μm.

https://doi.org/10.7554/eLife.42253.015

Altogether, these results suggest that ULK1-mediated phosphorylation of SEC23B promotes both its localization to the ERGIC and its autophagic function, possibly at the expense of its secretory function.

Next, we used a CRISPR/Cas9-dependent strategy to generate A375 cells in which all three alleles of SEC23B were mutated to SEC23B(S186A) (Figure 5—figure supplement 1B). In contrast to wild-type SEC23B, levels of SEC23B(S186A) did not increase upon starvation (Figure 5E). Significantly, automated quantification of endogenous LC3 showed that, in contrast to parental cells, nutrient deprivation-induced autophagy was strongly reduced in SEC23B(S186A) knock-in cells, while basal autophagy remained unperturbed (Figure 5F). Finally, in response to starvation, p62 was degraded in parental cells but not in SEC23B(S186A) knock-in cells (Figure 5E).

The above results indicate that the presence of Ser186 in SEC23B and, presumably, its phosphorylation and consequent stabilization are required for the proper induction of autophagy in response to starvation.

SEC24A/B, but not SEC24C/D, specifically associate with phosphorylated SEC23B and contribute to autophagy

Vertebrates express four SEC24 paralogs (Fromme et al., 2008), but it is not known whether any of them contribute to the regulation of the autophagic flux. We observed no difference in binding of the phospho-mimetic SEC23B(S186D) mutant with SEC24A and SEC24B (Figure 3—figure supplement 1D). Because the region of SEC23B that binds FBXW5 and SEC24 proteins overlaps, we investigated whether ULK1-dependent phosphorylation of SEC23B, in addition to blocking its interaction with FBXW5, affects SEC23B association with the other SEC24 paralogs. We observed that whereas both wild-type SEC23B and SEC23B(S186A) associated with all four SEC24 paralogs, SEC23B(S186D) only interacted with SEC24A and SEC24B (Figure 6A–B). Similarly, SEC24B, but not SEC24C, interacted with endogenous phosphorylated SEC23B (Figure 6C). Accordingly, molecular dynamics simulations showed that, in contrast to what observed with SEC23B(S186D) and SEC24A, there was a significant loss in the average number of contacts between SEC23B(S186D) and SEC24C, compared to contacts observed with wild-type SEC23B (Figure 6D). We also performed Molecular Mechanics/Generalized Born Surface Area calculations, which predicted a significantly higher value of binding free energy for the SEC23B(S186D)-SEC24C mutant dimer, resulting in the following trend: SEC23B-SEC24A ~ SEC23B-SEC24C SEC23B(S186D)-SEC24A << SEC23B(S186D)-SEC24C (Figure 6D). These observations indicate a lack of stability of the SEC23B(S186D)-SEC24C system compared to the other three, in agreement with our experimental results.

SEC24A/B, but not SEC24C/D, specifically associate with phosphorylated SEC23B and contribute to autophagy.

(A) HEK293T cells were transfected with an EV, SF-SEC23B, or SF-SEC23B mutants. Twenty-four hours after transfection, cells were treated with MLN4924 for 4 hr before harvesting them for affinity-purification (AP) with Streptactin (ST) beads and immunoblotting. (B) HEK293T cells were transfected with an EV or SF) SEC23B(S186D) in combination with either HA-tagged SEC24 paralogs as indicated. Twenty-four hours after transfection, cells were treated with MLN4924 for 4 hr before harvesting them for immunoprecipitation (IP) with anti-HA beads and immunoblotting. (C) HEK293T cells were transfected with either HA-tagged SEC24B or HA-tagged SEC24C together with SF-ULK1. Twenty-four hours after transfection, cells were treated with MLN4924 for 4 hr before harvesting them for immunoprecipitation (IP) with anti-HA beads and immunoblotting. (D) Evolution of the inter-molecular contacts between monomers in the four studied systems during 1 μs molecular dynamics simulations. Upper panel: SEC23B-SEC24C (red), SEC23B(S186D)-SEC24C (violet); bottom panel: SEC23B-SEC24A (red), SEC23B(S186D)-SEC24A (violet). Contacts were calculated as the number of heavy atom interacting pairs within a distance of 4.4 Å. (E) U-2OS cells stably expressing tandem fluorescent-tagged LC3 (pBabe-mCherry-EGFP-LC3) were transfected with a NT oligo or a FBXW5 siRNA oligo in combination with the indicated siRNA oligos. Forty-eight hours after transfection, cells were replated onto coverglass followed by fixation twenty-four hours after replating. Images of mCherry-EGFP-LC3 puncta underwent automated processing with at least 100 cells counted per sample. The data are presented as mean ±SD (right panel). Scale bar, 10 μm (F) A375 parental cells or SEC23B(S186D)-A357 knock-in cells were transfected with either HA-tagged SEC24B or HA-tagged SEC24C. Twenty-four hours after transfection, cells were either left untreated (UT) or starved with EBSS for two hours. Next, cells were fixed for immunofluorescence as indicated. Images were analysed by ImageJ with at least 100 cells counted per sample. Quantification of SEC24 overlapped with ERGIC53 was performed using the Pearson's correlation coefficient. The data are presented as mean ±SD (right panel). Scale bar, 10 μM.

https://doi.org/10.7554/eLife.42253.018

Altogether, these results suggest that upon serum starvation, the population of phosphorylated SEC23B preferentially associates with SEC24A and SEC24B.

Notably, co-depletion of both SEC24A and SEC24B, but not SEC24C and SEC24D, inhibited the increase in autophagic flux induced by the silencing of FBXW5 (Figure 6E and Figure 4—figure supplement 1B), similar to what we observed by co-depleting SEC23B together with FBXW5 (Figure 4C). We also evaluated by immunofluorescence the colocalization of SEC24 family members with ERGIC53. Upon starvation, SEC24B, but not SEC24C, colocalized more with ERGIC53 and this event was not observed in SEC23B(S186A) knock-in cells (Figure 6F), suggesting that the localization of SEC24B to the ERGIC is regulated by its association with phospho-SEC23B.

Thus, our data demonstrate the existence of a high degree of specificity among SEC24 paralogs with respect to their ability to contribute to the autophagic flux.

A human melanoma-associated mutation in SEC23B results in its stabilization and increased autophagy flux

By searching for mutations of SEC23B in publicly available human cancer databases, we found a mutation in human melanoma that converts S186 of SEC23B to asparagine (ID#: COSM5391641). We thus generated SEC23B(S186N), a mutant that mimics the cancer associated mutation, and observed a reduced binding to FBXW5, SEC24C, and SEC24D (Figure 7A) but not to SEC24A and SEC24B, similar to what observed with SEC23B(S186D) (Figure 6A). Moreover, SEC23B(S186N) displayed a longer half-life than wild-type SEC23B (Figure 6B). These results suggest that SEC23B(S186N), by being stable, may promote autophagy. To test this hypothesis, we used CRISPR/Cas9 to generate SEC23B(S186N) A375 melanoma cells (Figure 5—figure supplement 1B). Automated quantification of endogenous LC3 showed that, compared to parental cells, the SEC23B(S186N) knock-in cells displayed more autophagy during unstressed conditions (Figure 7C). The number of LC3 puncta were also increased in the presence of bafilomycin A1 (Figure 7C), indicating that SEC23B(S186N) increases the autophagic flux, and that the constitutive increase in LC3 puncta is not due to inhibition of the lysosome-dependent degradation of autophagosomes.

A human melanoma-associated mutation in SEC23B results in its stabilization and increased autophagy flux.

(A) HEK239T cells were transfected with either SF-SEC23B or SF-SEC23B(S186N). Twenty-four hours after transfection, cells were harvested for immunoprecipitation (IP) with FLAG-M2 beads and immunoblotted as indicated. (B) HEK293T cells were transfected with either Streptag-FLAG-tagged (SF) wild-type SEC23B (SF-SEC23B) or Streptag-FLAG-tagged (SF) SEC23B(S186N). Twenty-four hours after transfection, cells were treated with cycloheximide (CHX) for the indicated time and subjected to immunoblot analysis. (C) A375 parental cells or SEC23B(S186N)-A357 knock-in cells were treated with BafA1 and fixed for immunofluorescence. Images of endogenous LC3 puncta underwent automated processing with at least 100 cells counted per sample. The data are presented as mean ±SD (right panel). Scale bar, 10 μm.

https://doi.org/10.7554/eLife.42253.020

These results show that the SEC23B(S186N) mutation found in melanoma mimics SEC23B phosphorylated on Ser186 similarly to what observed for SEC23B(S186D). In fact, SEC23B(S186N) does not bind efficiently FBXW5, SEC24C, and SEC24D, and promotes autophagy, in agreement with a possible advantageous role resulting from increasing autophagic flux to ensure tumour cell homeostasis (Rybstein et al., 2018).

Discussion

SEC23B is an essential component of the COPII multi-subunit protein complex that is responsible for the transport of cargo proteins destined to be secreted (Fromme et al., 2008; Zanetti et al., 2011). Although COPII vesicles were originally thought to participate exclusively in the secretory pathway, growing evidence demonstrates that they also play an important role in controlling and executing the autophagic cascade (see Introduction). However, the molecular mechanisms that repurpose COPII for the autophagic process have remained largely unknown. In this study, we demonstrated that FBXW5 targets SEC23B for degradation to limit autophagy during basal, unperturbed conditions. Upon induction of autophagy by nutrient deprivation, ULK1 phosphorylates Ser186 in SEC23B, inhibiting its interaction with and degradation via FBXW5. The resulting increased levels of SEC23B are crucial for proper autophagic flux. We also found that phosphorylation of Ser186 in SEC23B inhibits its binding to SEC24C and SEC24D, but not SEC24A and SEC24B. Accordingly, depletion of SEC24A and SEC24B, but not SEC24C and SEC24D rescue the phenotype (i.e., the increase in autophagy during non-stressed conditions) induced by FBXW5 silencing. The fact that the phosphorylation of Ser186 inhibits the binding of SEC23B to SEC24C and SEC24D may explain, at least in part, the reduced secretion observed upon nutrient starvation. It would be interesting to understand whether Ser186 phosphorylation also modulates the GAP activity of SEC23B towards SAR1.

It has been suggested that autophagic COPII vesicles can be distinguished from trafficking COPII vesicles by their different site of generation, that is autophagic COPII vesicles are generated from the ERGIC rather than the ERES, and they are potent stimulators of LC3 lipidation in response to starvation (Ge et al., 2015; Ge et al., 2014). PI3KC3, which is activated by ULK1, is required for the relocation of COPII components to the ERGIC (Ge et al., 2015; Ge et al., 2014). However, the differential molecular composition between trafficking COPII vesicles and autophagic COPII vesicles remain largely unknown. Our results indicate that at least a subpopulation of autophagic COPII vesicles present at the ERGIC upon starvation contain SEC23B phosphorylated on Ser186 by ULK1. Moreover, we show that SEC24B, but not SEC24C, colocalizes more abundantly with the ERGIC, and this event depends on the presence of Ser186 in SEC23B. Interestingly, in yeast, phosphorylation of Sec24 by casein kinase one promotes its binding to autophagy factors to increase autophagosome abundance (Davis et al., 2016), which is reminiscent of what we found in human cells (i.e., phosphorylation of SEC23B by ULK1 induces the localization of SEC23B and, consequently, SEC24A and SEC24B to the ERGIC to promote the autophagic flux).

Recently, it has been shown that, in presence of nutrients, the basal activity of ULK1 mediates the phosphorylation of SEC16A (promoting the assembly of COPII complexes at the ERES) and that, in response to starvation, the activation of ULK1 induces the dissociation of SEC23A from SEC31A (inhibiting the secretory pathway) (Joo et al., 2016; Gan et al., 2017). Our study shows that, in the absence of nutrients, activated ULK1 promotes the phosphorylation of SEC23B on Ser186, inducing SEC23B accumulation and the formation of autophagic COPII vesicles. Moreover, the decrease in the abundance of the SEC23B-SEC24C/D complexes likely contributes to inhibit the secretory pathway.

Altogether, our results provide evidence for one of the molecular mechanisms by which ULK1 functions as a switch necessary to commit COPII to autophagy in response to starvation.

Materials and methods

Key resources table
Reagent type
(species)
or resource
DesignationSource or
reference
IdentifiersAdditional
information
AntibodyFBXW5This paperYenzyme(1:250 dilution for
western blotting)
AntibodyPhospho-
SEC23B (S186)
This paperYenzyme(1:200 dilution for
western blotting)
AntibodySEC23ARandy
Schekman’s lab
PMID: 24069399(1:1000 dilution for
western blotting)
AntibodySEC23BRandy
Schekman’s lab
PMID: 24069399(1:1000 dilution for
western blotting)
AntibodySEC24ARandy
Schekman’s lab
PMID: 18056412(1:2000 dilution for
western blotting)
AntibodySEC24BRandy
Schekman’s lab
PMID: 18056412(1:2000 dilution for
western blotting)
AntibodySEC24CRandy
Schekman’s lab
PMID: 18056412(1:2000 dilution for
western blotting)
AntibodySEC24DRandy
Schekman’s lab
PMID: 18056412(1:2000 dilution for
western blotting)
AntibodyFLAGSigma-AldrichCat# F7425(1:1000 dilution
for western blotting,
1:1000 for immunofluorescence)
AntibodyHACovanceCat# MMS-
101P
(1:1000 dilution for
western blotting)
AntibodyMYCSigma-Aldrich
Cat# M5546
(1:1000 dilution for
western blotting)
AntibodySAR1Thermo
Scientific
Cat# PA5
-27642
(1:5000 dilution for
western blotting)
AntibodyCUL1Life
Technologies
Cat# 322400(1:1000 dilution for
western blotting)
AntibodySKP1Michele
Pagano’s lab
Yenzyme(1:1000 dilution for
western blotting)
AntibodySEC13Bethyl
Laboratories
Cat# A303
-980A
(1:10,000 dilution for
western blotting)
AntibodySEC31ABethyl
Laboratories
Cat# A302
-336A
(1:10,000 dilution for
western blotting)
AntibodyULK1Cell Signaling
Technology
Cat# 8054 s(1:1000 dilution for
western blotting)
AntibodyPhospho-ULK1 (S757)Cell Signaling
Technology
Cat# 6888 s(1:1000 dilution for
western blotting)
AntibodyLC3BNovus
Biologicals
Cat# NB100
-2220
(1:10,000 dilution for
western blotting,
1:1000 for immunofluorescence)
AntibodyPhospho-
ATG13 (S318)
Rockland
Immunochemicals
Cat# 600–
401 C49S
(1:1000 dilution
for western blotting)
AntibodyPhospho-
Beclin-1 (S15)
AbbiotecCat# 254515(1:500 dilution
for western blotting)
AntibodyBeclin-1Santa Cruz
Biotechnology
Cat# SC-48381(1:1000 dilution
for western blotting)
AntibodyGAPDHCell Signaling
Technology
Cat# 97166S(1:10,000 dilution
for western blotting)
AntibodyACTINSigma-AldrichCat# A5441(1:10,000 dilution
for western blotting)
AntibodyTubulinSigma-AldrichCat# T6074
-200UL
(1:10,000 dilution
for western blotting)
AntibodyERGIC53Sigma-AldrichCat# E1031
-200UL
(1:5000 dilution for
western blotting,
1:1000 for immunofluorescence)
AntibodySEC16Bethyl
Laboratories
Cat# A300-648A(1:5000 dilution for
western blotting)
AntibodyFBXO11Novus
Biologicals
Cat# H00080204
-B01
(1:1000 dilution for
western blotting)
AntibodyFBXW9Michele
Pagano’s lab
(1:1000 dilution for
western blotting)
Peptide,
recombinant
protein
MG132Peptide
international
Cat# IZL-3175v(final concentration,
5 mM)
Chemical
compound,
drug
MLN4924Active BiochemCat# A-1139(final concentration,
1 mM)
Chemical
compound,
drug
CycloheximideSigma-AldrichCat# C7698-1G(final concentration,
50 ng/ml)
Chemical
compound,
drug
Bafilomycin A1Santa Cruz
Biotechnology
Cat# sc-201550A(final concentration,
0.1 mg/ml)
Chemical
compound,
drug
PolybreneSigma-AldrichCat# H9268-10G(final concentration,
8 μg/ml)
Chemical
compound,
drug
SBI-0206965Selleck
Chemicals
Cat# S7885(final concentration,
1 mM)
Peptide,
recombinant
protein
SEC23B
peptide
This paperYenzyme
Peptide,
recombinant
protein
SEC23B
phospho-
peptide
This paperYenzyme
Cell line (human)HEK293TATCCCat# CRL-3216
Cell line (human)U-2OSATCCCat# HTB-96
Cell line (human)RPE1-hTERTATCCCat# CRL-4000
Cell line (human)A375ATCCCat# CRL-1619
Cell line (human)HeLaATCCCat# CCL-2
Commercial
assay or kit
Pierce Gaussia
Luciferase Flash
Assay Kit
Thermo
Scientific
Cat# 16159
OthersiRNAs to
FBXW5 (#1)
DharmaconCat# J-013389
-05-0002
Oligonucleotides
OthersiRNAs to
FBXW5 (#2)
DharmaconCat# J-013389
-06-0002
Oligonucleotides
OthersiRNAs to
FBXW5 (#3)
DharmaconCat# J-013389
-07-0002
Oligonucleotides
OthersiRNAs to
FBXW5 (#4)
DharmaconCat# J-013389
-08-0002
Oligonucleotides
OthersiRNAs to
FBXW5
Santa Cruz
Biotechnology
Cat# sc-92629Oligonucleotides
OthersiRNAs to
SEC23A
DharmaconCat# M-009582
-00-0005
Oligonucleotides
OthersiRNAs to
SEC23B
DharmaconCat# M-009592
-01-0005
Oligonucleotides
OthersiRNAs to
ULK1
Santa Cruz
Biotechnology
Cat# sc-44182Oligonucleotides
OthersiRNAs to
ULK2
Santa Cruz
Biotechnology
Cat# sc-44183Oligonucleotides
OthersiRNAs to
SEC24A
DharmaconCat# L-024405
-01-0005
Oligonucleotides
OthersiRNAs to
SEC24B
DharmaconCat# L-008299
-02-0005
Oligonucleotides
OthersiRNAs to
SEC24C
DharmaconCat# L-008467
-02-0005
Oligonucleotides
OthersiRNAs to
SEC24D
DharmaconCat# L-008493
-01-0005
Oligonucleotides
OtherNon-targeting
siRNA
(CGUACGCGGAAUACUUCGA)
DharmaconOligonucleotides
Recombinant
DNA reagent
pCS2 + 3x
HA-Sec24A
Randy
Schekman’s lab
Recombinant
DNA reagent
pCS2 + 3x
HA-Sec24B
Randy
Schekman’s lab
Recombinant
DNA reagent
pCS2 + 3x
HA-Sec24C
Randy
Schekman’s lab
Recombinant
DNA reagent
pCS2 + 3x
HA-Sec24D
Randy
Schekman’s lab
Recombinant
DNA reagent
pBabe-puro-
mCherry-EGFP-LC3B
AddgeneCat# 22418
Recombinant
DNA reagent
pcdna6.2- myc
ULK1 wt
AddgeneCat# 27629
Recombinant
DNA reagent
pcdna6.2- myc
ULK1 k46I
AddgeneCat# 27630
Recombinant
DNA reagent
GFP-SAR1
(T39N)
Antonella
De Matteis’ lab
Recombinant
DNA reagent
GFP-SAR1
(H79G)
Antonella De
Matteis’ lab

Cell lines and drug treatments

Cell lines were purchased from ATCC and were routinely checked for mycoplasma contamination with the Universal Mycoplasma Detection Kit (ATCC 30–1012K). All cells were maintained in DMEM/GlutaMAX supplemented with 10% fetal bovine serum (FBS) and penicillin/streptomycin. For nutrient starvation, DMEM and FBS were removed and cells were grown in EBSS (Sigma) for the indicated times.

Biochemical methods

For immunoprecipitation, cell extracts were prepared using lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 0.5% NP-40, protease inhibitors, and phosphatase inhibitors), followed by incubation with Streptactin beads (IBA) or FLAG-M2 beads (Sigma) for 2 hr at 4°C. For immunoblotting, each sample was solubilized with lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 0.5% NP-40, protease inhibitors, and phosphatase inhibitors). Cell extracts were quantified using BCA protein assay kit (Pierce) and solubilized with LDS-sample buffer (Life technology) followed by boiling at 95°C for five minutes.

Immunofluorescence microscopy

Immunofluorescence microscopy was performed as described previously (Jeong et al., 2013). Briefly, cells were cultured on round coverglass in 24-well culture dishes. After the indicated treatments, cells were washed with PBS followed by fixation with either cold methanol or 4% PFA/PBS. Cells were then permeabilized for 15 min with 3% BSA in 0.5% Triton X-100/PBS. Primary antibodies were incubated for one hour at room temperature, and secondary antibodies conjugated to either Alexa Fluor 488 or Alexa Fluor 555 were incubated for one hour at room temperature in 3% BSA/0.1% Triton X-100/PBS. Coverglasses were mounted on slideglass using Pro-long Gold anti-fading reagent with DAPI (Molecular probes).

Affinity purification and mass spectrometry

Affinity purification and mass spectrometry were performed as described previously (Jeong et al., 2013). Briefly, Streptag-FLAG-tagged FBXW5 was transiently transfected into HEK293T cells. Cells were treated with MLN4924 for four hours prior to harvest, and then solubilized with lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 2 mM EDTA, 10% glycerol, 0.5% NP-40, protease inhibitors, and phosphatase inhibitors). Cell extracts were immunoprecipitated with either Streptactin beads (IBA) or FLAG-M2 beads (Sigma). Immunoprecipitation and subsequent mass spectrometry was carried out as previously described (Kuchay et al., 2017).

Plasmids, siRNA and shRNA

FBXW5 and SEC23B mutants were generated using KAPA HiFi polymerase (Kapabiosystems). All cDNAs were subsequently sequenced. ULK1 and ULK1(K46I) plasmids were purchased from Addgene. Sar1 plasmids were generously provided by Dr. Antonella De Matteis. SEC24A, B, C, and D plasmids were generously provided by Dr. Randy Schekman. ON-Target siRNAs targeting FBXW5, SEC23B, SEC24A, SEC24B, SEC24C, SEC24D were purchased from Dharmacon. The production of lentivirus was previously described (Jeong et al., 2013). ULK1 and ULK2 siRNA oligos and pooled FBXW5 siRNA oligos were purchased from Santa Cruz Biotechnology. Non-targeting siRNA oligo (CGUACGCGGAAUACUUCGA) served as a negative control.

Antibodies

An anti-FBXW5 antibody was generated by immunizing rabbits with FBXW5 peptides (Yenzyme) and affinity-purified using the same peptides immobilized on CNBr-sepharose. A rabbit polyclonal antibody against phospho-S186-SEC23B was generated and affinity purified by YenZym Antibodies. Anti- SEC23B, SEC24A, SEC24B, SEC24C, and SEC24D antibodies were provided by Dr. Randy Schekman. Mouse monoclonal antibodies were from Sigma-Aldrich (anti-FLAG M2), Covance (anti-HA), and Thermo Scientific (Sar1). Rabbit polyclonal antibodies were from Invitrogen (CUL1 and SKP1), Bethyl Laboratories, Inc. (Sec13 and SEC31A), Cell Signaling Technology (ULK1 and phospho-ULK1 (S757)), Novus Biological (LC3B), Rockland (phospho-ATG13 (S318)), and Abbiotec (phospho-Beclin-1 (S15).

In vitro kinase assay

Kinase and substrates were purified from HEK293T cells that had been transfected with plasmids expressing individual kinase and substrates. Kinase reaction buffer (KRB; 20 mM Tris, pH 7.5, 20 mM MgCl2, 25 mM β-glycerophosphate, 2 mM dithiothreitol and 100 μM sodium orthovanadate) were used to elute the purified proteins. Kinase and substrates were mixed and incubated at a final volume of 20 μL in KRB containing 20 μM ATP, 5 μg substrates at 30°C for 60 min. The reaction was stopped by the addition of sample buffer, boiled and analysed by immunoblot with phospho-SEC23B (Ser186) specific antibody.

Computational methods

The SEC24A and SEC24C monomers were extracted from the Protein Data Bank (PDB) (entries 3EGD and 3EH2, respectively), while SEC23B was modeled by homology from the PDB structure of SEC23A (entry 5KYN), including the missing residues from the crystal structure. The structure of the mutant SEC23B(S186D) was obtained using PyMol Mutagenesis Wizard tool (Edn. Version 1.7. Schrodinger, LLC, 2013). MD simulations were performed with the GROMACS v5.1 package (Abraham et al., 2015) using the Amber99SB force field (Hornak et al., 2006); the starting conformation for each dimer was modeled from the structure of the SEC23A-SEC24A dimer (PDB 3EGD). Only residues 120 to 405 from SEC23B, 502 to 742 from SEC24, and 173 to 419 from SEC24C were used in the simulations, as these are the domains involved in the dimer interaction, and we have seen that their structures were not influenced by the rest of the protein (data not shown). The systems were solvated with the SPCE water model in a triclinic box, extending at least 10 Å from every atom of the protein, and neutralized adding sufficient Na and Cl counter ions to reach 0.15 M concentration. Bond lengths were constrained using the LINCS algorithm allowing a 2fs time-step. Long-range electrostatics interactions were taken into account using the particle-mesh Ewald (PME) approach. The non-bonded cut-off for Coulomb and Van der Waals interactions were both 10 Å, and the non-bonded pair list was updated every 25 fs. Energy minimization was conducted through the steepest-descent algorithm, until the maximum force decayed to 1000 [kJ mol−1 nm−1]. The equilibration stage of the whole system consisted in 500 ps of NVT simulation followed by 500 ps of NPT simulation. Temperature was kept constant at 310 K using a modified Berendsen thermostat (Essmann et al., 1995) with a coupling constant of 0.1 ps. Constant pressure of 1 bar was applied in all directions with a coupling constant of 2.0 ps and a compressibility of 4.5 10−5 bar−1. Finally, the equilibrated systems were subjected to a 1 μs MD simulation run each at 310 K. Binding free energies were calculated using de MM-GBSA scheme (Genheden and Ryde, 2015) provided in the Amber16 package (Case et al., 2017) using the single trajectory approach. Hundred snapshots were collected at time intervals of 5 ns from the last 500 ns of the MD simulations, thus guaranteeing statistical independence (Anisimov and Cavasotto, 2011). The salt concentration was set to 0.150 M, and the dielectric constants to 80 and 1 for the solvent and the proteins, respectively.

CRISPR genome editing

To generate SEC23B S186A and S186N knock-in cells, an optimal gRNA target sequence closest to the genomic target site and a 2.1 kb homologous recombination (HR) donor template were designed using the Benchling CRISPR Genome Engineering tool. The HR donor template was designed to introduce alanine or asparagine substitutions at position S186, and a silent mutation to introduce a KpnI restriction site for genotyping. SEC23B gRNA target sequence (see; Figure 5—figure supplement 1B) was cloned into pSpCas9(BB)−2A-GFP (PX458), a gift from F. Zhang (Addgene plasmid no. 48138) (Ge et al., 2013). A375 cells were seeded into 10 cm dishes at approximately 70% confluency and transfected with 2.5 μg each of gRNA-containing PX458 plasmid and HR donor template, using lipofectamine 3000 (Life Technologies). The transfection was performed according to the manufacturer’s recommended protocol, using a 2:1 ratio of lipofectamine:DNA. Two days after transfection, GFP-positive cells were sorted using the Beckman Coulter MoFlo XDP cell sorter (100 μm nozzle), and 5,000 cells were plated on a 15 cm dish. About a week later, single-cell clones were picked, trypsinized in 0.25% Trypsin-EDTA for 5 min, and plated into individual wells of a 96-well plate for genotyping. Genomic DNA was collected using QuickExtract (Epicentre). Genotyping PCRs were performed with MyTaq HS Red Mix (Bioline), using primers surrounding the genomic target sites. The resulting PCR products were then sequenced and aligned to the corresponding wild-type template in Benchling to determine the presence of a recombination event.

Gaussia luciferase assay

To measure the activity of the secretory pathway in HEK293T cells transfected with various cDNAs, a pCMV-Gaussia luciferase plasmid (ThermoFisher Scientific) was co-transfected to be used as a reporter by measuring luciferase activity in the conditioned medium (Badr et al., 2007). The activity of the secreted Gaussia luciferase was measured using Pierce Gaussia luciferase Flash Assay Kit (ThermoFisher Scientific) according to the manufacturer’s instruction.

Membrane fractionation

Membrane fractionation was performed through a modified protocol based on Ge et al. (Ge et al., 2013). HeLa cells (ten 15 cm dishes) were cultured to 95% confluence. Cells were treated with 20 µg/ml digitonin (5 min on ice) in B88 (20 mM Hepes, pH 7.2, 250 mM sorbitol, 150 mM potassium acetate, and 5 mM magnesium acetate). Membranes were pelleted at 300xg, washed in B88, and incubated with 3 mM GMPPNP and purified human COPII proteins [10 µg SAR1B and 10 µg of either SEC23B or SEC23B(S186D)], which were purified as described (Kim et al., 2005). Membranes were incubated for 30 min at 37°C and subjected to sequential differential centrifugation at 1,000 × g (10 min), 3,000 × g (10 min), 25,000 × g (20 min) to collect the membranes sedimented at each speed. The 25,000 × g membrane pellet was suspended in 0.75 ml 1.25 M sucrose buffer and overlayed with 0.5 ml 1.1 M and 0.5 ml 0.25 M sucrose buffer (Golgi isolation kit; Sigma). Centrifugation was performed at 120,000 × g for 2 hr (TLS 55 rotor, Beckman), after which the interface between 0.25 M and 1.1 M sucrose (L fraction) was selected and suspended in 1 ml 19% OptiPrep for a step gradient containing 0.5 ml 22.5%, 1 ml 19% (sample), 0.9 ml 16%, 0.9 ml 12%, 1 ml 8%, 0.5 ml 5% and 0.2 ml 0% OptiPrep each. Each density of OptiPrep was prepared by diluting 50% OptiPrep (20 mM Tricine-KOH, pH 7.4, 42 mM sucrose and 1 mM EDTA) with a buffer containing 20 mM Tricine-KOH, pH 7.4, 250 mM sucrose and 1 mM EDTA. The OptiPrep gradient was centrifuged at 150,000 × g for 3 hr (SW 55 Ti rotor, Beckman) and subsequently fractions of 0.5 ml each, were collected from the top. The fractions were then analyzed by immunoblot.

Quantification and statistical analysis

Images were analyzed with an in-house developed python script. Cell counts were calculated by detecting nuclei labelled by DAPI in the blue channel of the image. First, a Gaussian smoothing was applied, and then an Otsu threshold to determine the nuclei mask. This was followed by watershed segmentation to separate touching nuclei, as well as filtering based on area to remove small spots. Partial nuclei touching image borders were included but were counted as a fraction based on the average nuclei size measured in the data set. The puncta (green channel) were detected using the Laplacian of Gaussian (LoG) blob detection algorithm as provided by the python package ‘scikit-image’ (van der Walt et al., 2014). Any blobs found in the regions covered by cell nuclei were ignored. The puncta area was calculated by finding the area of a circle with radius proportional to the standard deviation of the Gaussian kernel that detected the blob, as returned by the LoG algorithm. Prior to applying LoG, some images required a median filter for removal of speckle noise. Some very bright cells were removed from the analysis (identified nuclei and a surrounding area), since it was not possible to distinguish puncta in these areas. These cells were identified by finding outliers from the set of nuclei mean intensities of the image. All statistical analysis was performed with unpaired Student’s t test, and it is considered significant when the p value is less than 0.05. n.s., not significant. Data were expressed as mean ±SD of at least three independent experiments performed in triplicate.

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    492. RC Dickson
    493. M Diederich
    494. P Digard
    495. I Dikic
    496. SP Dinesh-Kumar
    497. C Ding
    498. WX Ding
    499. Z Ding
    500. L Dini
    501. JH Distler
    502. A Diwan
    503. M Djavaheri-Mergny
    504. K Dmytruk
    505. RC Dobson
    506. V Doetsch
    507. K Dokladny
    508. S Dokudovskaya
    509. M Donadelli
    510. XC Dong
    511. X Dong
    512. Z Dong
    513. TM Donohue
    514. KS Doran
    515. G D'Orazi
    516. GW Dorn
    517. V Dosenko
    518. S Dridi
    519. L Drucker
    520. J Du
    521. LL Du
    522. L Du
    523. A du Toit
    524. P Dua
    525. L Duan
    526. P Duann
    527. VK Dubey
    528. MR Duchen
    529. MA Duchosal
    530. H Duez
    531. I Dugail
    532. VI Dumit
    533. MC Duncan
    534. EA Dunlop
    535. WA Dunn
    536. N Dupont
    537. L Dupuis
    538. RV Durán
    539. TM Durcan
    540. S Duvezin-Caubet
    541. U Duvvuri
    542. V Eapen
    543. D Ebrahimi-Fakhari
    544. A Echard
    545. L Eckhart
    546. CL Edelstein
    547. AL Edinger
    548. L Eichinger
    549. T Eisenberg
    550. A Eisenberg-Lerner
    551. NT Eissa
    552. WS El-Deiry
    553. V El-Khoury
    554. Z Elazar
    555. H Eldar-Finkelman
    556. CJ Elliott
    557. E Emanuele
    558. U Emmenegger
    559. N Engedal
    560. AM Engelbrecht
    561. S Engelender
    562. JM Enserink
    563. R Erdmann
    564. J Erenpreisa
    565. R Eri
    566. JL Eriksen
    567. A Erman
    568. R Escalante
    569. EL Eskelinen
    570. L Espert
    571. L Esteban-Martínez
    572. TJ Evans
    573. M Fabri
    574. G Fabrias
    575. C Fabrizi
    576. A Facchiano
    577. NJ Færgeman
    578. A Faggioni
    579. WD Fairlie
    580. C Fan
    581. D Fan
    582. J Fan
    583. S Fang
    584. M Fanto
    585. A Fanzani
    586. T Farkas
    587. M Faure
    588. FB Favier
    589. H Fearnhead
    590. M Federici
    591. E Fei
    592. TC Felizardo
    593. H Feng
    594. Y Feng
    595. Y Feng
    596. TA Ferguson
    597. ÁF Fernández
    598. MG Fernandez-Barrena
    599. JC Fernandez-Checa
    600. A Fernández-López
    601. ME Fernandez-Zapico
    602. O Feron
    603. E Ferraro
    604. CV Ferreira-Halder
    605. L Fesus
    606. R Feuer
    607. FC Fiesel
    608. EC Filippi-Chiela
    609. G Filomeni
    610. GM Fimia
    611. JH Fingert
    612. S Finkbeiner
    613. T Finkel
    614. F Fiorito
    615. PB Fisher
    616. M Flajolet
    617. F Flamigni
    618. O Florey
    619. S Florio
    620. RA Floto
    621. M Folini
    622. C Follo
    623. EA Fon
    624. F Fornai
    625. F Fortunato
    626. A Fraldi
    627. R Franco
    628. A Francois
    629. A François
    630. LB Frankel
    631. ID Fraser
    632. N Frey
    633. DG Freyssenet
    634. C Frezza
    635. SL Friedman
    636. DE Frigo
    637. D Fu
    638. JM Fuentes
    639. J Fueyo
    640. Y Fujitani
    641. Y Fujiwara
    642. M Fujiya
    643. M Fukuda
    644. S Fulda
    645. C Fusco
    646. B Gabryel
    647. M Gaestel
    648. P Gailly
    649. M Gajewska
    650. S Galadari
    651. G Galili
    652. I Galindo
    653. MF Galindo
    654. G Galliciotti
    655. L Galluzzi
    656. L Galluzzi
    657. V Galy
    658. N Gammoh
    659. S Gandy
    660. AK Ganesan
    661. S Ganesan
    662. IG Ganley
    663. M Gannagé
    664. FB Gao
    665. F Gao
    666. JX Gao
    667. L García Nannig
    668. E García Véscovi
    669. M Garcia-Macía
    670. C Garcia-Ruiz
    671. AD Garg
    672. PK Garg
    673. R Gargini
    674. NC Gassen
    675. D Gatica
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    677. J Gavard
    678. E Gavathiotis
    679. L Ge
    680. P Ge
    681. S Ge
    682. PW Gean
    683. V Gelmetti
    684. AA Genazzani
    685. J Geng
    686. P Genschik
    687. L Gerner
    688. JE Gestwicki
    689. DA Gewirtz
    690. S Ghavami
    691. E Ghigo
    692. D Ghosh
    693. AM Giammarioli
    694. F Giampieri
    695. C Giampietri
    696. A Giatromanolaki
    697. DJ Gibbings
    698. L Gibellini
    699. SB Gibson
    700. V Ginet
    701. A Giordano
    702. F Giorgini
    703. E Giovannetti
    704. SE Girardin
    705. S Gispert
    706. S Giuliano
    707. CL Gladson
    708. A Glavic
    709. M Gleave
    710. N Godefroy
    711. RM Gogal
    712. K Gokulan
    713. GH Goldman
    714. D Goletti
    715. MS Goligorsky
    716. AV Gomes
    717. LC Gomes
    718. H Gomez
    719. C Gomez-Manzano
    720. R Gómez-Sánchez
    721. DA Gonçalves
    722. E Goncu
    723. Q Gong
    724. C Gongora
    725. CB Gonzalez
    726. P Gonzalez-Alegre
    727. P Gonzalez-Cabo
    728. RA González-Polo
    729. IS Goping
    730. C Gorbea
    731. NV Gorbunov
    732. DR Goring
    733. AM Gorman
    734. SM Gorski
    735. S Goruppi
    736. S Goto-Yamada
    737. C Gotor
    738. RA Gottlieb
    739. I Gozes
    740. D Gozuacik
    741. Y Graba
    742. M Graef
    743. GE Granato
    744. GD Grant
    745. S Grant
    746. GL Gravina
    747. DR Green
    748. A Greenhough
    749. MT Greenwood
    750. B Grimaldi
    751. F Gros
    752. C Grose
    753. JF Groulx
    754. F Gruber
    755. P Grumati
    756. T Grune
    757. JL Guan
    758. KL Guan
    759. B Guerra
    760. C Guillen
    761. K Gulshan
    762. J Gunst
    763. C Guo
    764. L Guo
    765. M Guo
    766. W Guo
    767. XG Guo
    768. AA Gust
    769. ÅB Gustafsson
    770. E Gutierrez
    771. MG Gutierrez
    772. HS Gwak
    773. A Haas
    774. JE Haber
    775. S Hadano
    776. M Hagedorn
    777. DR Hahn
    778. AJ Halayko
    779. A Hamacher-Brady
    780. K Hamada
    781. A Hamai
    782. A Hamann
    783. M Hamasaki
    784. I Hamer
    785. Q Hamid
    786. EM Hammond
    787. F Han
    788. W Han
    789. JT Handa
    790. JA Hanover
    791. M Hansen
    792. M Harada
    793. L Harhaji-Trajkovic
    794. JW Harper
    795. AH Harrath
    796. AL Harris
    797. J Harris
    798. U Hasler
    799. P Hasselblatt
    800. K Hasui
    801. RG Hawley
    802. TS Hawley
    803. C He
    804. CY He
    805. F He
    806. G He
    807. RR He
    808. XH He
    809. YW He
    810. YY He
    811. JK Heath
    812. MJ Hébert
    813. RA Heinzen
    814. GV Helgason
    815. M Hensel
    816. EP Henske
    817. C Her
    818. PK Herman
    819. A Hernández
    820. C Hernandez
    821. S Hernández-Tiedra
    822. C Hetz
    823. PR Hiesinger
    824. K Higaki
    825. S Hilfiker
    826. BG Hill
    827. JA Hill
    828. WD Hill
    829. K Hino
    830. D Hofius
    831. P Hofman
    832. GU Höglinger
    833. J Höhfeld
    834. MK Holz
    835. Y Hong
    836. DA Hood
    837. JJ Hoozemans
    838. T Hoppe
    839. C Hsu
    840. CY Hsu
    841. LC Hsu
    842. D Hu
    843. G Hu
    844. HM Hu
    845. H Hu
    846. MC Hu
    847. YC Hu
    848. ZW Hu
    849. F Hua
    850. Y Hua
    851. C Huang
    852. HL Huang
    853. KH Huang
    854. KY Huang
    855. S Huang
    856. S Huang
    857. WP Huang
    858. YR Huang
    859. Y Huang
    860. Y Huang
    861. TB Huber
    862. P Huebbe
    863. WK Huh
    864. JJ Hulmi
    865. GM Hur
    866. JH Hurley
    867. Z Husak
    868. SN Hussain
    869. S Hussain
    870. JJ Hwang
    871. S Hwang
    872. TI Hwang
    873. A Ichihara
    874. Y Imai
    875. C Imbriano
    876. M Inomata
    877. T Into
    878. V Iovane
    879. JL Iovanna
    880. RV Iozzo
    881. NY Ip
    882. JE Irazoqui
    883. P Iribarren
    884. Y Isaka
    885. AJ Isakovic
    886. H Ischiropoulos
    887. JS Isenberg
    888. M Ishaq
    889. H Ishida
    890. I Ishii
    891. JE Ishmael
    892. C Isidoro
    893. K Isobe
    894. E Isono
    895. S Issazadeh-Navikas
    896. K Itahana
    897. E Itakura
    898. AI Ivanov
    899. AK Iyer
    900. JM Izquierdo
    901. Y Izumi
    902. V Izzo
    903. M Jäättelä
    904. N Jaber
    905. DJ Jackson
    906. WT Jackson
    907. TG Jacob
    908. TS Jacques
    909. C Jagannath
    910. A Jain
    911. NR Jana
    912. BK Jang
    913. A Jani
    914. B Janji
    915. PR Jannig
    916. PJ Jansson
    917. S Jean
    918. M Jendrach
    919. JH Jeon
    920. N Jessen
    921. EB Jeung
    922. K Jia
    923. L Jia
    924. H Jiang
    925. H Jiang
    926. L Jiang
    927. T Jiang
    928. X Jiang
    929. X Jiang
    930. X Jiang
    931. Y Jiang
    932. Y Jiang
    933. A Jiménez
    934. C Jin
    935. H Jin
    936. L Jin
    937. M Jin
    938. S Jin
    939. UK Jinwal
    940. EK Jo
    941. T Johansen
    942. DE Johnson
    943. GV Johnson
    944. JD Johnson
    945. E Jonasch
    946. C Jones
    947. LA Joosten
    948. J Jordan
    949. AM Joseph
    950. B Joseph
    951. AM Joubert
    952. D Ju
    953. J Ju
    954. HF Juan
    955. K Juenemann
    956. G Juhász
    957. HS Jung
    958. JU Jung
    959. YK Jung
    960. H Jungbluth
    961. MJ Justice
    962. B Jutten
    963. NO Kaakoush
    964. K Kaarniranta
    965. A Kaasik
    966. T Kabuta
    967. B Kaeffer
    968. K Kågedal
    969. A Kahana
    970. S Kajimura
    971. O Kakhlon
    972. M Kalia
    973. DV Kalvakolanu
    974. Y Kamada
    975. K Kambas
    976. VO Kaminskyy
    977. HH Kampinga
    978. M Kandouz
    979. C Kang
    980. R Kang
    981. TC Kang
    982. T Kanki
    983. TD Kanneganti
    984. H Kanno
    985. AG Kanthasamy
    986. M Kantorow
    987. M Kaparakis-Liaskos
    988. O Kapuy
    989. V Karantza
    990. MR Karim
    991. P Karmakar
    992. A Kaser
    993. S Kaushik
    994. T Kawula
    995. AM Kaynar
    996. PY Ke
    997. ZJ Ke
    998. JH Kehrl
    999. KE Keller
    1000. JK Kemper
    1001. AK Kenworthy
    1002. O Kepp
    1003. A Kern
    1004. S Kesari
    1005. D Kessel
    1006. R Ketteler
    1007. IC Kettelhut
    1008. B Khambu
    1009. MM Khan
    1010. VK Khandelwal
    1011. S Khare
    1012. JG Kiang
    1013. AA Kiger
    1014. A Kihara
    1015. AL Kim
    1016. CH Kim
    1017. DR Kim
    1018. DH Kim
    1019. EK Kim
    1020. HY Kim
    1021. HR Kim
    1022. JS Kim
    1023. JH Kim
    1024. JC Kim
    1025. JH Kim
    1026. KW Kim
    1027. MD Kim
    1028. MM Kim
    1029. PK Kim
    1030. SW Kim
    1031. SY Kim
    1032. YS Kim
    1033. Y Kim
    1034. A Kimchi
    1035. AC Kimmelman
    1036. T Kimura
    1037. JS King
    1038. K Kirkegaard
    1039. V Kirkin
    1040. LA Kirshenbaum
    1041. S Kishi
    1042. Y Kitajima
    1043. K Kitamoto
    1044. Y Kitaoka
    1045. K Kitazato
    1046. RA Kley
    1047. WT Klimecki
    1048. M Klinkenberg
    1049. J Klucken
    1050. H Knævelsrud
    1051. E Knecht
    1052. L Knuppertz
    1053. JL Ko
    1054. S Kobayashi
    1055. JC Koch
    1056. C Koechlin-Ramonatxo
    1057. U Koenig
    1058. YH Koh
    1059. K Köhler
    1060. SD Kohlwein
    1061. M Koike
    1062. M Komatsu
    1063. E Kominami
    1064. D Kong
    1065. HJ Kong
    1066. EG Konstantakou
    1067. BT Kopp
    1068. T Korcsmaros
    1069. L Korhonen
    1070. VI Korolchuk
    1071. NV Koshkina
    1072. Y Kou
    1073. MI Koukourakis
    1074. C Koumenis
    1075. AL Kovács
    1076. T Kovács
    1077. WJ Kovacs
    1078. D Koya
    1079. C Kraft
    1080. D Krainc
    1081. H Kramer
    1082. T Kravic-Stevovic
    1083. W Krek
    1084. C Kretz-Remy
    1085. R Krick
    1086. M Krishnamurthy
    1087. J Kriston-Vizi
    1088. G Kroemer
    1089. MC Kruer
    1090. R Kruger
    1091. NT Ktistakis
    1092. K Kuchitsu
    1093. C Kuhn
    1094. AP Kumar
    1095. A Kumar
    1096. A Kumar
    1097. D Kumar
    1098. D Kumar
    1099. R Kumar
    1100. S Kumar
    1101. M Kundu
    1102. HJ Kung
    1103. A Kuno
    1104. SH Kuo
    1105. J Kuret
    1106. T Kurz
    1107. T Kwok
    1108. TK Kwon
    1109. YT Kwon
    1110. I Kyrmizi
    1111. AR La Spada
    1112. F Lafont
    1113. T Lahm
    1114. A Lakkaraju
    1115. T Lam
    1116. T Lamark
    1117. S Lancel
    1118. TH Landowski
    1119. DJ Lane
    1120. JD Lane
    1121. C Lanzi
    1122. P Lapaquette
    1123. LR Lapierre
    1124. J Laporte
    1125. J Laukkarinen
    1126. GW Laurie
    1127. S Lavandero
    1128. L Lavie
    1129. MJ LaVoie
    1130. BY Law
    1131. HK Law
    1132. KB Law
    1133. R Layfield
    1134. PA Lazo
    1135. L Le Cam
    1136. KG Le Roch
    1137. H Le Stunff
    1138. V Leardkamolkarn
    1139. M Lecuit
    1140. BH Lee
    1141. CH Lee
    1142. EF Lee
    1143. GM Lee
    1144. HJ Lee
    1145. H Lee
    1146. JK Lee
    1147. J Lee
    1148. JH Lee
    1149. JH Lee
    1150. M Lee
    1151. MS Lee
    1152. PJ Lee
    1153. SW Lee
    1154. SJ Lee
    1155. SJ Lee
    1156. SY Lee
    1157. SH Lee
    1158. SS Lee
    1159. SJ Lee
    1160. S Lee
    1161. YR Lee
    1162. YJ Lee
    1163. YH Lee
    1164. C Leeuwenburgh
    1165. S Lefort
    1166. R Legouis
    1167. J Lei
    1168. QY Lei
    1169. DA Leib
    1170. G Leibowitz
    1171. I Lekli
    1172. SD Lemaire
    1173. JJ Lemasters
    1174. MK Lemberg
    1175. A Lemoine
    1176. S Leng
    1177. G Lenz
    1178. P Lenzi
    1179. LO Lerman
    1180. D Lettieri Barbato
    1181. JI Leu
    1182. HY Leung
    1183. B Levine
    1184. PA Lewis
    1185. F Lezoualc'h
    1186. C Li
    1187. F Li
    1188. FJ Li
    1189. J Li
    1190. K Li
    1191. L Li
    1192. M Li
    1193. M Li
    1194. Q Li
    1195. R Li
    1196. S Li
    1197. W Li
    1198. W Li
    1199. X Li
    1200. Y Li
    1201. J Lian
    1202. C Liang
    1203. Q Liang
    1204. Y Liao
    1205. J Liberal
    1206. PP Liberski
    1207. P Lie
    1208. AP Lieberman
    1209. HJ Lim
    1210. KL Lim
    1211. K Lim
    1212. RT Lima
    1213. CS Lin
    1214. CF Lin
    1215. F Lin
    1216. F Lin
    1217. FC Lin
    1218. K Lin
    1219. KH Lin
    1220. PH Lin
    1221. T Lin
    1222. WW Lin
    1223. YS Lin
    1224. Y Lin
    1225. R Linden
    1226. D Lindholm
    1227. LM Lindqvist
    1228. P Lingor
    1229. A Linkermann
    1230. LA Liotta
    1231. MM Lipinski
    1232. VA Lira
    1233. MP Lisanti
    1234. PB Liton
    1235. B Liu
    1236. C Liu
    1237. CF Liu
    1238. F Liu
    1239. HJ Liu
    1240. J Liu
    1241. JJ Liu
    1242. JL Liu
    1243. K Liu
    1244. L Liu
    1245. L Liu
    1246. Q Liu
    1247. RY Liu
    1248. S Liu
    1249. S Liu
    1250. W Liu
    1251. XD Liu
    1252. X Liu
    1253. XH Liu
    1254. X Liu
    1255. X Liu
    1256. X Liu
    1257. Y Liu
    1258. Y Liu
    1259. Z Liu
    1260. Z Liu
    1261. JP Liuzzi
    1262. G Lizard
    1263. M Ljujic
    1264. IJ Lodhi
    1265. SE Logue
    1266. BL Lokeshwar
    1267. YC Long
    1268. S Lonial
    1269. B Loos
    1270. C López-Otín
    1271. C López-Vicario
    1272. M Lorente
    1273. PL Lorenzi
    1274. P Lõrincz
    1275. M Los
    1276. MT Lotze
    1277. PE Lovat
    1278. B Lu
    1279. B Lu
    1280. J Lu
    1281. Q Lu
    1282. SM Lu
    1283. S Lu
    1284. Y Lu
    1285. F Luciano
    1286. S Luckhart
    1287. JM Lucocq
    1288. P Ludovico
    1289. A Lugea
    1290. NW Lukacs
    1291. JJ Lum
    1292. AH Lund
    1293. H Luo
    1294. J Luo
    1295. S Luo
    1296. C Luparello
    1297. T Lyons
    1298. J Ma
    1299. Y Ma
    1300. Y Ma
    1301. Z Ma
    1302. J Machado
    1303. GM Machado-Santelli
    1304. F Macian
    1305. GC MacIntosh
    1306. JP MacKeigan
    1307. KF Macleod
    1308. JD MacMicking
    1309. LA MacMillan-Crow
    1310. F Madeo
    1311. M Madesh
    1312. J Madrigal-Matute
    1313. A Maeda
    1314. T Maeda
    1315. G Maegawa
    1316. E Maellaro
    1317. H Maes
    1318. M Magariños
    1319. K Maiese
    1320. TK Maiti
    1321. L Maiuri
    1322. MC Maiuri
    1323. CG Maki
    1324. R Malli
    1325. W Malorni
    1326. A Maloyan
    1327. F Mami-Chouaib
    1328. N Man
    1329. JD Mancias
    1330. EM Mandelkow
    1331. MA Mandell
    1332. AA Manfredi
    1333. SN Manié
    1334. C Manzoni
    1335. K Mao
    1336. Z Mao
    1337. ZW Mao
    1338. P Marambaud
    1339. AM Marconi
    1340. Z Marelja
    1341. G Marfe
    1342. M Margeta
    1343. E Margittai
    1344. M Mari
    1345. FV Mariani
    1346. C Marin
    1347. S Marinelli
    1348. G Mariño
    1349. I Markovic
    1350. R Marquez
    1351. AM Martelli
    1352. S Martens
    1353. KR Martin
    1354. SJ Martin
    1355. S Martin
    1356. MA Martin-Acebes
    1357. P Martín-Sanz
    1358. C Martinand-Mari
    1359. W Martinet
    1360. J Martinez
    1361. N Martinez-Lopez
    1362. U Martinez-Outschoorn
    1363. M Martínez-Velázquez
    1364. M Martinez-Vicente
    1365. WK Martins
    1366. H Mashima
    1367. JA Mastrianni
    1368. G Matarese
    1369. P Matarrese
    1370. R Mateo
    1371. S Matoba
    1372. N Matsumoto
    1373. T Matsushita
    1374. A Matsuura
    1375. T Matsuzawa
    1376. MP Mattson
    1377. S Matus
    1378. N Maugeri
    1379. C Mauvezin
    1380. A Mayer
    1381. D Maysinger
    1382. GD Mazzolini
    1383. MK McBrayer
    1384. K McCall
    1385. C McCormick
    1386. GM McInerney
    1387. SC McIver
    1388. S McKenna
    1389. JJ McMahon
    1390. IA McNeish
    1391. F Mechta-Grigoriou
    1392. JP Medema
    1393. DL Medina
    1394. K Megyeri
    1395. M Mehrpour
    1396. JL Mehta
    1397. Y Mei
    1398. UC Meier
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    1466. S Moreno
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    1469. Y Moriyasu
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    1473. J Moscat
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    1475. S Mostowy
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    1477. D Mottet
    1478. JC Mottram
    1479. CE Moussa
    1480. VE Mpakou
    1481. H Mukhtar
    1482. JM Mulcahy Levy
    1483. S Muller
    1484. R Muñoz-Moreno
    1485. C Muñoz-Pinedo
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    1490. IU Mysorekar
    1491. IR Nabi
    1492. M Nabissi
    1493. GA Nader
    1494. Y Nagahara
    1495. Y Nagai
    1496. K Nagata
    1497. A Nagelkerke
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    1502. H Nakatogawa
    1503. M Nanjundan
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    1505. NI Naqvi
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    1508. M Narita
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    1510. R Natarajan
    1511. LC Navegantes
    1512. ST Nawrocki
    1513. TY Nazarko
    1514. VY Nazarko
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    1516. LM Neri
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    1518. RT Netea-Maier
    1519. BM Neves
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    1528. I Nishino
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Decision letter

  1. Noboru Mizushima
    Reviewing Editor; The University of Tokyo, Japan
  2. Ivan Dikic
    Senior Editor; Goethe University Frankfurt, Germany

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "The ULK1-FBXW5-SEC23B nexus controls autophagy" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Ivan Dikic as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Catherine Rabouille (Reviewer #3).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

Summary:

The authors identified SEC23B as a substrate of the E3 ligase FBXW5. They found that FBXW5 leads to proteasomal degradation of SEC23B under normal conditions. In contrast, under starvation conditions, ULK1 phosphorylates SEC23B on Ser186, preventing recognition by FBXW5 and redirecting SEC23B to the ERGIC together with SEC24A/B. The data are clearly demonstrated, and the logic is easy to follow.

Essential revisions:

1) The authors propose that ULK1-mediated phosphorylation of SEC23B promotes its translocation to the ERGIC, but this is not directly shown. The authors should test whether the translocation of SEC23B to the ERGIC is indeed abolished in ULK1- or ULK1/2-knockdown cells.

2) The authors conclude that FBXW5-dependent degradation of SEC23B downregulates basal autophagy based on the results of the LC3 puncta formation and mCherry-GFP-LC3 assays. However, the authors should rule out the possibility that SEC23B only increases the level of LC3-II on autophagosomes by enhancing LC3 lipidation at the ERGIC rather than the efficiency of autophagosome formation (the extent of autophagic degradation). To confirm that FBXW5 knockdown and the expression of the SEC23(SD) mutant indeed increase the autophagic flux even under nutrient-rich conditions, the authors should determine whether degradation of autophagic cargos such as p62 is also activated.

3) Related to comment #2, if the expression of SEC23B(S186D) is indeed sufficient to induce autophagy, the data suggest that the primary function of the ULK1 complex is phosphorylation of SEC23B among many known substrates. To prove this hypothesis, it would be important to test whether the expression of SEC23B(S186D) can rescue autophagy in ULK1- or ULK1/2-double knockdown cells.

4) If the regulation of the level of SEC23B by FBXW5 is important for the control of autophagy, is overexpression of even wild-type SEC23B sufficient to induce autophagy?

5) The number of LC3 can be increased by not only autophagy activation but also inhibition of the lysosomal function. Given that overexpression of SEC23B(S186D) affects secretion (Supplementary Figure 3), the authors should rule out the possibility that it also affects the lysosomal function and thereby causes accumulation of LC3. The lysosomal function can be tested by monitoring maturation of cathepsins or degradation of the EGF receptor.

6) Subsection “Ser186 in SEC23B is necessary for its localization to the ERGIC and an efficient autophagic response upon nutrient deprivation”, first paragraph and Supplementary Figure 3D and E: The data on the secretory pathway by assaying secreted Gaussia Luciferase are critical and should be shown in the main figures rather than in the supplement. It could have been well the case that SEC23-P/SEC24A/B would lead to vesicles feeding autophagosomes and fueling general secretion.

7) Perhaps one important point to clarify is what happens to the secretory pathway and ER to Golgi transport with SEC23B-P/SEC24A/B? Is there hard evidence to exclude the notion of SEC23B-P/SEC24A/B during starvation does not also fuel secretion (except for the luciferase secretion that is inhibited)? Any specific cargo? Why would this complex SEC23B-P/SEC24A/B not support full COPII coat formation? The authors seem in doubt exemplified with the question mark in their model. Does SEC23-P/SEC24A/B also bind the other COPII subunits to form a full coated vesicle? This should be experimentally tested. What about the other isoforms of SEC23 and SEC24C/D?

https://doi.org/10.7554/eLife.42253.026

Author response

Essential revisions:

1) The authors propose that ULK1-mediated phosphorylation of SEC23B promotes its translocation to the ERGIC, but this is not directly shown. The authors should test whether the translocation of SEC23B to the ERGIC is indeed abolished in ULK1- or ULK1/2-knockdown cells.

We now show that ULK1 knockdown significantly inhibits the translocation of SEC23B to the ERGIC (new Figure 5B).

2) The authors conclude that FBXW5-dependent degradation of SEC23B downregulates basal autophagy based on the results of the LC3 puncta formation and mCherry-GFP-LC3 assays. However, the authors should rule out the possibility that SEC23B only increases the level of LC3-II on autophagosomes by enhancing LC3 lipidation at the ERGIC rather than the efficiency of autophagosome formation (the extent of autophagic degradation). To confirm that FBXW5 knockdown and the expression of the SEC23(SD) mutant indeed increase the autophagic flux even under nutrient-rich conditions, the authors should determine whether degradation of autophagic cargos such as p62 is also activated.

We now show that FBXW5 knockdown and the expression of the SEC23(S186D) mutant activate the degradation of p62 under nutrient-rich conditions (new Figure 4B and new Figure 4E).

3) Related to comment #2, if the expression of SEC23B(S186D) is indeed sufficient to induce autophagy, the data suggest that the primary function of the ULK1 complex is phosphorylation of SEC23B among many known substrates. To prove this hypothesis, it would be important to test whether the expression of SEC23B(S186D) can rescue autophagy in ULK1- or ULK1/2-double knockdown cells.

We now show that the expression of SEC23B(S186D) is unable to rescue autophagy in ULK1 knockdown cells (new Figure 4—figure supplement 1D). This is likely because ULK1 is necessary to phosphorylate many pro-autophagic substrates. Moreover, in presence of nutrients, the basal activity of ULK1 is necessary to promote the assembly of COPII complexes (Joo et al., 2016). Therefore, in the absence of ULK1, it is expected that neither secretory nor autophagic COPII vesicles are efficiently formed.

4) If the regulation of the level of SEC23B by FBXW5 is important for the control of autophagy, is overexpression of even wild-type SEC23B sufficient to induce autophagy?

We now show that overexpression of wild-type SEC23B to levels similar to those of the stable SEC23B(S186D) mutant is sufficient to induce autophagy (new Figure 4F).

5) The number of LC3 can be increased by not only autophagy activation but also inhibition of the lysosomal function. Given that overexpression of SEC23B(S186D) affects secretion (Supplementary Figure 3), the authors should rule out the possibility that it also affects the lysosomal function and thereby causes accumulation of LC3. The lysosomal function can be tested by monitoring maturation of cathepsins or degradation of the EGF receptor.

By monitoring the levels of p62 as well as pro-Cathepsin and active-Cathepsin, we now show that the overexpression of SEC23B(S186D) does not affect the lysosomal function (new Figure 4E and new Figure 4F).

6) Subsection “Ser186 in SEC23B is necessary for its localization to the ERGIC and an efficient autophagic response upon nutrient deprivation”, first paragraph and Supplementary Figure 3D and E: The data on the secretory pathway by assaying secreted Gaussia Luciferase are critical and should be shown in the main figures rather than in the supplement. It could have been well the case that SEC23-P/SEC24A/B would lead to vesicles feeding autophagosomes and fueling general secretion.

We have moved these figures to the main figures (now Figure 5C-D).

7) Perhaps one important point to clarify is what happens to the secretory pathway and ER to Golgi transport with SEC23B-P/SEC24A/B? Is there hard evidence to exclude the notion of SEC23B-P/SEC24A/B during starvation does not also fuel secretion (except for the luciferase secretion that is inhibited)? Any specific cargo? Why would this complex SEC23B-P/SEC24A/B not support full COPII coat formation? The authors seem in doubt exemplified with the question mark in their model. Does SEC23-P/SEC24A/B also bind the other COPII subunits to form a full coated vesicle? This should be experimentally tested. What about the other isoforms of SEC23 and SEC24C/D?

The phosphorylation of Ser186 does not increase the affinity of SEC23B to SEC24A and SEC24B. Rather, it inhibits the binding of SEC23B to SEC24C and SEC24D. We never claimed in the text that the binding of phosphorylated SEC23B to SEC24A and SEC24B reduces secretion. The lack of binding between phosphorylated SEC23B and SEC24C/D is striking and we speculate that this inhibition in binding may explain the reduced secretion. However, we have shown that SEC23B(S186D) (which mimics the phosphorylated form of SEC23B) binds to SAR1(H79G), a GTP-bound SAR1 mutant that is constitutively associated with COPII vesicles, but not with the cytosolic GDP-bound SAR1(T39N) mutant (Figure 3—figure supplement 1E). Moreover, SEC23B(S186D) also interacts with SEC13, SEC16, and SEC31 (new Figure 6A). In conclusion, we believe that, upon starvation, SEC23B-SEC24A/B promotes autophagy, but can possibly also sustain secretion since these proteins can bind other COPII subunits to form a full-coated vesicle. However, the decrease in the abundance of the SEC23B-SEC24C/D complexes likely contributes to a decrease in secretion. Concerning the other isoform of SEC23, Gan et al. (2017) have shown that ULK1 induces the dissociation of SEC23A from SEC31A in response to starvation, further inhibiting the secretory pathway. We now integrated better these points on the Discussion section. We also agree that the question mark going from the SEC23B-SEC24A/B to the “ER-to-Golgi trafficking”, which we drew in the old cartoon (old Supplementary Figure 4E) is confusing and reductionist. In fact, we believe that a simple cartoon cannot easily summarize the effects of FBXW5, ULK1, SEC23B phosphorylation, and SEC23B accumulation on both secretion and autophagy. Therefore, we eliminated the cartoon and its shortcomings from the supplementary figures. Finally, we believe that the identification of specific cargos goes beyond the scope of the current study.

https://doi.org/10.7554/eLife.42253.027

Article and author information

Author details

  1. Yeon-Tae Jeong

    1. Department of Biochemistry and Molecular Pharmacology, NYU School of Medicine, New York, United States
    2. Perlmutter NYU Cancer Center, NYU School of Medicine, New York, United States
    Contribution
    Conceptualization, Resources, Data curation, Software, Formal analysis, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1104-1161
  2. Daniele Simoneschi

    1. Department of Biochemistry and Molecular Pharmacology, NYU School of Medicine, New York, United States
    2. Perlmutter NYU Cancer Center, NYU School of Medicine, New York, United States
    Contribution
    Resources, Investigation, Visualization, Writing—review and editing
    Competing interests
    No competing interests declared
  3. Sarah Keegan

    1. Department of Biochemistry and Molecular Pharmacology, NYU School of Medicine, New York, United States
    2. Perlmutter NYU Cancer Center, NYU School of Medicine, New York, United States
    3. Institute for System Genetics, NYU School of Medicine, New York, United States
    Contribution
    Software, Formal analysis, Investigation
    Competing interests
    No competing interests declared
  4. David Melville

    Department of Molecular and Cellular Biology, Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, United States
    Contribution
    Resources, Formal analysis, Validation, Investigation, Visualization
    Competing interests
    No competing interests declared
  5. Natalia S Adler

    1. Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA), CONICET-Partner Institute of the Max Planck Society, Buenos Aires, Argentina
    2. Translational Medicine Research Institute (IIMT), CONICET, Facultad de Ciencias Biomédicas and Facultad deIngeniería, Universidad Austral, Pilar-Derqui, Argentina
    Contribution
    Formal analysis, Investigation, Visualization
    Competing interests
    No competing interests declared
  6. Anita Saraf

    The Stowers Institute for Medical Research, Kansas, United States
    Contribution
    Data curation, Formal analysis
    Competing interests
    No competing interests declared
  7. Laurence Florens

    The Stowers Institute for Medical Research, Kansas, United States
    Contribution
    Supervision, Writing—review and editing
    Competing interests
    No competing interests declared
  8. Michael P Washburn

    1. The Stowers Institute for Medical Research, Kansas, United States
    2. Department of Pathology and Laboratory Medicine, The University of Kansas Medical Center, Kansas, United States
    Contribution
    Supervision, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7568-2585
  9. Claudio N Cavasotto

    1. Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA), CONICET-Partner Institute of the Max Planck Society, Buenos Aires, Argentina
    2. Translational Medicine Research Institute (IIMT), CONICET, Facultad de Ciencias Biomédicas and Facultad deIngeniería, Universidad Austral, Pilar-Derqui, Argentina
    Contribution
    Software, Supervision, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1372-0379
  10. David Fenyö

    1. Department of Biochemistry and Molecular Pharmacology, NYU School of Medicine, New York, United States
    2. Perlmutter NYU Cancer Center, NYU School of Medicine, New York, United States
    3. Institute for System Genetics, NYU School of Medicine, New York, United States
    Contribution
    Software, Supervision, Writing—review and editing
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5049-3825
  11. Ana Maria Cuervo

    Department of Developmental and Molecular Biology, Institute for Aging Studies, Albert Einstein College of Medicine, Bronx, United States
    Contribution
    Resources, Supervision, Writing—review and editing
    Competing interests
    No competing interests declared
  12. Mario Rossi

    1. Department of Biochemistry and Molecular Pharmacology, NYU School of Medicine, New York, United States
    2. Instituto de Investigación en Biomedicina de Buenos Aires (IBioBA), CONICET-Partner Institute of the Max Planck Society, Buenos Aires, Argentina
    3. Perlmutter NYU Cancer Center, NYU School of Medicine, New York, United States
    Contribution
    Supervision, Writing—original draft, Writing—review and editing
    For correspondence
    mrossi@ibioba-mpsp-conicet.gov.ar
    Competing interests
    No competing interests declared
  13. Michele Pagano

    1. Department of Biochemistry and Molecular Pharmacology, NYU School of Medicine, New York, United States
    2. Perlmutter NYU Cancer Center, NYU School of Medicine, New York, United States
    3. Howard Hughes Medical Institute, New York University School of Medicine, New York, United States
    Contribution
    Conceptualization, Resources, Supervision, Funding acquisition, Writing—original draft, Project administration, Writing—review and editing
    For correspondence
    michele.pagano@nyumc.org
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3210-2442

Funding

National Institutes of Health (R01‐CA076584)

  • Michele Pagano

National Institutes of Health (R01‐GM057587)

  • Michele Pagano

Agencia Nacional de Promoción Científica y Tecnológica (PICT‐2014‐0458)

  • Mario Rossi

Agencia Nacional de Promoción Científica y Tecnológica (PICT2016‐2620)

  • Mario Rossi

Agencia Nacional de Promoción Científica y Tecnológica (PICT‐ 2014‐3599)

  • Claudio N Cavasotto

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

The authors thank A D Matteis and R Schekman for reagents; MP and YTJ are grateful to TM Thor and SO Hong, respectively, for continuous support. This work was funded by grants from the National Institute of Health (R01-CA076584 and R01-GM057587) to MP, and Agencia Nacional de Promoción Científica y Tecnológica-Argentina (PICT-2014–0458, PICT2016-2620) to MR, and (PICT-2014–3599) to CNC CNC thanks the National System of High-Performance Computing (Sistemas Nacionales de Computación de Alto Rendimiento, SNCAD) and the Computational Centre of High-Performance Computing (Centro de Computación de Alto Rendimiento, CeCAR) for granting use of their computational resources. MP is an Investigator with the Howard Hughes Medical Institute.

Senior Editor

  1. Ivan Dikic, Goethe University Frankfurt, Germany

Reviewing Editor

  1. Noboru Mizushima, The University of Tokyo, Japan

Publication history

  1. Received: September 25, 2018
  2. Accepted: December 27, 2018
  3. Accepted Manuscript published: December 31, 2018 (version 1)
  4. Version of Record published: January 29, 2019 (version 2)

Copyright

© 2018, Jeong et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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