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Sec24 phosphorylation regulates autophagosome abundance during nutrient deprivation

  1. Saralin Davis
  2. Juan Wang
  3. Ming Zhu
  4. Kyle Stahmer
  5. Ramya Lakshminarayan
  6. Majid Ghassemian
  7. Yu Jiang
  8. Elizabeth A Miller
  9. Susan Ferro-Novick  Is a corresponding author
  1. University of California, San Diego, United States
  2. Columbia University, United States
  3. University of Pittsburgh School of Medicine, United States
  4. MRC Laboratory of Molecular Biology, United Kingdom
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Cite this article as: eLife 2016;5:e21167 doi: 10.7554/eLife.21167

Abstract

Endoplasmic Reticulum (ER)-derived COPII coated vesicles constitutively transport secretory cargo to the Golgi. However, during starvation-induced stress, COPII vesicles have been implicated as a membrane source for autophagosomes, distinct organelles that engulf cellular components for degradation by macroautophagy (hereafter called autophagy). How cells regulate core trafficking machinery to fulfill dramatically different cellular roles in response to environmental cues is unknown. Here we show that phosphorylation of conserved amino acids on the membrane-distal surface of the Saccharomyces cerevisiae COPII cargo adaptor, Sec24, reprograms COPII vesicles for autophagy. We also show casein kinase 1 (Hrr25) is a key kinase that phosphorylates this regulatory surface. During autophagy, Sec24 phosphorylation regulates autophagosome number and its interaction with the C-terminus of Atg9, a component of the autophagy machinery required for autophagosome initiation. We propose that the acute need to produce autophagosomes during starvation drives the interaction of Sec24 with Atg9 to increase autophagosome abundance.

https://doi.org/10.7554/eLife.21167.001

eLife digest

When cells experience stressful conditions, such as a shortage of nutrients, they can digest their own material via a ‘self-eating’ process called autophagy and then recycle the products for further use. When autophagy is triggered, a new membrane structure called the autophagosome forms within the cell as it engulfs the material that is to be digested. The autophagosome delivers these materials to a compartment where they are broken down into smaller parts and the resulting raw materials are reused as needed.

The membranes that make up the autophagosome are derived from other membranes within the cell. These include small membrane-bound compartments called vesicles, which carry proteins from one part of the cell to another, or to the outside of the cell. COPII vesicles, for example, carry out the first transport step in the pathway that leads out of the cell – the so-called secretory pathway. Recently it was found that, when cells are starving, COPII vesicles can be diverted to the autophagy pathway and provide a source of membrane to build the autophagosome. However, it was not understood how the membrane of a COPII vesicle is reprogrammed so that it can interact with the cellular machinery that builds autophagosomes.

Using genetic and biochemical methods, Davis et al. have now teased apart the distinct roles of COPII vesicles in autophagy and the secretory pathway in budding yeast. The results show that a protein called Sec24, a component of the coat on the vesicles, interacts with another protein called Atg9, which is needed for the first steps of autophagosome formation. Davis et al. observed that Sec24 could be modified by the attachment of phosphate groups at a distinct site on the surface of Sec24. This modification promotes Sec24 to interact with Atg9 and increases the number of autophagosomes that form when cells are starving. Davis et al. also found that the enzyme casein kinase 1 is one of the enzymes responsible for attaching phosphate groups to Sec24.

Following on from this work, it will be important to test whether modification of vesicle coat proteins is a widespread mechanism for reprogramming membranes for different uses in other situations as well.

https://doi.org/10.7554/eLife.21167.002

Introduction

Autophagy is a highly conserved catabolic process that uses membrane traffic to target proteins and organelles for degradation. Basal levels of autophagy continuously replenish the cellular pool of amino acids and other metabolites to maintain homeostasis. However, when cells are starved for nutrients, autophagy is quickly upregulated. This upregulation leads to a dramatic intracellular reorganization to meet the high demand for membrane required to form autophagosomes, distinct organelles that target cellular components for degradation (Nakatogawa et al., 2009). Induction of autophagy leads to the formation of a double-membrane structure, called the isolation membrane, that forms adjacent to a pre-autophagosome structure (PAS) where autophagy related proteins (Atg) are recruited in a hierarchical manner (Nakatogawa et al., 2009). As the isolation membrane expands, it engulfs cytoplasmic proteins and organelles targeted for degradation before it seals to form an autophagosome. The autophagosome then fuses with the vacuole/lysosome, releasing its contents for degradation (Lamb et al., 2013; Nakatogawa et al., 2009). Although the assembly pathway of the Atg proteins is known, the mechanism by which membranes are directed to the autophagy pathway remains a central unanswered question in the field.

Autophagosome biogenesis has been linked to COPII vesicles and an ER subdomain called the ER exit sites (ERES) where COPII vesicles are formed (Graef et al., 2013; Suzuki et al., 2013; Tan et al., 2013; Ge et al., 2014; Wang et al., 2015; Lemus et al., 2016). How COPII vesicles, which are faithfully targeted to the Golgi, can be reprogrammed to function on an alternate trafficking pathway during nutrient deprivation remains enigmatic. COPII coated vesicle formation is initiated at the ER with the recruitment of an inner coat layer comprising the Sec23/Sec24 complex. Coat polymerization and vesicle budding occur when Sec23/Sec24 recruits a second complex (Sec13/Sec31) that forms the outer shell of the coat. Sec24, the major cargo adaptor of the COPII coat, recruits biosynthetic cargo and SNAREs (which mediate vesicle fusion) into vesicles that are delivered to the Golgi (Lord et al., 2013). After vesicle fission, the coat lingers on the vesicle to facilitate vesicle targeting to the Golgi (Cai et al., 2007; Lord et al., 2011). COPII vesicle budding mutants, as well as other mutants that disrupt ER-Golgi traffic, also disrupt autophagy (Hamasaki et al., 2003). However, a direct functional link between COPII vesicles, the COPII coat and autophagy has been difficult to demonstrate.

Multiple COPII coat subunits, including Sec23 and Sec24, are phosphorylated by Hrr25 (Bhandari et al., 2013; Lord et al., 2011), a kinase required for ER-Golgi traffic and autophagy (Lord et al., 2011; Murakami et al., 1999; Wang et al., 2015; Yu and Roth, 2002). Previously, we showed coat phosphorylation is required for COPII vesicle fusion (Lord et al., 2011; Wang et al., 2015). Given the recently identified role of COPII vesicles in autophagosome formation, and the observation that Hrr25 is required for autophagy, we asked if coat phosphorylation also functions to regulate vesicle traffic during autophagy. Here we find that phosphorylation of the membrane distal surface of Sec24 promotes the interaction of Sec24 with the C-terminus of Atg9, which is needed for autophagy. Failure to phosphorylate this Sec24 site leads to a decrease in autophagosome number, but not autophagosome expansion. This phosphorylation event is independent of the assembly of the Atg machinery at the PAS. Together these studies reveal a surprising role for coat phosphorylation in reprogramming core trafficking machinery to fulfill a separate function during starvation induced stress.

Results

Identification of Sec24 phosphorylation sites

To address whether coat phosphorylation allows COPII vesicles to function in autophagy versus ER-Golgi traffic, we purified the COPII inner coat from yeast cells induced for autophagy. Our analysis initially focused on Sec24 as it is the major COPII cargo adaptor. Using mass spectrometry, we identified 27 high confidence Sec24 phosphorylation sites in vivo (Supplementary file 1) and subsequently tested if they specifically affect autophagy but not ER-Golgi traffic. Many of the identified Sec24 phosphosites were conserved in the closely related paralog Iss1, which is also a cargo adaptor (Kurihara et al., 2000) (Supplementary file 1).

Two of the conserved Sec24 phosphosites (S730 and S735) map to a region of the protein that comprises one of the four well-characterized yeast cargo-binding sites (Miller et al., 2003, 2005; Pagant et al., 2015). The so-called A-site (Figure 1—figure supplement 1A) packages a SNARE, Sed5, needed for ER-Golgi traffic and autophagy (Miller et al., 2005; Mossessova et al., 2003; Tan et al., 2013), marking these residues as candidate regulatory sites. COPII vesicles formed in vitro with Sec24-S730A/S735A showed normal capture of Sed5 and other cargo, whereas those formed with Sec24-S730D/S735D contained reduced amounts of Sed5 and were modestly impaired in their fusion efficiency with the Golgi (Figure 1—figure supplement 1B,C). Additionally, a strain harboring the Sec24-S730D/S735D mutations had reduced autophagic activity, whereas the Sec24-S730A/S735A mutation had no effect (Figure 1—figure supplement 1D,E). Because the Sec24-S730D/S735D mutations had effects on both ER-Golgi traffic and autophagy, these sites are unlikely to specify the pathway the vesicle takes and instead highlight the importance of Sed5 packaging into COPII vesicles in both pathways. A second set of conserved phosphosites, S645/S678, located near a known cargo binding site in mammalian Sec24 (mSec24) (Figure 1A), did not disrupt the packaging of any cargo tested (Figure 1—figure supplement 2).

Figure 1 with 2 supplements see all
Identification of Sec24 phosphorylation sites required for autophagy.

(A) Ribbon diagram of Sec23 (lime) and Sec24 (lavender) with groups of key Sec24 phosphorylation sites (green, pink, and teal). The mSec24 membrin-binding site and conserved cargo binding B-site are colored light brown. (B) Sec24 phosphorylated residues conserved in Iss1 were mutated to alanine and introduced into sec24∆ and sec24∆iss1∆ deletion strains as described in the Materials and Methods. Sec24 and Iss1 were aligned using MAFFT alignment program and residues were considered conserved if either serine or threonine. Mutants in the sec24∆iss1∆ background were screened for autophagy defects 2 to 4 hr after nitrogen starvation using vacuolar alkaline phosphatase activity as a marker. Assays were performed in triplicate biological replicates and mutants were considered defective if p<0.05 using Student’s paired t-test. (C) Plasmids encoding SEC24 (WT) or mutant sec24 were expressed in SFNY2201 (left) or SFNY2202 (right) and grown on 5-FOA at 25°C to select against the WT balancing plasmid as described in the Materials and methods.

https://doi.org/10.7554/eLife.21167.003

A novel role for Sec24 during autophagy

Having ruled out known cargo-packaging sites on Sec24 as a functional switch for COPII vesicles from ER-Golgi traffic to autophagy, we next screened a panel of alanine mutations in the remaining phosphorylation sites conserved in Iss1. The effects of mutations in these sites on autophagy were monitored in a sec24Δiss1Δ double mutant background by assaying Pho8Δ60 activity. Pho8Δ60 is a cytosolic form of vacuolar alkaline phosphatase that is delivered to and activated in the vacuole when autophagy is induced (Klionsky, 2007). Only combinations of T324A, T325A and T328A were defective in autophagy (Figure 1B, red box). These three sites form a patch located on the membrane distal surface of Sec24 (Figure 1A), making them unlikely to regulate cargo packaging. None of the additional 13 phosphosites tested showed autophagy defects (Figure 1B).

When we dissected the role of individual residues on this membrane distal surface of Sec24, single and double mutant combinations of T324A/T325A/T328A showed a range of defects in Pho8Δ60 activity, with the triple alanine mutant showing the most dramatic defect (Figure 2A). We were unable to test the full range of phosphomimetic combinations, as some caused lethality (Figure 1C and Supplementary file 1). However, none of the viable phosphomimetic mutants showed a defect in autophagy (Figure 2B,C). Sec24-T324A/T325A/T328A (hereafter referred to as Sec24-3A) was further characterized because it had the most dramatic defect in Pho8Δ60 activity. To confirm that phosphorylation of the Sec24 membrane distal surface is required for autophagy, we examined a second autophagy marker, Atg8. Cytosolic GFP-Atg8 is lipidated and incorporated into membrane at the PAS before being delivered to the vacuole (Klionsky et al., 2007). Translocation of GFP-Atg8 to the vacuole, following nitrogen starvation, was significantly reduced in a strain expressing Sec24-3A (Figure 2D). This defect was confirmed using western blot analysis by monitoring the cleavage of GFP-Atg8 to GFP (Figure 2E). The GFP-Atg8 translocation defect was less severe in the presence of Iss1 (Figure 2D, right), suggesting functional complementation by the paralogous protein.

Figure 2 with 2 supplements see all
Phosphorylation of T324/T325/T328 in Sec24 is required for autophagy, but not ER-Golgi transport.

(A) Vacuolar alkaline phosphatase activity was assayed in lysates prepared from a sec24∆iss1∆ deletion strain harboring sec24 alanine mutations. The activity of wild-type (WT) 2 hr after starvation was set as 100% and 0 time-point values were subtracted. Averages and s.e.m. are shown for 3 (or four for T324A/T325A) biological replicates. p-values=0.006 (T324A), 0.012 (T325A), 0.009 (T328), 0.008 (T324A/T328A), 0.02 (T324A/T325A), 0.01 (T325A/T328A), 0.006 (T324A/T325A/T328A), Student’s paired t-test. (B, C) As in (A) except activity was assayed in extracts from phosphomimetic mutations in sec24∆iss1∆ (B) or sec24∆ (C) deletion strains. Averages and s.e.m. are shown for three biological replicates. p-values=0.88 (T325E), 0.78 (T328E), 0.32 (T324E/T328E), 0.26 (T324E/T325E), Student’s paired t-test. (D) The translocation of GFP-Atg8 to the vacuole was examined 1 hr after nitrogen starvation at 25°C in sec24∆iss1∆ and sec24∆ deletion strains in either the presence of WT Sec24 or Sec24-3A. Representative images (left) and quantification from 300 cells (right) are shown. Scale bar 2 µm. WT was set to 100% for each experiment and had an average vacuolar localization of 76% (sec24∆iss1∆) and 86% (sec24∆). Averages and s.e.m. are shown for three biological replicates. p-values=0.006 (sec24∆iss1∆), 0.02 (sec24∆), Student’s paired t-test. (E) Cleavage of GFP-Atg8 was examined in sec24∆iss1∆ cells expressing Sec24 or Sec24-3A 1 hr after starvation at 25°C (left). The ratio of free GFP to GFP-Atg8 was quantitated. The cleavage in WT was set to 1 (right). Averages and s.e.m. are shown for three biological replicates. p-value = 0.015, Student’s unpaired t-test. (F) sec24∆iss1∆ cells expressing Sec24 (lanes 1–4) or Sec24-3A (lanes 5–8) were pulse-labeled for 4 min and chased for the indicated times (left). The p1 (ER), p2 (Golgi) and m (vacuolar) forms of CPY are labeled. Quantitation of the ratio of p2/p1 CPY for the 5 and 10 min time points are shown (right). Averages and s.e.m. are shown for three biological replicates. p-values=0.08 (5 min), 0.66 (10 min), Student’s unpaired t-test. *p<0.05; **p<0.01.

https://doi.org/10.7554/eLife.21167.006

We next demonstrated that phosphorylation of the membrane distal sites on Sec24 is specifically required for autophagy by examining ER-Golgi traffic in cells containing Sec24-3A. The processing of carboxypeptidase Y (CPY), as it traffics from the ER (p1), Golgi (p2), and vacuole (m) is kinetically indistinguishable from wild-type (Figure 2F), demonstrating that phosphorylation of the Sec24 membrane distal patch regulates a novel function of Sec24 that is specific to autophagy. Supporting the model that a subpopulation of COPII vesicles is diverted from the secretory pathway during autophagy, ER to Golgi traffic was delayed, but not blocked, during starvation (Figure 2—figure supplement 1A). Expression of Sec24-3A did not rescue this delay even in an iss1∆ strain background (Figure 2—figure supplement 1B,C) suggesting that additional sites and/or factors also participate in reprogramming COPII vesicles during starvation.

Since Sec24 robustly co-purifies with Sec23, our mass spectrometry data also included information on Sec23, which contained three phosphosites on an alpha helix structurally equivalent to the Sec24 membrane distal patch (Figure 2—figure supplement 2A). Mutation of these Sec23 sites (T146/S147/S149) caused no defects in growth (Figure 2—figure supplement 2B), Pho8∆60 activity (Figure 2—figure supplement 2C) or translocation of GFP-Atg8 to the vacuole (Figure 2—figure supplement 2D), suggesting the effects we observed on autophagy are specific to Sec24 and its paralog, Iss1.

Phosphorylation of T324/T325/T328 regulates autophagosome number during starvation

We next wanted to address whether phosphorylation of the Sec24 membrane distal sites is specifically required for autophagosome formation during starvation-induced upregulation of autophagy. To begin to address this question, structured illumination microscopy (SIM) was used to examine GFP-Atg8 puncta formation in the absence (nutrient rich) or presence of rapamycin. GFP-Atg8 puncta mark autophagosomes, as well as the PAS and isolation membrane (Kirisako et al., 1999). In nutrient-rich media, autophagosome-like structures, called Cvt vesicles, form. These vesicles traffic a precursor form of aminopeptidase I (prApe1) to the vacuole in an Atg11-dependent manner (He et al., 2006), where it is proteolytically activated (mApe1). GFP-Atg8 puncta were smaller (Figure 3A), less numerous, and dependent on Atg11 (Figure 3B) in nutrient rich medium. Consistent with the possibility that the GFP-Atg8 puncta may represent Cvt vesicles, Atg11 was only required for their formation in rich medium and not in rapamycin-treated cells (compare Figure 3B,C).

Figure 3 with 1 supplement see all
Phosphorylation of Sec24 regulates autophagosome frequency during starvation.

(A) Representative images from WT cells expressing GFP-Atg8 treated with 400 ng/ml rapamycin for 1 hr at 25°C (left top) or untreated (left bottom). Deconvolved images are shown. Scale bar, 1 µm. 100 GFP-Atg8 puncta were measured in WT cells treated with or without rapamycin (right). Averages and s.e.m. are shown for three biological replicates; p=0.0002, Student’s unpaired t-test. (B) WT Sec24 and Sec24-3A expressed in the sec24Δiss1Δ deletion strain, and WT (+Atg11) and atg11Δ cells (-Atg11) expressing GFP-Atg8 were imaged and the number of puncta per cell was quantitated from 300 cells. Averages and s.e.m. are shown for three biological replicates; p-values=0.81 (Sec24-3A), 0.009 (-Atg11), Student’s unpaired t-test. (C) As in (B) except cells were treated with 400 ng/ml rapamycin for 1 hr at 25°C. Averages and s.e.m. are shown for three biological replicates; p-values=0.02 (Sec24-3A), 0.68 (-Atg11), Student’s unpaired t-test. (D) As in (C) only the diameter of 100 GFP-Atg8 puncta was measured from cells expressing WT Sec24 and Sec24-3A in sec24Δiss1Δ deletion strains treated with 400 ng/ml rapamycin for 1 hr at 25°C. Averages and s.e.m. are shown for four biological replicates; p-value = 0.057, Student’s unpaired t-test. (E) Representative images of autophagic bodies in cells expressing Sec24 (left) and Sec24-3A (right) in sec24Δiss1Δpep4Δ deletion strains after 1.5 hr of nitrogen starvation at 30°C. Scale bar represents 500 nm. (F) Histogram showing the distribution of the number of autophagic bodies per vacuole section in Sec24 and Sec24-3A. The number of autophagic bodies was quantitated for 78 vacuole sections for each strain (left). p-value = 0.00012; Mann-Whitney Test. Box plot of the number of autophagic bodies per vacuole section. Bars show data between the lower and upper quartiles, the median is a horizontal line within the box. Whiskers indicate the smallest and largest observations (right). (G) The diameter of autophagic bodies was determined. For Sec24 N = 398, for Sec24-3A N = 342. Averages with error bars as s.e.m. are shown. *p<0.05; **p<0.01; ***p<0.001.

https://doi.org/10.7554/eLife.21167.009

Cells expressing Sec24-3A were not defective in GFP-Atg8 puncta formation in nutrient rich medium (Figure 3B), however, fewer puncta formed in the rapamycin-treated Sec24-3A cells (Figure 3C). While Sec24-3A significantly reduced the number of puncta formed, it did not significantly affect their size (Figure 3D). Consistent with Sec24-3A not affecting Cvt vesicle formation, processing of Ape1 was unaffected in sec24 alanine mutants (Figure 3—figure supplement 1A). Similar results were obtained when we blocked COPII vesicle budding at 37°C in the temperature-sensitive mutant sec12-4 mutant (Figure 3—figure supplement 1B,C). The observation that COPII vesicles are not needed on the Cvt pathway is consistent with our earlier studies (Wang et al., 2015) and those of Ishihara et al. (2001)

We used a second assay to address the effect of Sec24-3A on the frequency of autophagosome formation during starvation. Autophagosome number and size can be assessed using transmission electron microscopy by analyzing autophagic bodies, fully formed autophagosomes that have fused with the vacuole. Upon deletion of the PEP4 gene, which encodes a protease that is required for the activation of multiple vacuolar hydrolases, autophagic bodies accumulate in the vacuole (Backues et al., 2014).

After starvation, fewer autophagic bodies accumulated in cells expressing Sec24-3A compared to WT Sec24 (Figure 3E,F). Although autophagic body number was significantly reduced, autophagic body size was not affected (Figure 3E,F,G). Together these findings indicate that phosphorylation of the Sec24 membrane distal sites regulates autophagosome number, while autophagosome size or expansion is largely unaffected. The role of Sec24 in autophagy is likely to be conserved in mammalian cells as T324 and T328 are conserved in mSec24A (Figure 4A).

Figure 4 with 1 supplement see all
The Sec23/Sec24 complex binds the C-terminus of Atg9.

(A) Structure of yeast Sec24 and mSec24a with conserved residues in membrane distal sites (red). (B) ypt7∆ cells expressing Atg9-13myc were grown in nutrient rich (SMD) or starvation (SD-N) media for 4 hr and Sec24 was immunoprecipitated and blotted for Atg9-13myc (left). Precipitated Atg9-13myc was quantitated and normalized to the amount of Sec24 in the precipitate. SMD was set as one for each experiment. ypt7∆ cells were used as autophagosomes fail to fuse with the vacuole in the absence of Ypt7 and accumulate in the depleted cells (Kirisako et al., 1999). Averages and s.e.m. are shown for four biological replicates, p-value = 0.037, Student’s unpaired t-test. (C) Schematic showing cytosolic domains of Atg9. (D) Equimolar amounts (200 nM) of purified GST, GST-Sec31 (aa878-1114) or GST-Atg9 fragments were incubated with 50 or 100 nM of Sec23/Sec24-His6. (E) Equimolar amounts (100 nM) of purified GST or GST-Atg9C was incubated with increasing amounts of Sec23/Sec24-His6. (F) Same as (D) except His6-Sec23 was used.

https://doi.org/10.7554/eLife.21167.011

Phosphorylation regulates the interaction of Sec24 with the C-terminus of Atg9

We previously proposed that COPII vesicles fuse with Atg9 vesicles at the PAS (Tan et al., 2013). When autophagy is induced, Atg9 vesicles traffic from the late Golgi to the PAS and localize adjacent to the ER exit sites that produce COPII vesicles (Graef et al., 2013; Suzuki et al., 2013; Yamamoto et al., 2012). Interestingly, similar to our observations with Sec24, Atg9 was recently shown to regulate autophagosome number, but not size (Jin et al., 2014). Proteomics also revealed an interaction between multiple COPII coat subunits and Atg9 in detergent lysates (Graef et al., 2013). However, it remained unclear from these studies which coat subunit mediated this interaction and whether it was required for autophagy. Therefore, we first confirmed that Sec24 co-precipitates with Atg9 and tested whether this interaction is regulated by starvation. Sec24 was immunoprecipitated from cells expressing Atg9-13myc that were grown in nutrient rich conditions (SMD) or starved for nitrogen (SD-N), and the precipitate was then blotted with anti-myc antibody. Atg9-13myc co-immunoprecipitated with Sec24, but not the pre-immune control (Figure 4B). Moreover, approximately 2.5-fold more Atg9-13myc co-immunoprecipitated with Sec24 from lysates prepared from nitrogen starved cells (Figure 4B), demonstrating that this interaction is upregulated during starvation.

Atg9 is a six transmembrane protein with N-terminal, C-terminal and core or middle (M) cytoplasmic domains (Figure 4C). While the middle and C-terminal domains are present in mammalian cells, the N-terminus is largely absent (Young et al., 2006). In order to determine which domain of Atg9 interacts with Sec24, the cytoplasmic domains of Atg9 were fused to GST and the purified fusion proteins were incubated in vitro with purified Sec23/Sec24 complex. The Sec23/Sec24 complex was used for these studies, since Sec24-His6 is unstable in the absence of Sec23. Sec24 was predominantly unphosphorylated, as it was purified without phosphatase inhibitors and stored in the freezer following purification. A GST fusion to a Sec31 fragment (aa878-1114), which was previously shown to interact with the Sec23/Sec24 complex (Bi et al., 2007), served as a positive control. The C-terminus of Atg9 bound to Sec23/Sec24, while the N-terminal and middle hydrophilic Atg9 domains did not (Figure 4D, Figure 4—figure supplement 1A). Furthermore, binding of Sec23/Sec24 to the C-terminus increased with increasing concentrations of the Sec23/Sec24 complex and appeared to be saturable (Figure 4E, Figure 4—figure supplement 1B). This interaction was also dependent on Sec24, as Sec23 alone did not interact with GST-Atg9C (Figure 4F, Figure 4—figure supplement 1C). As the crystal structure of Sec23 and Sec24 are identical whether they are in a complex or not, allosteric effects are unlikely (Bi et al., 2002).

Next we used the phosphomimetic mutations to ask if the interaction between Sec24 and Atg9 is enhanced by phosphorylation of the membrane distal sites. While the most dramatic effects on autophagy were seen with the sec24 triple alanine mutant (Figure 2), the sec24 triple phosphomimetic mutant is inviable. Therefore, to cover all three phosphosites of interest in our binding studies, we used Sec23/Sec24-T325E and Sec23/Sec24-T324E/T328E. Consistent with the notion that phosphorylation enhances the Sec24-Atg9 interaction, both T325E and T324E/T328E increased the interaction of Sec24 with GST-Atg9C (Figure 5A, Figure 5—figure supplement 1A).

To determine whether the Sec24-Atg9 interaction is regulated by phosphorylation in vivo, wild-type or mutant sec24 cells co-expressing Atg9-13myc were starved for nitrogen and Sec24 was immunoprecipitated in the presence of phosphatase inhibitors. Sec24-T324A/T325A (Figure 5B), but not Sec24-T324E/T325E (Figure 5—figure supplement 1B) disrupted the interaction of Sec24 with Atg9-13myc. This defect was more pronounced in Sec24-3A, where Sec24-T328 is also mutated (Figure 5C). Sec24-3A did not impair the trafficking of Atg9 to the PAS (Figure 5—figure supplement 2A,C), indicating that Sec24 does not regulate Atg9 traffic. Together these findings imply that the C-terminus of Atg9 interacts with phosphorylated Sec24 via its membrane distal surface after Atg9 has been recruited to the PAS.

Figure 5 with 5 supplements see all
Phosphorylation of the Sec24 membrane distal sites regulates the Sec24-Atg9 interaction.

(A) GST-Atg9C (200 nM) was incubated with 37.5 nM of WT Sec23/Sec24-His6, Sec23/Sec24-T325E-His6 or Sec23/Sec24-T324E/T328E-His6 (left). Ratio of Sec24 bound to GST-Atg9C was quantified from three biological replicates. Averages and s.e.m. are shown (right). WT Sec23/Sec24 was set as one for each experiment; p-value = 0.018 (T325E), 0.0008 (T324E/T328E), Student’s unpaired t-test. (B) Sec24 (WT) and Sec24-T324A/T325A or (C) Sec24-T324A/T325A/T328A (Sec24-3A) were immunoprecipitated from lysates expressing Atg9-13myc as described in the Materials and Methods. Precipitated Atg9-13myc was quantitated and normalized to the amount of Sec24 in the precipitate. WT Sec24 was set as one for each experiment. Averages and s.e.m. are shown for 4 (B) or 5 (C) biological replicates; p-value = 0.006 (B), 0.0008 (C) Student’s unpaired t-test. (D) Alignment of the region surrounding T324/T325/T328 (shown in red) with Sec24 orthologues. (E) Same as (B) except Sec24 was immunoprecipitated from WT or hrr25-5 lysates. Averages and s.e.m. are shown for five biological replicates. p-value = 0.0002, Student’s unpaired t-test. **p<0.01; ***p<0.01; ***p<0.001.

https://doi.org/10.7554/eLife.21167.013

Microscopy-based studies have linked ERES to autophagosome formation (Graef et al., 2013; Suzuki et al., 2013), however, the ERES are also a subdomain of the ER where COPII vesicles bud (Budnik and Stephens, 2009). To ask if Sec24-3A disrupts the formation of ERES, the localization of Sec13-GFP was examined in cells expressing wild-type Sec24 or Sec24-3A. Sec13-GFP, a component of the outer COPII coat, predominantly localizes to ERES (Shindiapina and Barlowe, 2010). ERES localization of Sec13-GFP was not affected by Sec24-3A in nutrient rich or starvation conditions, suggesting Sec24-3A does not disrupt ERES formation (Figure 5—figure supplement 3). Thus, our findings imply that the major function of the Sec24 membrane distal surface is to regulate the interaction of COPII vesicles with the Atg machinery.

Hrr25 regulates autophagy via Sec24

Hrr25, the only kinase known to phosphorylate Sec24 in yeast, is required for COPII vesicle fusion in ER-Golgi traffic and autophagy (Lord et al., 2011; Wang et al., 2015). Recent studies, however, have suggested Hrr25 has an additional role in autophagy, upstream of COPII vesicle fusion at the PAS (Wang et al., 2015). Specifically, we found that while COPII vesicles accumulate at the PAS in some mutants that disrupt autophagy, they failed to accumulate in the hrr25-5 mutant. Additionally, epistasis studies revealed Hrr25 acts upstream of the key autophagy kinase, Atg1 (Wang et al., 2015).

Interestingly, Sec24-T328 fits the CK1 consensus motif (pS/pT-X-X-S/T, Figure 5D) (Knippschild et al., 2005) and was found with low confidence to be phosphorylated by Hrr25 in vitro. Consistent with a role for Hrr25 in phosphorylating the Sec24 membrane distal patch, less Atg9 co-immunoprecipitated with Sec24 from an hrr25-5 mutant lysate (Figure 5E, Figure 5—figure supplement 4). Additionally, like the sec24 triple mutant (Figure 3E,F,G), we observed a reduction in autophagic body number, but not size in the hrr25-5 mutant using transmission electron microscopy (Figure 5—figure supplement 5). To determine if Hrr25 regulates the Sec24-Atg9 interaction via Sec24 phosphorylation, we asked whether the Sec24 phosphomimetic mutations could rescue the Sec24-Atg9 interaction defect in hrr25-5. Sec24 T325/T328 was chosen for this analysis as it contains T328, which is phosphorylated by Hrr25. WT Sec24, Sec24 T325A/T328A or Sec24 T325E/T328E was ectopically expressed in hrr25-5 cells containing Atg9-13myc and Sec24 was immunoprecipitated. Sec24 T325E/T328E significantly rescued the Sec24-Atg9 interaction in hrr25-5, whereas Sec24 T325A/T328A did not (Figure 6A). Additionally, Sec24 T325E/T328E alleviated the autophagy defect in hrr25-5 as the vacuolar localization of GFP-Atg8 induced by starvation was partially rescued by Sec24 T325E/T328E, but not Sec24 T325A/T328A (Figure 6B). To confirm the fluorescence results, cleavage of GFP-Atg8 was also examined. Sec24 T325E/T328E almost fully rescued the GFP cleavage defect in hrr25-5, while Sec24 T325A/T328A had no effect (Figure 6C, Figure 6—figure supplement 1). Therefore, although Hrr25 is required for COPII vesicle fusion (Lord et al., 2011; Wang et al., 2015), phosphorylation of Sec24 is a primary function of this kinase during starvation induced autophagy. These findings also indicate the Sec24-Atg9 interaction is needed for autophagy.

Figure 6 with 4 supplements see all
Hrr25 regulates autophagy via phosphorylation of the Sec24 membrane distal sites.

(A) Sec24 was immunoprecipitated from WT or hrr25-5 cells expressing Atg9-13myc and either WT Sec24 or Sec24 T325A/T328A or Sec24 T325E/T328E. Precipitated Atg9-13myc was quantitated and normalized to the amount of Sec24 in the precipitate. WT was set as one for each experiment. Averages and s.e.m. are shown for three biological replicates. p-value = 0.01 (hrr25-5 + T325E/T328E), 0.81 (hrr25-5 + T325A/T328A), Student’s unpaired t-test. (B) Vacuolar localization of GFP-Atg8 was examined 2 hr after nitrogen starvation at 37°C in WT or hrr25-5 cells expressing either WT Sec24 or Sec24 T325A/T328A or Sec24 T325E/T328E. Scale bar, 2 µm (left). Over 300 cells were quantitated from three biological replicates. WT was set as 100% for each experiment. Averages and s.e.m. are shown. p-value = 0.006 (hrr25-5 + T325E/T328E), 0.17 (hrr25-5 + T325A/T328A), Student’s unpaired t-test. (C) Cleavage of GFP-Atg8 in hrr25-5 cells expressing WT Sec24 or Sec24 T325A/T328A or Sec24 T325E/T328E were examined 2 hr after nitrogen starvation at 37°C (left). The ratio of GFP to GFP-Atg8 was quantitated from three biological replicates. The cleavage in WT was set to 1. Averages and s.e.m. are shown. p-value = 0.03 (hrr25-5 + T325E/T328E), 0.99 (hrr25-5 + T325A/T328A), Student’s unpaired t-test. *p<0.05; **p<0.01.

https://doi.org/10.7554/eLife.21167.019

Despite the role of Hrr25 in regulating the Atg9-Sec24 interaction, neither Hrr25 expression, nor activity was altered upon nitrogen starvation (Figure 6—figure supplement 2). However, as CK1 kinases act preferentially on substrates that are already phosphorylated at the N-3 position (Figure 5D) (Knippschild et al., 2005), during autophagy Hrr25 may work with other kinases that create additional Sec24 phosphosites. To determine if Hrr25 mediated phosphorylation of Sec24 is required for Atg complex assembly (Figure 5—figure supplement 2B), we visualized the PAS recruitment of a member of each of the major groups of Atg proteins required for autophagosome formation in the hrr25-5 and sec24 triple alanine mutants. We found no effect of hrr25-5 (Figure 6—figure supplement 3) or Sec24-3A (Figure 5—figure supplement 2A,C) on the PAS recruitment of Atg2, Atg5, Atg9, Atg13 or Atg14. Consistent with our findings, blocking COPII vesicle formation in the sec12-4 mutant also had no effect on the PAS localization of Atg2 and Atg14 (Figure 6—figure supplement 4). Thus, while COPII vesicles are needed for autophagy, they are dispensable for the assembly of the Atg hierarchy. Together these findings demonstrate that Sec24 phosphorylation does not indirectly affect autophagy by disrupting the trafficking of Atg proteins to the PAS.

Discussion

Although significant progress has been made in defining the upstream events leading to the assembly of Atg proteins at the PAS (Nakatogawa et al., 2009), the membrane rearrangements that occur during autophagy remain poorly understood. Here we show that phosphorylation of a conserved regulatory domain of the major COPII cargo adaptor Sec24 reprograms the function of COPII vesicles by modulating its interaction with the C-terminus of Atg9, a key component of the autophagy machinery. The C-terminus of Atg9 is present in vertebrates and is essential for autophagy (He et al., 2008; Young et al., 2006). Atg9 functions early in autophagosome initiation (Suzuki et al., 2007; Yamamoto et al., 2012) and affects autophagosome number, but not size (Jin et al., 2014). Similarly, failure to phosphorylate the Sec24 regulatory sites, which disrupts the Sec24-Atg9 interaction, specifically affects autophagosome number. Consistent with our proposal that autophagosome formation requires the fusion of COPII vesicles with Atg9 vesicles (Tan et al., 2013; Figure 7), we see an accumulation of COPII coated structures at the PAS in the atg9Δ mutant (Figure 7—figure supplement 1).

Figure 7 with 1 supplement see all
Phosphorylation of a conserved regulatory surface of Sec24 enhances the ability of the COPII coat to recognize the autophagy machinery.

Phosphorylation of the Sec24 membrane distal sites regulates the interaction of Sec24 with the C-terminus of Atg9. During starvation, the Sec24-Atg9 interaction is needed to increase autophagosome number. If Sec24 is not phosphorylated, it is unable to efficiently interact with Atg9 and COPII vesicles traffic to the Golgi.

https://doi.org/10.7554/eLife.21167.024

For technical reasons, we have been unable to directly address if the Sec24 membrane distal sites are phosphorylated as a consequence of inducing autophagy. However, given that these sites are only required for autophagosome formation during starvation-induced autophagy, and both phosphorylation and autophagy enhance the Sec24-Atg9 interaction, it seems likely the membrane distal Sec24 surface is specifically phosphorylated when autophagy is induced. As autophagosomes form in proximity to each other in nutrient rich and starved cells, the regulated phosphorylation of Sec24 would prevent the inappropriate fusion of COPII vesicles with Atg9 membranes during constitutive growth.

Phosphorylation of the Sec24 membrane distal surface is regulated at least in part by Hrr25. We have shown Hrr25 is required for autophagy and functions upstream of COPII vesicle delivery to the PAS (Wang et al., 2015), but how Hrr25 regulates COPII vesicles on this pathway has been unclear. The findings we present here show that, during autophagy, Hrr25 acts through Sec24 by regulating its interaction with Atg9. As Hrr25 has been linked to the TOR network (Breitkreutz et al., 2010), a key negative regulator of autophagy (Loewith and Hall, 2011), it is tempting to speculate that Hrr25 works with one or more kinases that act downstream of Tor during nutrient deprivation. We propose that the heightened need to rapidly produce autophagosomes during starvation leads to phosphorylation of the Sec24 membrane distal surface, which enhances the Sec24-Atg9 interaction, resulting in an increase in autophagy to maintain homeostasis (Figure 7). These events are independent of the assembly of the Atg hierarchy that occurs when autophagy is induced.

Previous work has established that ERES and COPII vesicles are required for autophagy in both yeast and mammalian cells (Ge et al., 2014; Graef et al., 2013; Lemus et al., 2016; Suzuki et al., 2013; Tan et al., 2013; Wang et al., 2015). However, because it has been difficult to fully tease apart the function of COPII vesicles on the secretory pathway from their role in autophagy, their contribution to autophagy has been problematic to address. Our finding that phosphorylation of a novel conserved regulatory surface of Sec24 is specifically required for autophagy and not ER-Golgi transport conclusively demonstrates that COPII vesicles play a direct role in autophagy, rather than an indirect role in maintaining the trafficking of autophagy machinery through the secretory pathway. Additionally, we can now ascribe an autophagy-specific role to COPII vesicles in enhancing autophagosome number during nutrient deprivation, which is likely to be conserved.

In mammalian cells, nucleation of autophagosomes occurs in the vicinity of the ER and the Atg9 compartment (Karanasios et al., 2016). Interestingly, membrane fractionation identified the ER-Golgi intermediate compartment (ERGIC), which resides adjacent to the ER, as the site of lipidation of the Atg8 homologue LC3 (Ge et al., 2014). During starvation, the ERES were found to translocate to the ERGIC fraction, and although the ERGIC is typically a location of COPI vesicle budding (Lorente-Rodríguez and Barlowe, 2011), COPII vesicle budding from this ERGIC fraction was shown to be required for LC3 lipidation (Ge et al., 2014). These observations suggested that, in higher eukaryotes, the COPII vesicles used in autophagy are spatially separated from those that traffic to the Golgi. The findings we report here could explain how these spatially segregated COPII vesicles engage the Atg machinery.

Other components of the secretory pathway have also been implicated in autophagy (Amaya et al., 2015; Geng et al., 2010; Nair et al., 2011). It remains to be seen whether these components directly contribute membrane for autophagosome formation or if they only affect the trafficking of autophagy machinery, such as Atg9, to sites of autophagosome formation. It is known that another component of the autophagy machinery, Atg8, controls autophagosome size, but not number (Xie et al., 2008). However, it is unclear if Atg8 is sufficient to drive autophagosome expansion or if additional membranes are required for this event.

In conclusion, our findings highlight an unexpected role for phosphorylation in regulating the reorganization of membrane trafficking pathways during starvation and demonstrate that the COPII coat is a key target of this regulation. The findings we present here ascribe a new role for Sec24 and the COPII coat, and provide a possible explanation for why the coat is retained on the vesicle subsequent to vesicle scission (Cai et al., 2007; Lord et al., 2011). Identification of a cellular mechanism that redirects the flow of membrane during autophagy makes it possible to now study how these complex membrane rearrangement events culminate in the formation of a distinct organelle, the autophagosome. Future work will be needed to determine if the diverted COPII vesicles contain autophagy-specific cargo, or if the role of these vesicles in autophagy is solely to bring certain lipids and SNAREs to this pathway.

Materials and methods

Media, growth conditions and yeast strains

Strains used in this study are listed in Supplementary file 2. Yeast cells were grown at 25°C in rich media (YPD: 1% yeast extract, 2% peptone, and 2% dextrose) or synthetic minimal media (SMD: 0.67% yeast nitrogen base, 2% dextrose and auxotrophic amino acids as needed). Nitrogen starvation was induced in synthetic minimal medium lacking nitrogen (SD-N: 0.17% yeast nitrogen base without amino acids, 2% dextrose). For galactose induction of Sec24, the growth conditions are described in the next section. For solid media, agar was added to a final concentration of 2%.

Mass spectrometry

Sec24-His6 was purified from SFNY2181 (Supplementary file 2) as described previously (Kurihara et al., 2000) with the following modifications. Cells were grown overnight to log phase in SC-Ura-Leu medium with 10% glycerol as the carbon source, and induced at a starting OD600 of 0.6–0.8 with 0.2% galactose for 5 hr at 30°C. These growth conditions were found to induce autophagy in the absence of rapamycin. Cells were lysed in approximately 20 ml HSLB (0.75 M potassium acetate, 50 mM HEPES pH 7.0, 0.1 mM EGTA, 20% glycerol) with protease and phosphatase inhibitor cocktails (Sigma, St. Louis, MO). The cleared lysate was incubated with Ni-NTA beads for 1 hr at 4°C and washed with 20 ml B-II and 20 ml B-III (Kurihara et al., 2000). Sample preparation for mass-spectrometry was carried out as described before (Guttman et al., 2009), then liquid chromatography coupled tandem mass spectrometry analysis (LC-MS/MS) was performed as described previously (Meyer et al., 2014). The high confidence Sec24 mass spectrometry data from three runs are compiled in Supplementary file 3. Note, potential sites buried in the Sec24 structure were not analyzed further.

Generation of sec24 mutants

Mutations in pSFN1915 (SEC24, HIS3, CEN) were made using the QuikChange Site-directed mutagenesis kit (Agilent technologies, Santa Clara, CA) and all mutations were confirmed by DNA sequencing. Plasmids were then introduced into SFNY2201 and SFNY2202 (Supplementary file 2) and grown for two rounds on 5-fluoroorotic acid (5-FOA) plates at 25°C to select against pLM22 (SEC24, URA3, CEN). To observe growth defects, sec24 mutants were compared to WT SEC24 at 25°C after two rounds of 5-FOA. For purification of mutant coat proteins, the mutations were made on pSFNB1895 (GAL1-SEC24-His, LEU2, CEN) and co-transformed with pSFNB1894 (GAL1-SEC23, URA3, CEN) into SFNY2367 (Supplementary file 2).

Analysis of phosphosites on the Sec23/Sec24 structure

All structures were accessed through the protein data bank (PDB) and analyzed with PyMol software. The Sec23/Sec24 structure, PDB ID 1M2V, was reported in a previous study (Bi et al., 2002). The Sec24 structure with the Sed5 peptide, PDB ID 1PD0, was reported in a previous study (Mossessova et al., 2003).

In vitro vesicle budding and fusion assays

COPII proteins (Sar1, Sec23/Sec24 and Sec13/Sec31) were purified (Miller et al., 2003) and used to generate vesicles from microsomal membranes prepared as described (Barlowe et al., 1994). Vesicle budding assays were performed as described previously (Miller et al., 2002). Vesicle fusion was monitored by measuring α−1,6-mannose modification of 35S-labeled pro-α-factor as described previously (Barlowe et al., 1994).

Pho8∆60 assay

Alkaline phosphatase assays were performed as previously described (Klionsky, 2007). Cells were grown overnight at 25°C to log phase, OD600 between 0.7 and 1.0, washed with 10 ml SD-N medium and incubated in SD-N medium for 2 to 4 hr at 25°C or 37°C to induce autophagy. 2.5 OD600 units of cells were collected and washed, and lysed in 250 µl of lysis buffer (20 mM PIPES pH 7.2, 0.5% TritonX-100, 50 mM KCl, 100 mM potassium acetate, 10 mM MgS04, 10 µM ZnSO4, and 1 mM PMSF) using glass beads. Lysates, with a protein concentration around 0.5 mg/ml, were spun for 5 min at 13,000 rpm, and 100 µl of lysate was assayed at 37°C in 400 µl reaction buffer (1.25 mM p-nitrophenyl phosphate, 250 mM Tris-HCl pH 8.5, 0.4% Triton X-100, 10 mM MgSO4, and 10 µM ZnSO4). The reaction was stopped with 500 µl of stop buffer (1 M glycine/KOH pH 11.0), and the OD400 value was determined. The data were normalized to protein concentration using the Bradford method and IgG as a standard.

Fluorescence microscopy

For GFP-Atg8 vacuolar localization, cells were grown overnight at 25°C in SC-Ura to early log phase, OD600 between 0.6 and 1.0. Cells were washed and resuspended in SD-N and incubated for 1 hr at 25°C (Sec24-3A), 30 min at 37°C (Sec24-S730/S735), or 2 hr at 37°C (hrr25-5 mutant). For co-localization of Ape1-RFP with Atg proteins, cells were grown overnight to early log phase at OD6000.6–1.0 and treated with 400 ng/mL rapamycin for 1 hr at 25°C (Sec24-3A), or starved for nitrogen for 2 hr at 37°C (hrr25-5 and sec12-4 mutants). Cells were then visualized at 25°C with a Zeiss Axio Imager Z1 fluorescence microscope using a 100×1.3 NA oil-immersion objective. Images were captured with a Zeiss AxioCam MRm digital camera and analyzed with AxioVision software.

To examine GFP-Atg8 puncta by structured illumination (SIM) microscopy, cells were grown overnight at 25°C in SC-Ura to early log phase, OD600 between 0.6 and 1.0. For autophagy induction, cells were treated with 400 ng/ml rapamycin for 1 hr at 25°C or for 1 hr at 37°C for temperature-sensitive mutants. Cells were pelleted and incubated in 3.7% formaldehyde for 30 min at 25°C and visualized on an Applied Precision DeltaVision OMX Super Resolution System using an Evolve 512 EMCCD camera. The data was acquired and processed using Delta Vision OMX Master Control software and SoftWoRx reconstruction and analysis software. To determine autophagosome size, deconvolved images were analyzed with Image J software.

GFP-Atg8 cleavage assay and Ape1 processing

To observe cleavage of GFP-Atg8 during autophagy, cells were grown overnight to early log phase, washed, resuspended in SD-N and incubated for 1 hr at 25°C (Sec24-3A) or 2 hr at 37°C (hrr25-5 mutant). The cells (2.5 OD600 units) were then pelleted, resuspended in 0.1 M NaOH and incubated for 5 min at room temperature. The samples were spun and heated in sample buffer for 5 min at 95°C before SDS PAGE. To monitor Ape1 processing, cells were grown in nutrient rich media to early log phase and lysed as described above.

CPY pulse chase

Cells were grown overnight in minimal media at 25°C to early log phase and 16 OD600 units of cells were pelleted and resuspended in 3.6 ml of fresh minimal media. For starved samples, cells were washed and shifted to SD-N for 1 hr at 25°C. 16 OD600 units of cells were then pelleted and resuspended in 3.6 ml of fresh SD-N. Cells were pulse labeled with 400 µCi of S35-methionine for 4 min at 25°C, and 700 µl of the cell suspension was removed and added to 700 µl of ice-cold 20 mM sodium fluoride/sodium azide (0 min time-point). 250 µl of chase mix (250 mM methionine, 250 mM cysteine) was added to the remaining sample, and then 700 µl of cells were removed at 5, 10 and 15 min. Cells were pelleted and washed with 1 ml of cold 10 mM sodium fluoride/sodium azide, resuspended in 150 µl spheroplasting buffer (1.4 M sorbitol, 100 mM sodium phosphate pH 7.5, 0.35% β-mercaptoethanol and 0.2 mg/ml zymolyase) and incubated at 37°C for 45 min. Spheroplasts were spun for 3 min at 6500 rpm and heated for 5 min at 95°C in 100 µl 1% SDS. 900 µl of PBS plus 2% Triton X-100 was added to the lysates before they were spun for 15 min at 14,000 rpm. CPY antibody (3 µl of anti-Rabbit serum prepared against CPY) was added to 920 µl of cleared lysate and incubated for 1 hr at 4°C with rotation. 50 µl of 50% Protein-A sepharose was added and incubated for 1 hr. The protein-A beads were washed twice with 1 ml of PBS, followed by two washes with 1 ml of 1% β-mercaptoethanol and heated in 70 µl of 1x sample buffer for 5 min at 95°C. Samples were normalized to cpm in the cell lysate, then loaded onto an 8% SDS-PAGE gel and processed for autoradiography. Protein bands were quantified using Image J software.

Electron microscopy

Cells were grown overnight in YPD to an OD600 of 1.0 and shifted to SD-N for 1.5 hr at 30°C (Sec24-3A) or 1.5 hr at 37°C (hrr25-5 mutant). 30 OD600 units of cells were pelleted, resuspended in 1 mL of 1.5% KMnO4 and incubated for 30 min at 4°C with nutation. Cells were then pelleted and resuspended in 1 mL of 1.5% KMnO4 and incubated overnight at 4°C with nutation. Samples were dehydrated in ethanol, embedded in Durcupan epoxy resin (Sigma-Aldrich) and sectioned at 60 nm on a Leica UCT ultramicrotome. Sections were picked up on Formvar and carbon-coated copper grids and stained with 2% uranyl acetate for 5 min and Sato's lead stain for 1 min. Grids were viewed using a Tecnai G2 Spirit BioTWIN transmission electron microscope equipped with an Eagle 4k HS digital camera (FEI, Hilsboro, OR). Autophagic body number and size were determined with Adobe Photoshop and Image J software as described previously (Backues et al., 2014).

Co-immunoprecipitation of Sec24 and Atg9

Cells were grown overnight to early log phase. For starvation, cells were shifted to SD-N for 4 hr at 30°C, or for the hrr25-5 mutant 2 hr at 37°C. 100 OD600 units of cells were pelleted, resuspended in 2 ml of spheroplasting buffer (1.4 M sorbitol, 100 mM sodium phosphate pH 7.5, 0.35% β-mercaptoethanol and 0.5 mg/ml zymolyase) and incubated for 30 min at 37°C. Spheroplasts were loaded on top of a 5 ml sorbitol cushion (1.7 M sorbitol, 100 mM HEPES pH 7.2) and spun for 5 min at 3000 rpm. Cells were lysed in 1 ml of lysis buffer II (20 mM Hepes pH 7.4, 150 mM NaCl, excess protease inhibitors (Roche, Switzerland and Sigma mix), phosphatase inhibitors (Sigma)) with a dounce homogenizer on ice. Cell debris was cleared by a 10 min spin at 500 xg. When cross-linking was performed, lysates were incubated on ice with 100 mM dithiobis (succinimidyl propionate) for 30 min. To quench excess crosslinker, 100 mM Tris pH 7.6 was added and incubated for 15 min on ice. Triton X-100 was added to a final concentration of 1% and incubated on ice for 30 min followed by a 15 min spin at 15,000xg. To immunoprecipitate Sec24, 2 mg of lysate was incubated with 10 µl of Sec24 antibody (rabbit polyclonal prepared against GST-Sec24) or 10 µl of pre-immune serum for 2 hr at 4°C with rotation. 50 µl of 50% protein A-sepharose was added and incubated for 45 min at 4°C with rotation. The protein-A beads were pre-incubated with 1 mg/ml BSA for 30 min at 4°C before they were added to the sample to reduce background. The beads were then washed five times with 1 ml of lysis buffer with 1% Triton X-100 and heated in 40 µl of 3x sample buffer for 5 min at 95°C. Note that similar amounts of Atg9-13myc co-immunoprecipitated with Sec24 without the use of crosslinker.

Purification of fusion proteins from bacteria for in vitro binding studies

To induce expression of GST fusion proteins, bacterial cells were incubated overnight at 18°C with 0.5 mM isopropyl β-D-1-thiogalactopyranoside. Cells were collected, resuspended in 1x phosphate-buffered saline (PBS) with 1 mM DTT and protease inhibitors, then sonicated for a total of 2 min with 15 s on/off bursts on ice. Triton X-100 was added to a final concentration of 1% and lysates were incubated on ice for 15 min. The lysates were cleared by a 15 min centrifugation at 15,000 rpm. The supernatant was incubated with 1 mL of 50% glutathione sepharose beads (GE Healthcare, United Kingdom) that was prewashed with PBS for 1 hr at 4°C with rotation before the beads were washed extensively with PBS and stored at 4°C.

His6-Sec23 was purified as described above for the GST fusion proteins, except cells were lysed in 50 mM Hepes pH 7.2, 150 mM NaCl, 15 mM Imidazole, 1 mM DTT with protease inhibitors and incubated with Ni2+-NTA resin (Qiagen, Germany). Protein was eluted from the resin with 50 mM Hepes pH 7.2, 150 mM NaCl, 250 mM Imidazole.

In vitro bindings with GST-Atg9

Equimolar amounts (0.2 µM) of GST fusion proteins were incubated with rotation in binding buffer (50 mM HEPES pH 7.2, 150 mM NaCl, 1% Triton X-100, 1 mM MgCl2, 1 mM EDTA, 1 mM DTT, protease inhibitors) for 4 hr at 4°C with increasing amounts of His6-Sec23 that was purified from bacteria or Sec23/Sec24-His6 purified from yeast as described before (Miller et al., 2002). The beads were washed 3–4 times with binding buffer and eluted in 50 µL of sample buffer by heating for 5 min at 95°C.

Kinase assay

Cells expressing Hrr25-HA were grown overnight in SC-Ura to early log phase, OD6000.6–0.8, then washed and shifted to SD-N medium for 1 hr at 25°C to induce autophagy. The cells were harvested by centrifugation, washed with 20 mM Tris pH 7.4, resupsended in 5 ml of spheroplasting buffer and incubated at 37°C for 30 min. Spheroplasts were pelleted through a 10 ml sorbitol cushion, resuspended in 5 ml of lysis buffer III (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 5 mM EDTA, 1 mM PMSF, 1% Triton X-100, 1X protease inhibitor mixture (Roche)) and lysed with a dounce homogenizer on ice. Lysates were then cleared by a 15 min centrifugation at 14,000 rpm. To immunoprecipitate Hrr25-HA, lysates were incubated with 20 µl anti-HA resin (Sigma) for 2 hr at 4°C with rotation. The beads were washed three times with lysis buffer and two times with kinase buffer (50 mM HEPES pH 7.4, 5 mM MgCl2, 0.2% NP-40 and 1 mM DTT). The kinase activity of immunopurified Hrr25-HA was assayed in a 50 µl reaction volume using 1 µg of myelin basic protein (MBP) as substrate as described before (Wang et al., 2013).

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Decision letter

  1. Noboru Mizushima
    Reviewing Editor; The University of Tokyo, Japan

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.

[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]

Thank you for choosing to send your work, "Sec24 phosphorylation regulates autophagosome abundance during nutrient deprivation", for consideration at eLife. Your initial submission has been assessed by Randy Schekman in consultation with three reviewers. Although the work is of interest, we regret to inform you that the findings at this stage are too preliminary for further consideration at eLife.

The reviews were mixed but the general opinion is that the work is interesting but not yet complete. Given the nature of the additional work described in the reviews below, the feeling among the referees is that more than two months of additional experimental effort would be required to satisfy the concerns and thus we must decline to publish this work now but certainly encourage you to return a new version when you have completed the additional work. We will endeavor to seek the views of the same reviewers on such a resubmission.

Please note that we aim to publish articles with a single round of revision that would typically be accomplished within two months. This means that work that has potential, but in our judgement would need extensive additional work, will not be considered for in-depth review. We do not intend any criticism of the quality of the data or the rigor of the science. We wish you good luck with your work and we hope you will consider eLife for future submissions.

Reviewer #1

The first part that describe the identification of Sec24 phosphorylation sites (T324/T325/T328) specifically important for autophagy is clear. However, the second part proposing that Hrr25-mediated phosphorylation of Sec24 reprograms COPII vesicles to function in autophagy is too preliminary. As the interaction of COPII subunits and Atg9 is not novel, the data presented here represent a limited advance and rather preliminary to support the model shown in Figure 6 (Figure 1 is not immediately relevant to this study). It is also unclear how the Sec24-Atg9 interaction regulates the number, rather than the size, of the autophagosome.

1) It is unclear how Sec24 phosphorylation regulates the COPII-Atg9 vesicle interaction as shown in the model in Figure 6. To examine the requirement of the Sec24-Atg9 binding in autophagosome biogenesis, it is important to identify the binding sites in both Sec24 and Atg9 and generate binding-deficient mutants.

2) The association of the COPII vesicles and Atg9 vesicles should be extensively investigated using biochemical methods and dependency on the Sec24 phosphorylation and the Sec24-Atg9 binding should also be tested.

3) Figure 3D: Showing autophagy-dependent cleavage of GFP-Atg8 by immunoblotting will complement the results.

4) It is not clear whether Hrr25 contributes to autophagy indeed through Sec24 phosphorylation. This can be tested by expression of the phosphomimetic Sec24 SE mutant in hrr25-5 mutant cells. The binding between Sec24 SE and Atg9 should be tested in hrr25 mutant cells (Figure 5F).

Reviewer #2

My opinion is mixed. The study provides a nice assessment of phosphosites in Sec24 and mutation of the distal threonine residues (324, 328, 328) clearly influence autophagosome abundance. However, I felt the evidence that starvation activates Hrr25 to directly phosphorylate these residues and that phosphorylated Sec24 then reprograms COPII vesicles for targeting to Atg9 containing membranes was a bit preliminary or overstated. At this stage I don't think it is up to par for eLife but I could have missed something.

From the consultation session:

In reading over your reviews we appear to see similar strengths but share some of the same concerns. I think a current limitation is in clearly placing Sec23-Sec24 activity in autophagosome biogenesis. Phosphorylation of Sec24 appears to play a regulatory role but this could be in somehow re-configuring ERES or in recruiting factors or directing COPII vesicles to a specific target. To devise assays/methods to address this issue is likely to take more than a couple of months which is why my enthusiasm was moderate.

Reviewer #3

The membrane supplying autophagosome biogenesis has been difficult to clarify. Recent evidence in yeast and mammals indicate the involvement of COPII vesicles as a contribution to the autophagic membrane. However, how COPII vesicles are diverted from ER-Golgi trafficking to support autophagosome biogenesis was not clear. Some previous evidence shed light on the possible mechanism. For example, the Ge L et al. indicated a spatial regulation by generating the COPII from the ERGIC to separate from the COPII for trafficking which is from the ERES (Ge L et al. eLife 2014) in mammalian cells. Yeast cells lack a special ERGIC. Nonetheless, COPII is still essential for autophagosome biogenesis as shown by the last author's work as well as the Ohsumi group. Since there is no obvious solution to spatially separate the autophagic COPII from the ER-Golgi trafficking COPII in yeast, different mechanisms should be involved. Based on the authors' previous work, they hypothesized that phosphorylation of COPII components should be a reasonable mechanism accounting for the diversion of COPII to autophagy. In the current study, the authors analyzed the phosphorylation of Sec24 because Graef et al. found an association between Sec24 and Atg9 in a previous large scale proteomic analysis (Graef et al. MBoC 2013). They identified the phosphorylation sites of Sec24 and created different loss or gain of function mutations. They found that three membrane distal phosphorylation sites are required for the number of autophagosomes formed during starvation possibly due to their association with Atg9. The Hrr25 is the kinase required for Sec24 phosphorylation and the proposed process shown in the final model. In general, the story is a nice extension of their previous studies. The new advance is to employ the phosphorylation to explain the selection of COPII function. More experiments are necessary to validate the process.

1) It is not clear at which stage does the Sec24 phosphorylated COPII act. Although the authors showed no effect on Atg9 on PAS, other marker should also be tested such as Atg14, Atg1, Atg17, Atg2, Atg18 and Atg5. Also, it is not shown if Sec24 mutations, such as the 3A or 3E that affects autophagy, also influence the localization of Sec24 on the PAS.

2) The binding between Sec24 and Atg9 is weak. And the consequence of the interaction is not clear without more biochemical data and interaction site information. The author should find the cytosolic domain of Atg9 that associates with Sec24 and validate with in vitro pull down. Based on this notion, single mutation of this Atg9 domain should also achieve a similar effect of abolishment of Sec24 phosphorylation. Or, the author could express a peptide either from Atg9 or Sec24 to compete off the interaction to further confirm.

3) Although the model of COPII-Atg9 vesicle fusion looks reasonable and has appeared many times in many of their previous papers, it is not supported by any direct evidence. in vitro fusion should be performed or the fusion should be analyzed in vivo by live cell imaging. The Sec24 mutants should be employed as negative controls.

From the consultation session:

The work is preliminary although has some interesting points. Considering their previous work about the negative effect of SEC23 phosphorylation on autophagy (Wang J et al. JCB 2005) while here it is positive effect of SEC24 phosphorylation on autophagy, the role of COPII phosphorylation and how Hrr25 regulates the specific phosphorylation of SEC23 and SEC24 on different sites and different conditions is really complicated. Although it may take more than 2 months to address these concerns, I believe the authors should be able to address the major concerns of the reviewers to improve the current work. In addition, the mechanism of how Hrr25 selects SEC23 and SEC24 as well as specific phosphorylation sites on SEC24 that specifically affect autophagy pathway and ATG9 association is very important and will increase the impact of the study.

[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]

Thank you for submitting your article "Sec24 phosphorylation regulates autophagosome abundance during nutrient deprivation" for consideration by eLife. Your article has been favorably evaluated by Randy Schekman (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. The reviewers have opted to remain anonymous.

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:

This group previously reported that Hrr25 is required for autophagy, but what this kinase phosphorylates and how it contributes to autophagosome formation have been unknown. In this study, the authors report that phosphorylation of T324, T325, and T328 of the COPII subunit Sec24 is required for autophagy. Mechanistically, phosphorylation of these residues promotes the interaction between the C-terminus of Atg9 and Sec24, which is important for the increase in the number rather than the size of autophagosomes. Based on these results, the authors propose that COPII vesicles are reprogrammed to be used for autophagosome formation by Hrr25-mediated Sec24 phosphorylation in response to nitrogen starvation.

This is a resubmitted version and the manuscript has been significantly improved. In particular, the experiments in Figure 5A showing that the interaction of phosphomimic Sec24 (T324E/T328E) with Atg9 is stronger than that of non-phosphorylated Sec24 and those in Figure 6 using T325E/T328E in hrr25-5 mutant cells have strengthened the authors' conclusion that Hrr25-mediated phosphorylation of Sec24 is important for binding with Atg9 and autophagy. However, there are several points that need to be addressed, some of which were raised by a new reviewer.

Essential revisions:

1) Figure 3A. It is hard to tell that the GFP-Atg8 structures indeed represent autophagosomes rather than precursor structures (phagophores) and the PAS. The size of phagophores and the PAS are smaller than that of closed autophagosomes. It would be ideal to measure the size of GFP-Atg8 structures in ypt7Δ cells, in which closed autophagosomes should accumulate.

2) Related to the above criticism, it is also difficult to distinguish small autophagosomes from the PAS under nutrient-rich conditions. The presence of small GFP-Atg8 puncta does not necessarily mean that Cvt vesicles are normally formed because they may merely represent the PAS. Thus, the authors' proposal that COPII vesicles contribute to autophagosome formation under starvation but not nutrient-rich conditions (Cvt vesicle formation) should be carefully reevaluated. In fact, it was reported that COPII mutants are defective in the Cvt pathway (Reggiori et al. Mol Biol Cell 15, 2189, 2004). The Cvt pathway should be directly examined by seeing the maturation of Ape1 in the 3A mutant.

3) It is difficult to conclude that COPII vesicles localize to the PAS by simply observing Sec13 puncta, most of which likely represent ER exit sites. It would be difficult to distinguish the ERESs and COPII vesicles by fluorescence microscopy. Other groups concluded that the PAS forms in the vicinity of the ER exit site based on results of similar experiments.

4) The title and/or Abstract should provide a clear indication of the biological system under investigation (i.e., species name or broader taxonomic group, if appropriate). Please revise your title and/or abstract with this advice in mind.

https://doi.org/10.7554/eLife.21167.029

Author response

[Editors’ note: the author responses to the first round of peer review follow.]

[…]

Reviewer #1

The first part that describe the identification of Sec24 phosphorylation sites (T324/T325/T328) specifically important for autophagy is clear. However, the second part proposing that Hrr25-mediated phosphorylation of Sec24 reprograms COPII vesicles to function in autophagy is too preliminary. As the interaction of COPII subunits and Atg9 is not novel, the data presented here represent a limited advance and rather preliminary to support the model shown in Figure 6 (Figure 1 is not immediately relevant to this study). It is also unclear how the Sec24-Atg9 interaction regulates the number, rather than the size, of the autophagosome.

We agree that Figure 1 is not entirely central to the final conclusions of our study and have moved these data to the supplement (see Figure 1—figure supplement 1). Speaking to the novelty of the Atg9/Sec24 interaction, we note that although an earlier proteomics study linked Atg9 to multiple COPII coat subunits, it was not shown which COPII coat subunit drives this interaction. Furthermore, and most importantly, the significance of this interaction was not explored in the earlier study. Specifically, it was not demonstrated that the COPII-Atg9 interaction is required for autophagy. We now provide additional data that supports a role for Hrr25 in modulating autophagy via the Sec24 phosphosites (i.e. the suppression of Hrr25 autophagy defects by the Sec24 phosphomimetic mutations). Thus, our data shows the Atg9-COPII interaction is mediated by Sec24, regulated by the CK1 kinase Hrr25, and used as a mechanism to commit a COPII vesicle to the macroautophagy pathway. This is a novel discovery that contributes to our understanding of how membrane traffic is regulated during autophagy.

1) It is unclear how Sec24 phosphorylation regulates the COPII-Atg9 vesicle interaction as shown in the model in Figure 6. To examine the requirement of the Sec24-Atg9 binding in autophagosome biogenesis, it is important to identify the binding sites in both Sec24 and Atg9 and generate binding-deficient mutants.

We have now performed in vitro binding experiments with His-tagged Sec24 and Sec23 using fragments of Atg9 fused to GST. Monomeric Sec24 is unstable in the absence of Sec23, so we used purified Sec23/Sec24 complex to demonstrate a direct interaction between Atg9 and Sec23/Sec24. Additionally, using bacterially-expressed monomeric Sec23, to rule out an interaction between Atg9 and Sec23, allows us to conclude that the Atg9-COPII coat interaction requires Sec24. We note that the crystal structure of Sec23 and Sec24 are identical whether they are in complex with each other or not, making allosteric effects entirely unlikely. We further defined the site of interaction with Atg9 by domain dissection. Atg9 is a multi-spanning transmembrane protein that contains cytoplasmic N-(Atg9N; aa 2-318) and C- (Atg9C; aa 747- 997) termini. The middle of the protein is membrane-bound with a small domain (Atg9M; aa 395-534) that faces the cytosol. We fused the three cytoplasmic domains of Atg9 to GST, purified them from bacteria and performed in vitro binding experiments with purified Sec23/Sec24 complex. We found that the Sec23/Sec24 complex bound in vitro to GST-Atg9C. Importantly, binding is specific, appears to be saturable and dependent on the presence of Sec24 in the complex (Figure 4C-F). We also showed Sec24 phosphorylation enhances the interaction of the Sec23/Sec24 complex with Atg9 (Figure 5A). Therefore, our data implies the sec24 triple alanine and double alanine mutants are binding deficient mutants (Figure 5B, 5C).

We also made truncations of the N and C terminal domains of Atg9, as well as portions of the core middle domain, to examine the interaction of Sec24 with Atg9 in vivo. Unfortunately, truncated Atg9 rapidly degraded during the cell lysis procedure used for immunoprecipitation. We encountered similar problems when we expressed fragments of Sec24 in yeast. Nonetheless, the sum total of our findings show that phosphorylation of the conserved membrane distal α helix of Sec24 increases the binding affinity of the Sec23/Sec24 complex with the C-terminus of Atg9 in vitro and in vivo.

2) The association of the COPII vesicles and Atg9 vesicles should be extensively investigated using biochemical methods and dependency on the Sec24 phosphorylation and the Sec24-Atg9 binding should also be tested.

We agree that understanding the association of Atg9 vesicles with COPII vesicles is an important question. We have now extensively studied the interaction of Sec24 with domains of Atg9 (described in Point 1 above, also see new data in Figures 4, 5A and the corresponding supplemental data). We initially attempted to form Atg9 vesicles in vitro and monitor their fusion with COPII vesicles. However, the yield of Atg9 vesicles formed in vitro was very low, and the Atg8 lipidation reaction is not very efficient in yeast, leading us to abandon this approach. More recently, in collaboration with Thomas Wollert’s lab, we have been examining the interaction of COPII-coated liposomes with liposomes containing Atg9. Consistent with our in vitro binding data (Figure 4D), preliminary experiments using the Atg9 core incorporated into liposomes (aa 281-779 in Figure 4C) showed no specific interaction with coated vesicles containing Sec24. These experiments were complicated by the lipid composition used in the Atg9 vesicle preparation, and the affinity of each component for lipids. We are thus continuing to collaborate with Thomas’s lab to better dissect the membrane-related effects of Sec24 and Atg9, but this avenue goes beyond the scope of this manuscript.

3) Figure 3D: Showing autophagy-dependent cleavage of GFP-Atg8 by immunoblotting will complement the results.

We now include a gel to show the cleavage of GFP-Atg8 in the sec24 triple alanine mutant (Figure 2E).

4) It is not clear whether Hrr25 contributes to autophagy indeed through Sec24 phosphorylation. This can be tested by expression of the phosphomimetic Sec24 SE mutant in hrr25-5 mutant cells. The binding between Sec24 SE and Atg9 should be tested in hrr25 mutant cells (Figure 5F).

This was an excellent suggestion. We now show that Sec24 T325E/T328E, but not Sec24 T325A/T328A, partially restores the interaction of Sec24 with Atg9 in the hrr25-5 mutant (see Figure 6A). Furthermore, since demonstrating that Hrr25 contributes to autophagy through Sec24 phosphorylation is a key point in the manuscript, we have also added additional data that strengthen this point. We now show that Sec24 T325E/T328E, but not Sec24 T325A/T328A, largely restores the autophagy defect in the hrr25-5 mutant (see Figure 6B, C). We demonstrate this in two ways: by analyzing the translocation of GFP-Atg8 to the vacuole (Figure 6B), and by examining the cleavage of GFP-Atg8 in the vacuole (Figure 6C and Figure 6—figure supplement 1).

Additionally, we measured the accumulation of autophagic bodies in the hrr25-5 mutant by transmission electron microscopy (Figure 5—figure supplement 5). This analysis revealed that like the sec24 triple alanine mutant, the hrr25-5 mutant accumulates less autophagic bodies than wild- type, indicating that there is a reduction in the number of autophagosomes that form in the absence of Hrr25 kinase activity. Furthermore, as we found with the sec24 triple alanine mutant, the loss of Hrr25 activity does not affect autophagosome size.

Reviewer #2

My opinion is mixed. The study provides a nice assessment of phosphosites in Sec24 and mutation of the distal threonine residues (324, 328, 328) clearly influence autophagosome abundance. However, I felt the evidence that starvation activates Hrr25 to directly phosphorylate these residues and that phosphorylated Sec24 then reprograms COPII vesicles for targeting to Atg9 containing membranes was a bit preliminary or overstated. At this stage I don't think it is up to par for eLife but I could have missed something.

From the consultation session:

In reading over your reviews we appear to see similar strengths but share some of the same concerns. I think a current limitation is in clearly placing Sec23-Sec24 activity in autophagosome biogenesis. Phosphorylation of Sec24 appears to play a regulatory role but this could be in somehow re-configuring ERES or in recruiting factors or directing COPII vesicles to a specific target. To devise assays/methods to address this issue is likely to take more than a couple of months which is why my enthusiasm was moderate.

We agree that placing Sec24 phosphorylation at a specific point in the autophagy cascade is difficult. Therefore, we have used several approaches to address this question. We included new data that suggests Sec24 phosphorylation does not re-configure ERES (Figure 5—figure supplement 3). We also show Sec24 phosphorylation and Hrr25 do not indirectly disrupt autophagy by blocking the assembly of the Atg machinery at the PAS (Figure 5—figure supplement 2; Figure 6—figure supplement 3). Additionally, we now demonstrate that Sec24 phosphorylation directly regulates the interaction of the Sec23/Sec24 complex with Atg9 (Figure 5A) and this interaction is needed for autophagy (Figure 6). Our results indicate Sec24 interacts with Atg9 after Atg9 traffics to the PAS (Figure 5—figure supplement 2). Absent from a complete in vitro reconstitution, we believe our efforts to place these regulatory events in context are extensive, and are consistent with the model presented in Figure 7.

Reviewer #3

The membrane supplying autophagosome biogenesis has been difficult to clarify. Recent evidence in yeast and mammals indicate the involvement of COPII vesicles as a contribution to the autophagic membrane. However, how COPII vesicles are diverted from ER-Golgi trafficking to support autophagosome biogenesis was not clear. Some previous evidence shed light on the possible mechanism. For example, the Ge L et al. indicated a spatial regulation by generating the COPII from the ERGIC to separate from the COPII for trafficking which is from the ERES (Ge L et al. eLife 2014) in mammalian cells. Yeast cells lack a special ERGIC. Nonetheless, COPII is still essential for autophagosome biogenesis as shown by the last author's work as well as the Ohsumi group. Since there is no obvious solution to spatially separate the autophagic COPII from the ER-Golgi trafficking COPII in yeast, different mechanisms should be involved. Based on the authors' previous work, they hypothesized that phosphorylation of COPII components should be a reasonable mechanism accounting for the diversion of COPII to autophagy. In the current study, the authors analyzed the phosphorylation of Sec24 because Graef et al. found an association between Sec24 and Atg9 in a previous large scale proteomic analysis (Graef et al. MBoC 2013). They identified the phosphorylation sites of Sec24 and created different loss or gain of function mutations. They found that three membrane distal phosphorylation sites are required for the number of autophagosomes formed during starvation possibly due to their association with Atg9. The Hrr25 is the kinase required for Sec24 phosphorylation and the proposed process shown in the final model. In general, the story is a nice extension of their previous studies. The new advance is to employ the phosphorylation to explain the selection of COPII function. More experiments are necessary to validate the process.

We would like to clarify that our motivation for studying Sec24 phosphorylation predates the proteomics study that linked multiple COPII coat subunits to Atg9. Since we first reported the COPII coat has targeting functions (Cai et al., 2007 Nature vol445: 941-944), we have been trying to understand the targeting function of the coat. Our interest in understanding the role of coat phosphorylation began when we reported that Sec23 and Sec24 are phosphorylated by Hrr25 (Lord et al., 2011 Nature vol 473:181-186). We began analyzing the role of Sec24 and Sec23 phosphosites in autophagy when we found Hrr25 acts in macroautophagy (Wang et al. 2015 J Cell Biol vol 210:273-285).

Furthermore, we believe the mechanism we describe here is likely to be conserved from yeast to higher eukaryotes. The phosphosites in Sec24 are conserved and Sec24 binds to the C- terminus of Atg9, which is conserved in vertebrates. It is also well known that the functions of Hrr25 are conserved from yeast to man. Our studies differ from the Ge et al. work in that they describe a molecular mechanism by which COPII vesicles recognize the Atg machinery. Even if the COPII vesicles used in autophagosome formation are spatially separated from the vesicles that traffic between the ER and Golgi in mammals, the vesicles used in autophagy have to recognize the Atg machinery. We have added a new section in the Discussion to address how our work relates to Ge et al. 2014.

1) It is not clear at which stage does the Sec24 phosphorylated COPII act. Although the authors showed no effect on Atg9 on PAS, other marker should also be tested such as Atg14, Atg1, Atg17, Atg2, Atg18 and Atg5. Also, it is not shown if Sec24 mutations, such as the 3A or 3E that affects autophagy, also influence the localization of Sec24 on the PAS.

As described above in the response to reviewer #2, our data now show that Sec24 (see new data in Figure 5—figure supplement 2) and Hrr25 (see new data in Figure 6—figure supplement 3) are not required for the assembly of the Atg machinery. To show this, we tested a member of each of the groups in the Atg hierarchy (see asterisk in each group in Figure 5—figure supplement 2B) and demonstrated they are all recruited normally to the PAS in the sec24 triple alanine and hrr25-5 mutants. We also found that the recruitment of Atg14 and Atg2 are normal in the sec12-4 mutant at its restrictive temperature of 37oC (Figure 6—figure supplement 4). Therefore, our data with the sec12 mutant are consistent with the data we obtained with the sec24 triple alanine and hrr25-5 mutants.

With regard to the second part of this concern (i.e. Also, it is not shown if Sec24 mutations, such as the 3A or 3E that affects autophagy, also influence the localization of Sec24 on the PAS), as we previously reported for hrr25-5, we did not see an effect on the localization of COPII vesicles at the PAS with Sec24-3A. But to properly interpret this experiment, we need to do an epistasis experiment in the sec24Δiss1Δ strain background with an atg mutant that disrupts localization (see Figure Supplement 5 in Wang et al. 2015 J Cell Biol vol 210:273-285). Despite our continued efforts, we have been unable to make this mutant. However, we now show that ERES are not disrupted in the sec24 triple alanine mutant (Figure 5—figure supplement 3). The ERES were marked by Sec13, not Sec24, for this experiment as it simplified the experiment (i.e. simpler than adding the triple alanine mutations on Sec24-3X-GFP and integrating tagged Sec24 into the genome). ERES in the triple sec24 phosphomimetic mutant could not be examined as this strain is inviable.

2) The binding between Sec24 and Atg9 is weak. And the consequence of the interaction is not clear without more biochemical data and interaction site information. The author should find the cytosolic domain of Atg9 that associates with Sec24 and validate with in vitro pull down. Based on this notion, single mutation of this Atg9 domain should also achieve a similar effect of abolishment of Sec24 phosphorylation. Or, the author could express a peptide either from Atg9 or Sec24 to compete off the interaction to further confirm.

As described in detail above to reviewer #1 (see comment #1), we now map the domain in Atg9 that interacts with Sec24.

3) Although the model of COPII-Atg9 vesicle fusion looks reasonable and has appeared many times in many of their previous papers, it is not supported by any direct evidence. in vitro fusion should be performed or the fusion should be analyzed in vivo by live cell imaging. The Sec24 mutants should be employed as negative controls.

We agree that examination of the interaction between COPII vesicles and Atg9 vesicles is a next key question. However, as described above (see comment #2 to Reviewer #1), this is a complex problem to dissect and remains beyond the scope of the current study. Please note that we provide support for the proposal that COPII vesicles fuse with Atg9 vesicles in Figure 7—figure supplement 1 and paragraph #1 in the Discussion. While this experiment does not provide direct proof for our model, it does support our model as only some mutants that disrupt autophagy lead to the accumulation of COPII vesicles at the PAS when autophagy is blocked (also see the last section of the Results entitled “Hrr25 regulates autophagy via Sec24”). Live cell imaging experiments are unlikely to be productive in yeast given their small size and the difficulty in tracking puncta that change composition over time. However, we are currently doing experiments with mammalian cells to support our model and these experiments may be feasible in the future, but are currently beyond the scope of this manuscript.

From the consultation session:

The work is preliminary although has some interesting points. Considering their previous work about the negative effect of SEC23 phosphorylation on autophagy (Wang J et al. JCB 2005) while here it is positive effect of SEC24 phosphorylation on autophagy, the role of COPII phosphorylation and how Hrr25 regulates the specific phosphorylation of SEC23 and SEC24 on different sites and different conditions is really complicated. Although it may take more than 2 months to address these concerns, I believe the authors should be able to address the major concerns of the reviewers to improve the current work. In addition, the mechanism of how Hrr25 selects SEC23 and SEC24 as well as specific phosphorylation sites on SEC24 that specifically affect autophagy pathway and ATG9 association is very important and will increase the impact of the study.

We agree that understanding how Hrr25 specifically interacts with Sec23 and Sec24 is a key question. We now use complementation experiments to provide additional evidence that Hrr25 acts in autophagy via phosphorylation of the Sec24 membrane distal regulatory surface (Figure 6). These results clarify our earlier finding that Hrr25 is a positive regulator of autophagy (Wang et al. 2015 J Cell Biol vol 210:273-28).

We previously reported that phosphomimetic (but not alanine) mutations at two sites (S742 and T747) in Sec23 disrupt ER-Golgi traffic and autophagy (Lord et al., 2011 Nature vol 473:181-186; Wang et al. 2015 J Cell Biol vol 210:273-28). These phosphomimetic mutations were shown to interfere with the binding of TRAPP (Ypt1/Rab1 GEF) to Sec23 (Lord et al., 2011 Nature vol 473:181-186). Technically, these previous data showed that dephosphorylation (not phosphorylation) of Sec23 is required for both autophagy and ER-Golgi traffic.

[Editors' note: the author responses to the re-review follow.]

[…]

Essential revisions:

1) Figure 3A. It is hard to tell that the GFP-Atg8 structures indeed represent autophagosomes rather than precursor structures (phagophores) and the PAS. The size of phagophores and the PAS are smaller than that of closed autophagosomes. It would be ideal to measure the size of GFP-Atg8 structures in ypt7Δ cells, in which closed autophagosomes should accumulate.

In response to this point, I wrote to the editorial office stating “we believe we already addressed concern #1 by the EM analysis we performed (Figure 3E, F, G).” We received a positive response (below) that we have addressed by changes to the figures and text. We hope this clarifies our conclusions.

Response from editors:

"We understand that the EM data convincingly show the decrease in the number not size of autophagic bodies in sec24 triple alanine mutant cells. However, the fluorescence data do not support this conclusion as explained in the previous comments. If the authors wish to retain the fluorescence data, it is important to state that the GFP-Atg8 structures represent either the PAS, phagophore, or autophagosome, and it is difficult to conclude that the size of autophagosome is not affected. For example, the Y-axis of Figure 3A-D cannot be "autophagosome number/diameter". These should be "GFP-Atg8 puncta number/diameter (/cell)".

We believe the SIM data contributes to our study, so we have retained the data, but we have changed the Y-axis in Figure 3A-D and Figure 3—figure supplement 1 to "GFP-Atg8 puncta number/diameter (/cell)". The text and legends have been changed accordingly. We now make it clear that the GFP-Atg8 puncta could represent either the PAS, phagophores or autophagosomes.

2) Related to the above criticism, it is also difficult to distinguish small autophagosomes from the PAS under nutrient-rich conditions. The presence of small GFP-Atg8 puncta does not necessarily mean that Cvt vesicles are normally formed because they may merely represent the PAS. Thus, the authors' proposal that COPII vesicles contribute to autophagosome formation under starvation but not nutrient-rich conditions (Cvt vesicle formation) should be carefully reevaluated. In fact, it was reported that COPII mutants are defective in the Cvt pathway (Reggiori et al. Mol Biol Cell 15, 2189, 2004). The Cvt pathway should be directly examined by seeing the maturation of Ape1 in the 3A mutant.

We have directly examined the Cvt pathway, by examining Ape1 maturation, and now show there is no processing defect in the sec24 alanine mutants (see Figure 3—figure supplement 1A). In the isogenic wild-type strain we use, Ape1 is partially processed. Nonetheless, it is clear that the sec24 alanine mutants process Ape1 as well as wild-type. We also note that, while Reggioro et al. 2004 (MBoC 15, 2189) concluded that COPII vesicles are required on the Cvt pathway, Ishihara et al. 2001 (MBoC 12, 3690) disagree. Their EM analysis showed Cvt vesicle formation occurs normally in a sec12 mutant. This is consistent with our observations.

3) It is difficult to conclude that COPII vesicles localize to the PAS by simply observing Sec13 puncta, most of which likely represent ER exit sites. It would be difficult to distinguish the ERESs and COPII vesicles by fluorescence microscopy. Other groups concluded that the PAS forms in the vicinity of the ER exit site based on results of similar experiments.

This is a good point and we should have been more careful with our phrasing. We now refer to Sec13 puncta as COPII coated structures, which includes ERES and COPII vesicles (see first paragraph in the Discussion).

4) The title and/or Abstract should provide a clear indication of the biological system under investigation (i.e., species name or broader taxonomic group, if appropriate). Please revise your title and/or abstract with this advice in mind.

The species is now mentioned in the Abstract.

https://doi.org/10.7554/eLife.21167.030

Article and author information

Author details

  1. Saralin Davis

    Department of Cellular and Molecular Medicine, University of California, San Diego, San Diego, United States
    Contribution
    SD, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9834-8683
  2. Juan Wang

    Department of Cellular and Molecular Medicine, University of California, San Diego, San Diego, United States
    Contribution
    JW, Acquisition of data, Analysis and interpretation of data
    Competing interests
    The authors declare that no competing interests exist.
  3. Ming Zhu

    Department of Cellular and Molecular Medicine, University of California, San Diego, San Diego, United States
    Contribution
    MZ, Acquisition of data, Analysis and interpretation of data
    Competing interests
    The authors declare that no competing interests exist.
  4. Kyle Stahmer

    Department of Biological Sciences, Columbia University, New York, United States
    Contribution
    KS, Acquisition of data, Analysis and interpretation of data
    Competing interests
    The authors declare that no competing interests exist.
  5. Ramya Lakshminarayan

    Department of Biological Sciences, Columbia University, New York, United States
    Contribution
    RL, Acquisition of data, Analysis and interpretation of data
    Competing interests
    The authors declare that no competing interests exist.
  6. Majid Ghassemian

    1. Department of Chemistry and Biochemistry, University of California, San Diego, San Diego, United States
    2. Biomolecular and Proteomics Mass Spectrometry Facility, University of California, San Diego, San Diego, United States
    Contribution
    MG, Acquisition of data, Analysis and interpretation of data
    Competing interests
    The authors declare that no competing interests exist.
  7. Yu Jiang

    Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, United States
    Contribution
    YJ, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  8. Elizabeth A Miller

    1. Department of Biological Sciences, Columbia University, New York, United States
    2. MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
    Contribution
    EAM, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  9. Susan Ferro-Novick

    Department of Cellular and Molecular Medicine, University of California, San Diego, San Diego, United States
    Contribution
    SF-N, Conception and design, Analysis and interpretation of data, Drafting or revising the article
    For correspondence
    sfnovick@ucsd.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8714-7352

Funding

National Institute of General Medical Sciences (GM114111)

  • Saralin Davis
  • Juan Wang
  • Susan Ferro-Novick

National Cancer Institute (CA169186)

  • Yu Jiang

National Institute of General Medical Sciences (GM085089)

  • Kyle Stahmer
  • Ramya Lakshminarayan
  • Elizabeth A Miller

National Institute of General Medical Sciences (GM115422)

  • Saralin Davis
  • Juan Wang
  • Susan Ferro-Novick

Medical Research Council (MC_UP_1201/10)

  • Elizabeth A Miller

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

Acknowledgements

We thank Deepali Bhandari for her participation in the mass spectrometry analysis of Sec24 in the initial stages of the project, Wenyun Zhou for technical assistance in the construction of sec24 mutants, Chris Lord for guidance with the in vitro transport studies, Jodi Nunnari for the pFA6a-3xyEGFP-CaURA3 construct; Claudine Kraft for the plasmid used to express GST-Atg9C, and Seth Field for comments during the preparation of the manuscript. We also thank Ying Jones in the Electron Microscopy facility in the Department of Cellular and Molecular Medicine at UCSD, headed by Dr. Marilyn Farquhar, for EM sample preparation, and Dan Klionsky for protocols on the preparation and analysis of the EM data. We acknowledge the UCSD School of Medicine microscopy core for use of the Applied Precision Delta Vision OMX Super Resolution System. This work was supported by NIGMS and NCI under award numbers R01GM114111 and R01GM115422 to SF-N, R01GM085089 and MC_UP_1201/10 to EAM, and R21CA169186 to Y Jiang.

Reviewing Editor

  1. Noboru Mizushima, The University of Tokyo, Japan

Publication history

  1. Received: September 1, 2016
  2. Accepted: November 14, 2016
  3. Accepted Manuscript published: November 18, 2016 (version 1)
  4. Version of Record published: December 8, 2016 (version 2)

Copyright

© 2016, Davis 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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