Molecular mechanism to target the endosomal Mon1-Ccz1 GEF complex to the pre-autophagosomal structure

  1. Jieqiong Gao
  2. Lars Langemeyer
  3. Daniel Kümmel
  4. Fulvio Reggiori
  5. Christian Ungermann  Is a corresponding author
  1. University of Osnabrück, Germany
  2. University Medical Center Groningen, University of Groningen, Netherlands

Abstract

During autophagy, a newly formed double membrane surrounds its cargo to generate the so-called autophagosome, which then fuses with a lysosome after closure. Previous work implicated that endosomal Rab7/Ypt7 associates to autophagosomes prior to their fusion with lysosomes. Here, we unravel how the Mon1-Ccz1 guanosine exchange factor (GEF) acting upstream of Ypt7 is specifically recruited to the pre-autophagosomal structure under starvation conditions. We find that Mon1-Ccz1 directly binds to Atg8, the yeast homolog of the members of the mammalian LC3 protein family. This requires at least one LIR motif in the Ccz1 C-terminus, which is essential for autophagy but not for endosomal transport. In agreement, only wild-type, but not LIR-mutated Mon1-Ccz1 promotes Atg8-dependent activation of Ypt7. Our data reveal how GEF targeting can specify the fate of a newly formed organelle and provide new insights into the regulation of autophagosome-lysosome fusion.

https://doi.org/10.7554/eLife.31145.001

eLife digest

Autophagy is a word derived from the Greek for “self-eating”. It describes a biological process in which a living cell breaks down its own material to release their chemical building blocks that can then be used to make new molecules. Autophagy is often triggered when a cell becomes damaged or when nutrients are in short supply. The hallmark of autophagy is the formation of structures called autophagosomes. These structures capture the cellular material, fuse with other compartments in the cell – namely endosomes in animals and vacuoles in yeast – and then deliver the material inside, ready to be broken down.

For an autophagosome to fuse to an endosome or a vacuole, small proteins of the Rab protein family must be located on the surface of the autophagosome. Rab proteins are recruited to this surface by enzymes known as GEFs. However it remains unclear how most GEFs get to the surface of a compartment within the cell to begin with.

The Mon1-Ccz1 complex is a GEF that occurs in yeast and animals, including fruit flies and humans. It is found on endosomes, and was recently shown to also localize to autophagosomes. Now, Gao et al. report that, in yeast, the Mon1-Ccz1 complex binds directly to a protein named Atg8. This protein is anchored on to the surface of autophagosomes, and is closely related to other proteins in animal cells.

Gao et al. discovered that this specific GEF binds to Atg8 via at least one binding site on its Ccz1 component. This binding site is only needed for the GEF to localize to the autophagosomes; the Mon1-Czz1 complex can still bind to endosomes without it. Blocking the GEF from binding to Atg8 stopped the autophagosomes from fusing with vacuoles.

These findings reveal how a GEF can be targeted to two distinct compartments in the cell: endosomes and autophagosomes. Further work is now needed to understand how this process is regulated by the availability of nutrients or damage to the cell, to ensure that autophagy is only triggered under the right conditions. Also, because cancer cells often rely on autophagy to survive, the molecules that regulate this process could represent possible targets for new anticancer drugs.

https://doi.org/10.7554/eLife.31145.002

Introduction

Macroautophagy, called here autophagy, is an important quality control pathway, during which cellular material such as organelles and cytosolic components are engulfed by a double-membrane vesicles, the autophagosome (Shibutani and Yoshimori, 2014; Mizushima et al., 2011). In both yeast and mammals, autophagosome formation is a complex process that begins with the assembly of the phagophore or isolation membrane. Once complete, the autophagosome first fuses with endosomes to form an amphisome and then with lysosomes in mammalian cells, while it directly fuse with the lysosome-like vacuole in yeast (Lamb et al., 2013; Chen and Klionsky, 2011).

How autophagosomes become fusion competent with lysosomes is still poorly understood. Like for other fusion events, autophagosome fusion with vacuoles or endosomes requires SNAREs, Rab GTPases (Rabs) and the HOPS tethering complex (Reggiori and Ungermann, 2017; Barr, 2013; Kümmel and Ungermann, 2014). Rabs have a central role in this fusion cascade. They are held soluble in the cytosol by the GDP-dissociation inhibitor (GDI) proteins, which bind GDP-loaded Rabs. Once on membranes, a Rab-specific guanine nucleotide exchange factor (GEF) converts Rabs into their active GTP-form (Barr, 2013). This allows their interaction with effectors such as tethering factors (Kümmel and Ungermann, 2014). The Rab7 GTPase is required for the fusion of endosomes with lysosomes and lysosomal transport (Nordmann et al., 2012). In yeast, the Rab7-homolog Ypt7 binds to the HOPS tethering complex in this process, which in turn supports SNARE assembly and fusion. Rab7 as well as Ypt7 are also required for fusion of autophagosomes with endosomes (Gutierrez et al., 2004; Ganley et al., 2011; McEwan et al., 2015) and detected on autophagosomes (Hegedűs et al., 2016).

The conserved Mon1-Ccz1 GEF complex triggers endosomal maturation by activating Ypt7 (or Rab7 in metazoans) primarily on late endosomes (Nordmann et al., 2010; Gerondopoulos et al., 2012; Singh et al., 2014; Cui et al., 2014), but likely also on autophagosomes (Hegedűs et al., 2016). In agreement with this notion, it has been shown that yeast Mon1-Ccz1 is essential for autophagy progression (Wang et al., 2002). As Mon1-Ccz1 can interact with Rab5-GTP, Rab5 may promote Rab7 recruitment to endosomes, possibly with support by the local generation of phosphatidylinositol-3-phosphate (PI-3-P) (Singh et al., 2014; Hegedűs et al., 2016; Cui et al., 2014). It remains unresolved, however, how Mon1-Ccz1 is specifically targeted to autophagosomes to trigger SNARE-mediated fusion (Figure 1A). The SNAREs involved in this event have been implicated in previous studies (Darsow et al., 1997; Fischer von Mollard and Stevens, 1999; Dilcher et al., 2001; Sato et al., 1998; Reggiori and Ungermann, 2017).

Figure 1 with 2 supplements see all
Mon1-Ccz1 and Ypt7 localize to autophagosomes during starvation.

(A) Working model of autophagosome-vacuole fusion. Red lines indicate the involved SNAREs Vam3, Vam7, Vti1, and Ykt6. Ypt7 is shown bound to the HOPS complex. For details see text. (B–H) Localization of Atg8 relative to Ccz1 and Ypt7 during growth and nitrogen starvation. Cells expressing mCherry-tagged Atg8 or GFP-tagged Ccz1 or Ypt7 were grown in YPD (normal, +N) or in synthetic medium without nitrogen (SD-N, labeled as N-starved) for 2 hr and analyzed by fluorescence microscopy and showed via individual slices. Size bar, 5 µm. (F–I) Percentage of Ccz1 puncta (F) or Ypt7 puncta (I) co-localizing with Atg8 under both conditions. Atg8 dots (n ≥ 50), Ccz1 dots (n ≥ 300) and Ypt7 dots (n ≥ 200) were quantified by Image J. Error bars represent standard deviation (SD). (K) Relocalization of Ccz1 during starvation. Time course of mCherry-tagged Vps21 and Atg8 relative to GFP-tagged Ccz1. Error bars represent SD.

https://doi.org/10.7554/eLife.31145.003
Figure 1—source data 1

Quantification of percentage of Ccz1 puncta or Ypt7 puncta co-localizing with Atg8 during growth and nitrogen starvation for Figure 1F,I.

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Figure 1—source data 2

Quantification of percentage of Ccz1 puncta co-localizing with Atg8 or Vps21 during growth and nitrogen starvation in different time points for Figure 1K.

https://doi.org/10.7554/eLife.31145.007
Figure 1—source data 3

Quantification of ALP activity for nitrogen starvation 2 hr and 4 hr in wild-type and vps21∆ cells.

https://doi.org/10.7554/eLife.31145.008

Atg8 is one of 16 conserved autophagy-related (Atg) proteins, which are essential for autophagosome formation, and it possesses six mammalian homologues (Shpilka et al., 2012). Members of the Atg8/LC3 protein family are conjugated to phosphatidylethanolamine (PE) at the autophagosome membrane, and interact with several Atg proteins via a LC3 interacting region (LIR motif) to control both maturation and fusion (Wild et al., 2014; Nakatogawa et al., 2007; Klionsky and Schulman, 2014; Abreu et al., 2017). Here, we demonstrate that Atg8 recruits the endosomal GEF Mon1-Ccz1 to the pre-autophagosomal structure. Mutants in a LIR motif present in the Ccz1 C-terminal do not impair GEF activity or endosomal function, but block autophagosome fusion with vacuoles. Our data thus reveal how a GEF can mark two different organelles with the same Rab for fusion via distinct mechanisms.

Results

To determine how yeast autophagosomes are specifically decorated with Ypt7, we analyzed the subcellular distribution of both Mon1 and Ccz1 as the GEF complex formed by these two proteins (Nordmann et al., 2010). In particular, we co-localize GFP-tagged Mon1 and Ccz1 with mCherry-tagged Atg8, an autophagosome marker protein (Suzuki et al., 2007), in wild type cells in growing and nitrogen starvation conditions, which induce autophagy. In yeast, autophagosomes form at the pre-autophagosomal assembly site proximal to the ER and vacuole, which is visible as a single dot-like structure by fluorescence microscopy (Klionsky et al., 2016; Graef et al., 2013; Suzuki et al., 2013; Mari and Reggiori, 2010). Ccz1 and Mon1 were found in distinct puncta, likely endosomes (Rana et al., 2015; Nordmann et al., 2010), which were not co-localizing with the Atg8 puncta in nutrient-rich conditions (Figure 1B; Figure 1—figure supplement 1A). After nitrogen starvation, however, Atg8 labeled the vacuole lumen in wild-type cells as expected (Rieter et al., 2013). This made it impossible to localize Ccz1 or Mon1 to autophagosomes under these conditions, because of their rapid fusion with the vacuole upon completion (Geng et al., 2008). We therefore employed different strategies to block fusion of autophagosomes with vacuoles to determine whether Ypt7, Ccz1, and Mon1 transiently co-localize with Atg8. Deletion of the vacuolar Qa-SNARE Vam3, or temperature sensitive mutants of either Vam3 or the HOPS subunit Vps11 block fusion processes with the vacuole (Darsow et al., 1997; Peterson and Emr, 2001). When cells with these mutations were starved, we indeed observed an accumulation of Atg8-positive autophagosomes, and both Ccz1 and Mon1 were markedly co-localizing with them (Figure 1C–E, quantified in F; Figure 1—figure supplement 1B,C). Likewise, a fraction of Ypt7 colocalized with Atg8 in vam3∆ cells only during starvation (Figure 1G–I). In agreement with this, purified autophagosomes contained both Ypt7 and Mon1-Ccz1 on their surface (Gao and Ungermann, in preparation). Furthermore, we analyzed GFP-Ypt7 in cells overexpressing Ape1. Ape1 overexpression results in the formation of a giant Ape1 oligomer, which is too large to be closed by the isolation membrane marked by mCherry-Atg8 (Suzuki et al., 2013). We found that Ypt7 localizes on the cup-shaped isolation membrane concentrated in a dot in wild-type and vam3∆ cells (Figure 1—figure supplement 1). These data support our interpretation that Ypt7 is present on the autophagosomal membrane. To determine whether starvation promotes the redistribution of Ccz1 to autophagosomes relative to endosomes, we monitored Ccz1 co-localization with Atg8 or Vps21, an endosomal marker protein (Cabrera et al., 2013), over time. Indeed, the fraction of Ccz1 in Vps21-positive organelles decreased, while the localization to Atg8-positive puncta increased during the monitored time period (Figure 1K). As recently published (Zhou et al., 2017), we found the vps21∆ mutant displays impaired autophagy as monitored by the processing of initially cytosolic Pho8∆60 in the vacuole lumen (Figure 1—figure supplement 2E). We also noticed that Ccz1 is cytosolic in vps21∆ cells before and after starvation, which did not allow us to detect this protein on autophagosomal structures (Figure 1—figure supplement 2A–D). It is possible that the localization of Mon1-Ccz1 to endosomes is a prerequisite for a later the movement of the GEF complex to autophagosomes during starvation.

These data suggest that the Mon1-Ccz1 complex is specifically recruited to autophagosomes. To monitor the potential contribution of Atg proteins, including Atg8, in targeting Mon1-Ccz1 and Ypt7 to autophagosomes, we selected the precursor Ape1 oligomer (Kim et al., 1997), a specific autophagosomal cargo behaving similar to Atg8 under starvation conditions, for a small colocalization screen. In wild-type cells and in agreement with the data obtained using mCherry-Atg8 (Figure 1B,G; Figure 1—figure supplement 1A), the starvation-induced co-localization with Ape1 was observed for Ypt7 but not for Ccz1 (Figure 2A,B; Figure 2—figure supplements 1 and 2). To clarify the contribution of known Atg proteins in this process, we generated double knock out mutants lacking VAM3 and selected ATG genes and repeated the assay. In vam3atg1∆ cells as in vam3∆ cells Ccz1 and Ypt7 both robustly colocalized with Ape1 upon nutrient deprivation (Figure 2A,B; Figure 2—figure supplements 1 and 2). However, all the mutants blocking Atg8 conjugation to PE such as those lacking the components of the conjugation machinery or Atg8 itself, abolished colocalization of Ccz1 and Ypt7 with Ape1. Interestingly, the deletion of Atg14, a subunit of the PI-3-kinase I complex required for autophagy (Kihara et al., 2001), did not impair colocalization of Ccz1 and Ape1 on autophagosomes (Figure 2A; Figure 2—figure supplement 1), though affected Ypt7 colocalization with Ape1 (Figure 2B; Figure 2—figure supplement 2). Colocalization of Ape1 relative to Atg8 was not affected in the atg14 mutant (Figure 2—figure supplement 2). This suggests that PI-3-P is not a main determinant for Mon1-Ccz1 targeting to autophagosomes, though might support its activity and/or recruitment of Ypt7.

Figure 2 with 3 supplements see all
Atg8 binds to Mon1-Ccz1 via the Ccz1 C-terminal part.

(A–B) Atg8 is required for localization of Ccz1 to autophagosomes. Graphs show percentage of colocalization of Ccz1 puncta (A) or Ypt7 puncta (B) relative to Ape1 puncta in wild-type and the different mutants. Cells were grown and analyzed as in Figure 1. Ape1 dots (n ≥ 50), Ccz1 dots (n ≥ 300), and Ypt7 dots (n ≥ 200) were quantified by Image J. Error bars represent SD. (C–E) Interaction analysis of Atg8 with Mon1-Ccz1. (C) Immunoprecipitation of TAP-tagged Ccz1 from wild-type and atg4∆ strain co-expressing GFP-Atg8. The strain was grown in YPD or in SD-N for 3 hr before preparing cell extracts. GFP-Atg8 was subsequently immunoprecipitated using GFP-trap beads. Finally, immunoprecipitates were analyzed by Western blotting against GFP and CbP-tag. The graph is the quantification of three independent experiments, where the interaction observed in unstarved cells from wild-type is set as 1. Error bars are SD. (D) Interaction of Atg8 with Mon1-Ccz1 or purified Ccz1. TAP-tagged proteins (shown as purified proteins on Coomassie stained gels to left) were incubated with GST, GST-ubiquitin, and GST-Atg8 immobilized on GSH-Sepharose. Eluted proteins were resolved by SDS-PAGE and analyzed by Western blotting against the CbP-tag (top) or by Coomassie staining (bottom). Load, 10%. (E) Interaction of Atg8 mutants with Mon1-Ccz1. Analysis was done as in (D) with the indicated GST-tagged Atg8 truncation mutants. (F) Interaction of Mon1-Ccz1∆C with Atg8. Mon1-Ccz1∆C was purified as wild-type and analyzed for interaction with GST-tagged Atg8 as before. Top, Western blot against the CbP tag. A star indicates the additional decoration of GST-Atg8 by the antibody; bottom, Coomassie staining and quantification of three experiments. (G) Requirements of Mon1 and Ccz1 domains for autophagy. The indicated truncations were analyzed in cells expressing mCherry-tagged Atg8. Vacuoles were stained with CMAC, and cells grown in SD-N medium were then analyzed by fluorescence microscopy as in Figure 1B. Size bar, 5 µm.

https://doi.org/10.7554/eLife.31145.009
Figure 2—source data 1

Quantification of percentage of Ccz1 puncta or Ypt7 puncta co-localizing with Ape1 during growth and nitrogen starvation for Figure 2A,B.

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Figure 2—source data 2

Quantification of the interaction between Atg8 and Mon1-Ccz1 during growth and nitrogen starvation from wild-type and atg4∆ cells for Figure 2C.

https://doi.org/10.7554/eLife.31145.014
Figure 2—source data 3

Quantification of interaction of Mon1-Ccz1∆C with Atg8 for Figure 2F.

https://doi.org/10.7554/eLife.31145.015
Figure 2—source data 4

Quantification of ALP activity for nitrogen starvation 3 hr in wild-type and Atg8 I21R mutant cells.

https://doi.org/10.7554/eLife.31145.016

Taken together these observations indicate that Mon1-Ccz1 recruitment onto autophagosome requires Atg8. To determine whether Mon1-Ccz1 binds to Atg8 in vivo, we immunoprecipitated GFP-tagged Atg8 from wild-type and atg4∆ cells co-expressing Ccz1-TAP. Atg4 is required for processing of Atg8 prior to its lipidation on the preautophagosomal structure (Chen and Klionsky, 2011). In agreement with our previous finding, we observed an interaction of Ccz1 with Atg8 in wild-type cells, which was greatly enhanced when cells were starved prior to lysis. In contrast, no interaction was observed in atg4∆, supporting our notion that Ccz1 binds to lipidated Atg8 on autophagosomal structures in vivo (Figure 2C). We next investigated whether Mon1-Ccz1 could bind to Atg8 directly. We thus incubated purified Mon1-Ccz1 with immobilized GST-Atg8 or ubiquitin, and detected robust binding only to Atg8 (Figure 2D). Atg8 recognizes LIR motifs via its N-terminal helices (Klionsky and Schulman, 2014). We therefore tested if truncation mutants of Atg8 lacking the 8 or 24 N-terminal residues still bind Mon1-Ccz1. Importantly, binding was now lost strongly suggesting that Mon1-Ccz1 specific association to Atg8 is mediated by one or more LIR motifs (Figure 2E). To further test whether this interaction depends on the Ccz1 LIR motif(s), we generated an Atg8 I21R mutant, which blocks the binding pocket for the crucial W0 LIR motif residue (Noda et al., 2008). We observed no binding between Atg8 I21R and Ccz1 (Figure 2E), indicating that Atg8 indeed recognizes a LIR motif in Ccz1. This Atg8 mutant functions in non-selective autophagy (Figure 2—figure supplement 3A–C), yet has some defect in selective autophagy when we followed ApeI processing during starvation (Figure 2—figure supplement 3D). It thus behaves like previously characterized mutants at this site (Noda et al., 2008).

We then asked which part of Mon1-Ccz1 binds to Atg8. Mon1 and Ccz1 interact with each other via their conserved longin domains (Nordmann et al., 2010; Cabrera et al., 2014), which form a common interface that is required for specific Ypt7 activation (Kiontke et al., 2017). However, Mon1 has some additional 150 residues at the N-terminus of its longin domain, and both Mon1 and Ccz1 have C-terminal domains, whose structure and function is so far unresolved. We therefore generated N- and C-terminal truncation mutants of both proteins and monitored localization and autophagy. In starvation conditions, GFP-tagged truncation mutants of Mon1 expressed in the mon1∆ background did not impair vacuole morphology or starvation-induced Atg8 trafficking to the vacuole lumen (Figure 2G). We noted though that GFP-Mon1 localization was more strongly impaired in the N-terminal than the C-terminal truncation. In contrast, deletion of the C-terminal domain of GFP-tagged Ccz1 resulted in fragmented vacuoles, even though Ccz1 was still localized to distinct puncta that did not co-localize with Atg8 (Figure 2G). We thus asked whether Ccz1 alone might be able to directly interact with purified Atg8. Although purified Mon1-Ccz1 as well as Ccz1 alone were able to bind GST-Atg8 (Figure 2D), a mutant complex of Mon1 with Ccz1∆C showed strongly reduced interaction (Figure 2F). Altogether, these observations suggest that the C-terminal part of Ccz1 directs the GEF complex to Atg8-positive autophagosomes.

Identification of putative LIR motifs in Ccz1

To determine the direct binding site in the Ccz1 C-terminal, we compared the C-termini of multiple Ccz1 homologs. As metazoan Ccz1 is shorter than yeast Ccz1, we narrowed our search on the conserved fragment and identified the putative LIR motifs (https://ilir.warwick.ac.uk; Figure 3A). We generated the corresponding mutants by changing the aromatic W0 and the hydrophobic L4 residues into alanines. Among the nine mutants (Figure 3A), two showed impaired GFP-Ccz1 localization to mCherry-Atg8-positive autophagosomes under nitrogen starvation conditions, that is Y236A V239A (named LIR1) and Y445A L448A (LIR2) (Figure 3B,C,E). These two motifs are highly conserved across species (Figure 3B). However, we noticed that trafficking of mCherry-tagged Atg8 to the vacuole was not totally compromised in the single mutants at normal growth temperature (Figure 3C). We therefore combined both mutations and nitrogen starved the cells. This resulted in a complete block of autophagy in the double mutant as shown by defects in Atg8 delivery and processing in the vacuole (Figure 3C and F), but also vacuole morphology (Figures 3C and 4D). Under these conditions, numerous mCherry-Atg8-positive autophagosomes accumulated in the cytoplasm, consistent with a defect in fusion with vacuoles. The LIR1,2 mutant behaves thus as the Ccz1∆C mutant, and is likewise compromised in both autophagy and vacuole biogenesis in general (Figures 2F and 3C,F).

Identification of the LIR motifs in Ccz1 required for function.

(A) Schematic representation of potential LIR motifs of the C-terminal part of Ccz1. Blue and red indicates all LIR motifs analyzed, red the motifs that also impair Ccz1 localization. (B) Alignments of the potential Ccz1 LIR motifs Mm: Mus musculus, Hs: Homo sapiens, Cg: Candida glabrata, Lt: Lachancea thermotolerans, Nd: Naumovozyma dairenensis, Ka: Kazachstania Africana. (C–D) Effect of LIR mutants on localization, autophagy and vacuole morphology. Analysis was done as in Figure 1B–H. CMAC staining was done for 15 min before analysis. Cells were grown either at 30°C or 37°C during growth or starvation. Size bar, 5 µm. (E) Quantification of Atg8 dots per cell from images in (C–D). Error bars represent SD. (F) Analysis of autophagy over time. Cells were grown at 30°C and incubated in starvation medium for the indicated time periods, then harvested, and proteins were analyzed by SDS-PAGE and Western blotting against GFP.

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Figure 3—source data 1

Quantification of Atg8 dots per cell from Ccz1 wild-type and LIR mutants for Figure 3E.

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LIR motifs in Ccz1 are required for Atg8 binding, but not for the endocytic pathway.

(A–C) Interaction of Ccz1 LIR mutants with Atg8. (A) Analysis of purified Mon1-Ccz1 wild-type and mutant complex by SDS-PAGE and Coomassie staining. All of strains were grown at 30°C for purification. (B–C) Mutations in the LIR motifs impair Mon1-Ccz1 interaction with Atg8. Interaction analysis was done as in Figure 2D, and proteins were analyzed by Western blotting (top) and Coomassie staining (bottom). (D) Comparison of vacuole morphology in LIR mutant cells. Cells were grown at 30 or 37°C in starvation medium, and vacuoles were then stained with CMAC. The number of vacuoles per cell was quantified as indicated. Error bars, SD. (E) Effect of LIR mutants on sorting of vacuolar hydrolases. The indicated cells were grown in starvation medium at the indicated temperature for 2 hr. Total cell lysates were generated and proteins were resolved on SDS-PAGE. Western blots were decorated against CPY and Tom40 (as loading control). (F) Endocytosis analysis in LIR mutants. The indicated cells expressing Mup1-GFP were grown in the absence (-Met) of methionine in minimal medium to an OD600 of 1.0 at the 23°C. Where indicated, methionine was added after the temperature shift to 37°C, and cells were analyzed by fluorescence microscopy after 1 hr. Size bar, 5 µm.

https://doi.org/10.7554/eLife.31145.019
Figure 4—source data 1

Quantification of vacuole morphology in LIR mutant cells for Figure 4D.

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We therefore focused on the single mutants. We were wondering why the single LIR mutants were still functional, even though Ccz1 targeting seemed diminished. We considered the possibility that the LIR mutants may be impaired at higher temperature, and thus repeated the starvation assay at 37°C (Figure 3D). Although the wild-type cells were functional in autophagy, both LIR mutants now accumulated Atg8-positive autophagosomes in cells (Figure 3D, quantified in E).

To test if these LIR mutants indeed compromise binding to Atg8, we produced and used the mutants in Atg8 binding assays (see Figure 2D–F). Both LIR1 and the LIR1,2 double mutants could be purified as wild-type Mon1-Ccz1 from yeast, indicating that they were not destabilizing the complex (Figure 4A). However, they showed poor interaction with Atg8 (Figure 4B,C). As we encounter major problems in the purification of the Mon1-Ccz1 complex with LIR2, we did not further pursue it in our in vitro analyses. Nonetheless, these data agree with a model, where one and possibly two Ccz1 LIR motifs are required for the recruitment of Mon1-Ccz1 to Atg8.

The Ccz1 LIR motifs are not required for endosomal trafficking

Our data suggest an important function of the one and possibly two LIR motifs in directing Mon1-Ccz1 to autophagosomes. As vacuole morphology of the LIR1 and LIR2 mutants was only mildly impaired during heat stress (Figures 3D and 4D), we asked if endosomal trafficking was functional in these mutants. The vacuolar hydrolase carboxypeptidase Y (CPY), which is normally sorted from the Golgi via the endosome to the vacuole, is lost from cells in mutants impaired in vacuole biogenesis such as vps39∆ or the temperature sensitive mutant vps11-1 at 37°C (Figure 4E). Likewise, ccz1∆ cells have less intracellular CPY. However, both LIR mutants in Ccz1 were entirely unperturbed also at elevated temperature or when cells were starved. As a second assay, we traced the sorting of the methionine transporter Mup1 from the plasma membrane to the vacuole (Arlt et al., 2015). In both wild-type cells and the LIR mutants, Mup1-GFP was mainly at the plasma membrane in the absence of methionine, but was efficiently sorted to the vacuole lumen when methionine was added after the temperature shift to 37°C (Figure 4F). This sorting remained unaffected at higher temperatures as well. We therefore conclude that the LIR1 and LIR2 mutants selectively disable Mon1-Ccz1 targeting to autophagosomes, whereas endosomal function of Mon1-Ccz1 remains unperturbed under the same conditions.

Atg8 specifies Mon1-Ccz1 function on autophagosomal membranes

Our data imply that Atg8 is indeed a primary determinant to recruit Mon1-Ccz1 to autophagosomes. We used our Mon1-Ccz1 LIR1 mutant to directly test this hypothesis as this was the best behaving complex. From previous in vitro experiments with purified organelles and proteins we have learned that mutations can compromise protein function in vitro much more clearly than in vivo (Bröcker et al., 2012; Ungermann et al., 1999). We therefore took advantage of GEF assay that we developed before to monitor Mon1-Ccz1 activity on membrane-bound Ypt7 (Cabrera et al., 2014). C-terminally His-tagged Ypt7 was preloaded with the MANT-GDP nucleotide, which looses fluorescence when exchanged for non-fluorescent GTP. In the presence of liposomes carrying the His-interacting DOGS-NTA lipid, and the nucleotide exchange reaction is strongly enhanced when Mon1-Ccz1 is also recruited onto the liposome surface (Cabrera et al., 2014). Using this assay, we compared wild-type and LIR1 mutated Mon1-Ccz1 complex (Figure 5A). Both complexes had similar activity for Ypt7 (Figure 5A,D). We then lowered the Mon1-Ccz1 concentration in our assay to test whether Mon1-Ccz1 targeting and function could depend on membrane-bound Atg8. Indeed, membrane-targeted Atg8-His, but not soluble Atg8, stimulated the GTP exchange reaction (Figure 5B,D), presumably due to its ability to recruit the GEF complex to membranes. In contrast, the Mon1-Ccz1 LIR mutant did not respond to the addition of Atg8 (Figure 5C,D). Our data thus show that membrane-bound Atg8 can recruit Mon1-Ccz1 to membranes to promote Ypt7 activation.

Functional reconstitution of Atg8-dependent GEF activity of Mon1-Ccz1.

(A) GEF activity of wild-type and mutant Mon1-Ccz1 complex. GEF activity was monitored by displacement of MANT-GDP from Ypt7 using a microplate reader (see Materials and methods). Assay was carried out with liposomes capable of binding His-tagged Ypt7 (Cabrera et al., 2014). Without GTP, blue line; without GEF, black line; wt refers to different concentrations of Mon1-Ccz1, LIR1 to the Mon1-Ccz1 mutant complex. (B–D) Effect of membrane-bound Atg8 or soluble Atg8 on GEF activity. Analysis was carried out as in (A) with reduced Mon-Ccz1 concentrations and upon addition of His-tagged Atg8 at the indicated concentrations. (E) Quantification of the rate constants of wild-type and mutant Mon1-Ccz1 complex in the presence and absence of Atg8 for Figure 5B–C. Rate constants were calculated based on the initial slope of the GEF curve (n = 3) (Kiontke et al., 2017; Langemeyer et al., 2014). Error bars, SD. (F) Model of Mon1-Ccz1 recruitment to the autophagosome and endosomes. For details see text.

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Figure 5—source data 1

GEF activity of wild-type and mutant Mon1-Ccz1 complex for Figure 5A.

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Figure 5—source data 2

Effect of membrane-bound Atg8 on GEF activity for Figure 5B,C.

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Figure 5—source data 3

Effect of soluble Atg8 on GEF activity for Figure 5D.

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Figure 5—source data 4

Quantification of the rate constants of wild-type and mutant Mon1-Ccz1 complex in the presence and absence of Atg8 for Figure 5E.

https://doi.org/10.7554/eLife.31145.025

Discussion

Previous studies showed that artificial redirection of GEFs can redirect different Rabs to other membranes (Blümer et al., 2013; Gerondopoulos et al., 2012), yet the molecular determinants that target GEFs to their correct membrane are only partially known and rarely experimentally validated. Autophagosomes form de novo and finally fuses with lysosomes (Reggiori and Ungermann, 2017). Like maturing endosomes, autophagosomes need to acquire the machinery to allow their fusion with lysosomes, including the Rab7/Ypt7 GTPase. Here we have uncovered a simple molecular mechanism that specifically targets the GEF Mon1-Ccz1 onto the surface of autophagosomes. The Ccz1 subunit has at least one conserved C-terminal LIR motif, which directly binds to the LC3 homolog Atg8. Once on autophagosomes, Mon1-Ccz1 recruits and activates the Rab7-like Ypt7 from the cytosol, which in turn can bind the HOPS tethering complex to trigger SNARE-mediated fusion. We indeed found recent evidence that Mon1-Ccz1 is sufficient to activate Ypt7, which was provided in a soluble complex with GDI, and thus triggered fusion in a reconstituted assay (Langemeyer et al., 2018). Similarly, the TRAPP GEF complexes could activate their corresponding Rab-GDI complexes on membranes (Thomas and Fromme, 2016). In agreement with this interpretation, only wild type but not LIR-mutated Mon1-Ccz1 strongly promotes Ypt7 activation in the presence of membrane-localized Atg8 (Figure 5E).

Our data imply that lipidated Atg8 is a specific determinant to redirect Mon1-Ccz1 to autophagosomes. In addition to Atg8, PI-3-P may support re-localization to both endosomes and autophagosomes (Hegedűs et al., 2016; Cabrera et al., 2014). Indeed, deletion of Atg14 in Drosophila fat cells appears to affect autophagosome fusion in addition to altering the biogenesis of these vesicles (Hegedűs et al., 2016). The generation of autophagosomal PI-3-P is required for multiple events, including efficient Atg8 lipidation (Shibutani and Yoshimori, 2014). Interestingly, Mon1-Ccz1 localization to Atg8 positive dots was not impaired if synthesis of the autophagosome-specific PI-3-P pool was blocked by atg14 deletion (Figure 2C). We consider it therefore unlikely that PI-3-P synthesis is a primary factor for Mon1-Ccz1 localization to autophagosomes. In contrast, our analysis suggests that PI-3-P may be critical for Mon1-Ccz1 activity, which could explain the defect in Ypt7 localization to autophagosomes of the atg14Δ mutant. Alternatively, PI-3-P might directly support the recruitment of Ypt7, even though we have evidence that Mon1-Ccz1 activity is most critical in this process (Langemeyer et al., 2018). How the reported PI-3-P binding (Lawrence et al., 2014; Cabrera et al., 2014) affects Mon1-Ccz1 function needs to be further dissected. Future studies will also need to explore how Mon1-Ccz1 is timely and spatially recruited to autophagosomes.

Importantly, our study reveals that Mon1-Ccz1 is functional in the endocytic pathway, when its LIR motifs are singularly mutated. This provides further evidence that Mon1-Ccz1 has a dual role and two different targeting mechanisms for two distinct organelles. By identifying the LIR mutants, we established one of the few conditions that might accumulate fully assembled autophagosomes, which are incompetent of fusing with vacuoles, while maintaining endosome-vacuole fusion and thus vacuoles functional.

In mammalian cells, additional proteins such as PLEKHM1 (McEwan et al., 2015) have been identified as factors involved in the fusion between autophagosomes and lysosomes. Interestingly, PLEKHM1 directly binds LC3-like proteins and Rab7, and could thus support HOPS-mediated tethering and fusion of autophagosomes with lysosomes. We believe that GEFs such as Mon1-Ccz1 are the most critical factors to confine Rab localization and thus determine organelle identity. The cooperation with LC3-like proteins could then provide a combinatorial code to target GEFs and additional fusion factors to autophagosomes. Interestingly, Atg8 is not homogenously distributed over the surface of forming autophagosomes (Graef et al., 2013), and could potentially cluster fusion factors to facilitate their cooperation during fusion. How Atg8 recycling and fusion are then coordinated (Abreu et al., 2017), it is yet another exciting riddle to be dissected. At least Mon1-Ccz1 localization to autophagosomes might be dispensable, once Rab7/Ypt7 is recruited and bound to HOPS.

Recent work of us and others revealed that GEFs can recruit Rab GTPases from the GDI complex to membranes (Langemeyer et al., 2018)(Thomas and Fromme, 2016). The identification of Atg8 as a determinant for Mon1-Ccz1 localization to autophagosomes provides the first example of how a GEF can be diverted to a different location. Differential spatiotemporal recruitment of GEFs allows cells to operate distinct pathways, such as autophagy and endosomal maturation, depending on their metabolic needs while employing the same machinery. For endosomal localization, Rab5-GTP has been suggested as a Mon1-Ccz1 interactor based on yeast-two-hybrid interactions (Li et al., 2015; Cui et al., 2014; Singh et al., 2014; Kinchen and Ravichandran, 2010). Future studies will need to dissect if this order of events can be indeed recapitulated in vitro and how further endosomal and autophagosomal factors specify GEF localization.

Materials and methods

Yeast strains and molecular biology

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Strains and plasmids used in this study are listed in Supplementary file 1 and 2, respectively. Deletions and tagging of genes were done by homologous recombination of respective PCR fragments (Janke et al., 2004; Puig et al., 1998). Mon1 and Ccz1 mutants were generated by QuikChange mutagenesis (Stratagene, La Jolla, CA). Mon1 and Ccz1 truncation mutants have been published (Kiontke et al., 2017). Plasmids encoding GST-Atg8 and Atg8-His6 were kindly provided by Ivan Dikic (Goethe University School of Medicine, Frankfurt am Main, Germany), and Sascha Martens (University of Vienna, Austria), respectively.

Tandem affinity purification

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Tandem affinity purification was performed as described (Bröcker et al., 2012; Lürick et al., 2017). Six liters of culture in YPG were grown at 30°C to OD600 of 6, and cells were harvested and lysed in lysis buffer (300 mM NaCl, 50 mM HEPES-NaOH, pH 7.4, 1.5 mM MgCl2, 1 × FY protease inhibitor mix (Serva, Germany), 0.5 mM PMSF and 1 mM DTT). Lysates were centrifuged for 1 hr at 100,000 g, and the cleared supernatant was incubated with IgG Sepharose beads (GE Healthcare, Penzberg, Germany) for 2 hr at 4°C. Beads were collected by centrifugation at 800 g for 2 min, and washed with ice cold 15 ml lysis buffer containing 0.5 mM DTT and 10% glycerol. Bound proteins were eluted by TEV cleavage overnight at 4°C. Purified proteins were analyzed on SDS-PAGE.

E.coli protein expression and purification

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Atg8 was purified from E. coli BL21 (DE3) Rosetta cells. Cells were grown to an OD600 of 0.6 and induced with 0.5 mM IPTG overnight at 16°C. Cells were lysed in lysis buffer (50 mM HEPES/NaOH, pH 7.5, 150 mM NaCl, 1 mM PMSF, 1x protease inhibitor cocktail (1x = 0.1 mg/ml of leupeptin, 1 mM o-phenanthroline, 0.5 mg/ml of pepstatin A, 0.1 mM Pefabloc)). Lysates were centrifuged for 20 min at 30,000 g, and the cleared supernatant was incubated with Glutathione Sepharose (GSH) beads (for GST-tagged proteins) or Ni-NTA beads (for His-tagged proteins) for 1 hr at 4°C on a nutator. Beads were washed with 20 ml cold lysis buffer (GSH-beads) or lysis buffer containing 20 mM imidazole (Ni-NTA beads). Bound proteins were eluted with buffer containing 15 mM reduced glutathione (GSH-beads) or buffer containing 300 mM imidazole (Ni-NTA beads). Buffer was exchanged to 50 mM HEPES/NaOH, pH 7.4, 150 mM NaCl, and 10% glycerol by using a NAP-10 column (GE Healthcare, Penzberg, Germany).

GST pull down binding assays

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To perform GST pull down binding assays, GST or GST-fused Atg8 wild type or Atg8 mutants or ubiquitin were used as bait, and Mon1-Ccz1 was used as a prey. GST or GST-tagged proteins (150 µg) were simultaneously incubated with GSH-beads for 1 hr at 4°C on a rotating wheel. Beads were washed three times with buffer (150 mM NaCl, 50 mM HEPES/NaOH, pH 7.4, 1.5 mM MgCl2, 0.1% NP-40), and the GSH-bound proteins were then incubated with Mon1-Ccz1 (25 µg) for 2 hr at 4°C on a rotating wheel. Beads were again washed three times in buffer. Bound proteins were eluted by boiling in SDS-sample buffer, resolved on SDS gels, and either analyzed by Coomassie Blue staining or immunoblotting with anti-CbP antibodies (Lürick et al., 2017).

Light microscopy and image analysis

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Yeast cells were first cultured in YPD media to log phase, and then switched to synthetic minimal medium lacking nitrogen (SD-N) for the indicated times to induce starvation. For CMAC staining of vacuoles, cells were incubated with 0.1 CMAC for 15 min at 30°C and subsequent washed with medium. Cells were imaged on a Deltavision Elite imaging system based on an inverted microscopy, equipped with 100x NA 1.49 and 60x NA 1.40 objectives, a sCMOS camera (PCO, Kelheim, Germany), an InsightSSI illumination system, and SoftWoRx software (Applied Precision, Issaquah, WA). Stacks of 6 or 8 images with 0.2 µm spacing were taken for constrained-iterative deconvolution (SoftWoRx) and quantification.

GEF assay on multilamellar vesicles (MLVs)

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GEF assays were performed as described (Nordmann et al., 2010; Cabrera et al., 2014). 60 pmoles Atg8-His were incubated with 60 µl multilamellar vesicles (MLVs, 15 mM) of the following composition (palmitoyloleoyl phosphatidylcholine, 84 mol%, palmitoyloleoyl phosphatidylcholine 10 mol%, DOGS-NTA (1,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1-carboxypentyl)iminodiacetic acid)succinyl]), 6 mol%) for 15 min at 25°C. 500 pmoles Ypt7-His were preloaded with MANT-GDP, and incubated with MLVs for 5 min at 25°C before addition of the Mon1–Ccz1 complex. MANT fluorescence was detected in a SpectraMax M3 Multi-Mode Microplate Reader (Molecular Devices, Germany). Samples were excited at 355 nm and fluorescence was detected at 448 nm. After 20–30 min, 0.1 mM GTP was added to trigger the exchange reaction. The decrease of MANT-GDP fluorescence is an indicator of nucleotide exchange.

Giant Ape1 assay

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Yeast cells (carry the plasmid pRS315-CUP1pr-BFP-APE1) were grown overnight in SDC-LEU medium, then diluted to early log phase next morning. 250 µM CuSO4 was added to induce the giant Ape1 oligomer formation for 4 hr, and cultures were then switched to SD-N medium containing 250 µM CuSO4 for 1 hr to induce autophagy.

References

    1. Klionsky DJ
    2. Abdelmohsen K
    3. Abe A
    4. Abedin MJ
    5. Abeliovich H
    6. Acevedo Arozena A
    7. Adachi H
    8. Adams CM
    9. Adams PD
    10. Adeli K
    11. Adhihetty PJ
    12. Adler SG
    13. Agam G
    14. Agarwal R
    15. Aghi MK
    16. Agnello M
    17. Agostinis P
    18. Aguilar PV
    19. Aguirre-Ghiso J
    20. Airoldi EM
    21. Ait-Si-Ali S
    22. Akematsu T
    23. Akporiaye ET
    24. Al-Rubeai M
    25. Albaiceta GM
    26. Albanese C
    27. Albani D
    28. Albert ML
    29. Aldudo J
    30. Algül H
    31. Alirezaei M
    32. Alloza I
    33. Almasan A
    34. Almonte-Beceril M
    35. Alnemri ES
    36. Alonso C
    37. Altan-Bonnet N
    38. Altieri DC
    39. Alvarez S
    40. Alvarez-Erviti L
    41. Alves S
    42. Amadoro G
    43. Amano A
    44. Amantini C
    45. Ambrosio S
    46. Amelio I
    47. Amer AO
    48. Amessou M
    49. Amon A
    50. An Z
    51. Anania FA
    52. Andersen SU
    53. Andley UP
    54. Andreadi CK
    55. Andrieu-Abadie N
    56. Anel A
    57. Ann DK
    58. Anoopkumar-Dukie S
    59. Antonioli M
    60. Aoki H
    61. Apostolova N
    62. Aquila S
    63. Aquilano K
    64. Araki K
    65. Arama E
    66. Aranda A
    67. Araya J
    68. Arcaro A
    69. Arias E
    70. Arimoto H
    71. Ariosa AR
    72. Armstrong JL
    73. Arnould T
    74. Arsov I
    75. Asanuma K
    76. Askanas V
    77. Asselin E
    78. Atarashi R
    79. Atherton SS
    80. Atkin JD
    81. Attardi LD
    82. Auberger P
    83. Auburger G
    84. Aurelian L
    85. Autelli R
    86. Avagliano L
    87. Avantaggiati ML
    88. Avrahami L
    89. Awale S
    90. Azad N
    91. Bachetti T
    92. Backer JM
    93. Bae DH
    94. Bae JS
    95. Bae ON
    96. Bae SH
    97. Baehrecke EH
    98. Baek SH
    99. Baghdiguian S
    100. Bagniewska-Zadworna A
    101. Bai H
    102. Bai J
    103. Bai XY
    104. Bailly Y
    105. Balaji KN
    106. Balduini W
    107. Ballabio A
    108. Balzan R
    109. Banerjee R
    110. Bánhegyi G
    111. Bao H
    112. Barbeau B
    113. Barrachina MD
    114. Barreiro E
    115. Bartel B
    116. Bartolomé A
    117. Bassham DC
    118. Bassi MT
    119. Bast RC
    120. Basu A
    121. Batista MT
    122. Batoko H
    123. Battino M
    124. Bauckman K
    125. Baumgarner BL
    126. Bayer KU
    127. Beale R
    128. Beaulieu JF
    129. Beck GR
    130. Becker C
    131. Beckham JD
    132. Bédard PA
    133. Bednarski PJ
    134. Begley TJ
    135. Behl C
    136. Behrends C
    137. Behrens GM
    138. Behrns KE
    139. Bejarano E
    140. Belaid A
    141. Belleudi F
    142. Bénard G
    143. Berchem G
    144. Bergamaschi D
    145. Bergami M
    146. Berkhout B
    147. Berliocchi L
    148. Bernard A
    149. Bernard M
    150. Bernassola F
    151. Bertolotti A
    152. Bess AS
    153. Besteiro S
    154. Bettuzzi S
    155. Bhalla S
    156. Bhattacharyya S
    157. Bhutia SK
    158. Biagosch C
    159. Bianchi MW
    160. Biard-Piechaczyk M
    161. Billes V
    162. Bincoletto C
    163. Bingol B
    164. Bird SW
    165. Bitoun M
    166. Bjedov I
    167. Blackstone C
    168. Blanc L
    169. Blanco GA
    170. Blomhoff HK
    171. Boada-Romero E
    172. Böckler S
    173. Boes M
    174. Boesze-Battaglia K
    175. Boise LH
    176. Bolino A
    177. Boman A
    178. Bonaldo P
    179. Bordi M
    180. Bosch J
    181. Botana LM
    182. Botti J
    183. Bou G
    184. Bouché M
    185. Bouchecareilh M
    186. Boucher MJ
    187. Boulton ME
    188. Bouret SG
    189. Boya P
    190. Boyer-Guittaut M
    191. Bozhkov PV
    192. Brady N
    193. Braga VM
    194. Brancolini C
    195. Braus GH
    196. Bravo-San Pedro JM
    197. Brennan LA
    198. Bresnick EH
    199. Brest P
    200. Bridges D
    201. Bringer MA
    202. Brini M
    203. Brito GC
    204. Brodin B
    205. Brookes PS
    206. Brown EJ
    207. Brown K
    208. Broxmeyer HE
    209. Bruhat A
    210. Brum PC
    211. Brumell JH
    212. Brunetti-Pierri N
    213. Bryson-Richardson RJ
    214. Buch S
    215. Buchan AM
    216. Budak H
    217. Bulavin DV
    218. Bultman SJ
    219. Bultynck G
    220. Bumbasirevic V
    221. Burelle Y
    222. Burke RE
    223. Burmeister M
    224. Bütikofer P
    225. Caberlotto L
    226. Cadwell K
    227. Cahova M
    228. Cai D
    229. Cai J
    230. Cai Q
    231. Calatayud S
    232. Camougrand N
    233. Campanella M
    234. Campbell GR
    235. Campbell M
    236. Campello S
    237. Candau R
    238. Caniggia I
    239. Cantoni L
    240. Cao L
    241. Caplan AB
    242. Caraglia M
    243. Cardinali C
    244. Cardoso SM
    245. Carew JS
    246. Carleton LA
    247. Carlin CR
    248. Carloni S
    249. Carlsson SR
    250. Carmona-Gutierrez D
    251. Carneiro LA
    252. Carnevali O
    253. Carra S
    254. Carrier A
    255. Carroll B
    256. Casas C
    257. Casas J
    258. Cassinelli G
    259. Castets P
    260. Castro-Obregon S
    261. Cavallini G
    262. Ceccherini I
    263. Cecconi F
    264. Cederbaum AI
    265. Ceña V
    266. Cenci S
    267. Cerella C
    268. Cervia D
    269. Cetrullo S
    270. Chaachouay H
    271. Chae HJ
    272. Chagin AS
    273. Chai CY
    274. Chakrabarti G
    275. Chamilos G
    276. Chan EY
    277. Chan MT
    278. Chandra D
    279. Chandra P
    280. Chang CP
    281. Chang RC
    282. Chang TY
    283. Chatham JC
    284. Chatterjee S
    285. Chauhan S
    286. Che Y
    287. Cheetham ME
    288. Cheluvappa R
    289. Chen CJ
    290. Chen G
    291. Chen GC
    292. Chen G
    293. Chen H
    294. Chen JW
    295. Chen JK
    296. Chen M
    297. Chen M
    298. Chen P
    299. Chen Q
    300. Chen Q
    301. Chen SD
    302. Chen S
    303. Chen SS
    304. Chen W
    305. Chen WJ
    306. Chen WQ
    307. Chen W
    308. Chen X
    309. Chen YH
    310. Chen YG
    311. Chen Y
    312. Chen Y
    313. Chen Y
    314. Chen YJ
    315. Chen YQ
    316. Chen Y
    317. Chen Z
    318. Chen Z
    319. Cheng A
    320. Cheng CH
    321. Cheng H
    322. Cheong H
    323. Cherry S
    324. Chesney J
    325. Cheung CH
    326. Chevet E
    327. Chi HC
    328. Chi SG
    329. Chiacchiera F
    330. Chiang HL
    331. Chiarelli R
    332. Chiariello M
    333. Chieppa M
    334. Chin LS
    335. Chiong M
    336. Chiu GN
    337. Cho DH
    338. Cho SG
    339. Cho WC
    340. Cho YY
    341. Cho YS
    342. Choi AM
    343. Choi EJ
    344. Choi EK
    345. Choi J
    346. Choi ME
    347. Choi SI
    348. Chou TF
    349. Chouaib S
    350. Choubey D
    351. Choubey V
    352. Chow KC
    353. Chowdhury K
    354. Chu CT
    355. Chuang TH
    356. Chun T
    357. Chung H
    358. Chung T
    359. Chung YL
    360. Chwae YJ
    361. Cianfanelli V
    362. Ciarcia R
    363. Ciechomska IA
    364. Ciriolo MR
    365. Cirone M
    366. Claerhout S
    367. Clague MJ
    368. Clària J
    369. Clarke PG
    370. Clarke R
    371. Clementi E
    372. Cleyrat C
    373. Cnop M
    374. Coccia EM
    375. Cocco T
    376. Codogno P
    377. Coers J
    378. Cohen EE
    379. Colecchia D
    380. Coletto L
    381. Coll NS
    382. Colucci-Guyon E
    383. Comincini S
    384. Condello M
    385. Cook KL
    386. Coombs GH
    387. Cooper CD
    388. Cooper JM
    389. Coppens I
    390. Corasaniti MT
    391. Corazzari M
    392. Corbalan R
    393. Corcelle-Termeau E
    394. Cordero MD
    395. Corral-Ramos C
    396. Corti O
    397. Cossarizza A
    398. Costelli P
    399. Costes S
    400. Cotman SL
    401. Coto-Montes A
    402. Cottet S
    403. Couve E
    404. Covey LR
    405. Cowart LA
    406. Cox JS
    407. Coxon FP
    408. Coyne CB
    409. Cragg MS
    410. Craven RJ
    411. Crepaldi T
    412. Crespo JL
    413. Criollo A
    414. Crippa V
    415. Cruz MT
    416. Cuervo AM
    417. Cuezva JM
    418. Cui T
    419. Cutillas PR
    420. Czaja MJ
    421. Czyzyk-Krzeska MF
    422. Dagda RK
    423. Dahmen U
    424. Dai C
    425. Dai W
    426. Dai Y
    427. Dalby KN
    428. Dalla Valle L
    429. Dalmasso G
    430. D'Amelio M
    431. Damme M
    432. Darfeuille-Michaud A
    433. Dargemont C
    434. Darley-Usmar VM
    435. Dasarathy S
    436. Dasgupta B
    437. Dash S
    438. Dass CR
    439. Davey HM
    440. Davids LM
    441. Dávila D
    442. Davis RJ
    443. Dawson TM
    444. Dawson VL
    445. Daza P
    446. de Belleroche J
    447. de Figueiredo P
    448. de Figueiredo RC
    449. de la Fuente J
    450. De Martino L
    451. De Matteis A
    452. De Meyer GR
    453. De Milito A
    454. De Santi M
    455. de Souza W
    456. De Tata V
    457. De Zio D
    458. Debnath J
    459. Dechant R
    460. Decuypere JP
    461. Deegan S
    462. Dehay B
    463. Del Bello B
    464. Del Re DP
    465. Delage-Mourroux R
    466. Delbridge LM
    467. Deldicque L
    468. Delorme-Axford E
    469. Deng Y
    470. Dengjel J
    471. Denizot M
    472. Dent P
    473. Der CJ
    474. Deretic V
    475. Derrien B
    476. Deutsch E
    477. Devarenne TP
    478. Devenish RJ
    479. Di Bartolomeo S
    480. Di Daniele N
    481. Di Domenico F
    482. Di Nardo A
    483. Di Paola S
    484. Di Pietro A
    485. Di Renzo L
    486. DiAntonio A
    487. Díaz-Araya G
    488. Díaz-Laviada I
    489. Diaz-Meco MT
    490. Diaz-Nido J
    491. Dickey CA
    492. Dickson RC
    493. Diederich M
    494. Digard P
    495. Dikic I
    496. Dinesh-Kumar SP
    497. Ding C
    498. Ding WX
    499. Ding Z
    500. Dini L
    501. Distler JH
    502. Diwan A
    503. Djavaheri-Mergny M
    504. Dmytruk K
    505. Dobson RC
    506. Doetsch V
    507. Dokladny K
    508. Dokudovskaya S
    509. Donadelli M
    510. Dong XC
    511. Dong X
    512. Dong Z
    513. Donohue TM
    514. Doran KS
    515. D'Orazi G
    516. Dorn GW
    517. Dosenko V
    518. Dridi S
    519. Drucker L
    520. Du J
    521. Du LL
    522. Du L
    523. du Toit A
    524. Dua P
    525. Duan L
    526. Duann P
    527. Dubey VK
    528. Duchen MR
    529. Duchosal MA
    530. Duez H
    531. Dugail I
    532. Dumit VI
    533. Duncan MC
    534. Dunlop EA
    535. Dunn WA
    536. Dupont N
    537. Dupuis L
    538. Durán RV
    539. Durcan TM
    540. Duvezin-Caubet S
    541. Duvvuri U
    542. Eapen V
    543. Ebrahimi-Fakhari D
    544. Echard A
    545. Eckhart L
    546. Edelstein CL
    547. Edinger AL
    548. Eichinger L
    549. Eisenberg T
    550. Eisenberg-Lerner A
    551. Eissa NT
    552. El-Deiry WS
    553. El-Khoury V
    554. Elazar Z
    555. Eldar-Finkelman H
    556. Elliott CJ
    557. Emanuele E
    558. Emmenegger U
    559. Engedal N
    560. Engelbrecht AM
    561. Engelender S
    562. Enserink JM
    563. Erdmann R
    564. Erenpreisa J
    565. Eri R
    566. Eriksen JL
    567. Erman A
    568. Escalante R
    569. Eskelinen EL
    570. Espert L
    571. Esteban-Martínez L
    572. Evans TJ
    573. Fabri M
    574. Fabrias G
    575. Fabrizi C
    576. Facchiano A
    577. Færgeman NJ
    578. Faggioni A
    579. Fairlie WD
    580. Fan C
    581. Fan D
    582. Fan J
    583. Fang S
    584. Fanto M
    585. Fanzani A
    586. Farkas T
    587. Faure M
    588. Favier FB
    589. Fearnhead H
    590. Federici M
    591. Fei E
    592. Felizardo TC
    593. Feng H
    594. Feng Y
    595. Feng Y
    596. Ferguson TA
    597. Fernández ÁF
    598. Fernandez-Barrena MG
    599. Fernandez-Checa JC
    600. Fernández-López A
    601. Fernandez-Zapico ME
    602. Feron O
    603. Ferraro E
    604. Ferreira-Halder CV
    605. Fesus L
    606. Feuer R
    607. Fiesel FC
    608. Filippi-Chiela EC
    609. Filomeni G
    610. Fimia GM
    611. Fingert JH
    612. Finkbeiner S
    613. Finkel T
    614. Fiorito F
    615. Fisher PB
    616. Flajolet M
    617. Flamigni F
    618. Florey O
    619. Florio S
    620. Floto RA
    621. Folini M
    622. Follo C
    623. Fon EA
    624. Fornai F
    625. Fortunato F
    626. Fraldi A
    627. Franco R
    628. Francois A
    629. François A
    630. Frankel LB
    631. Fraser ID
    632. Frey N
    633. Freyssenet DG
    634. Frezza C
    635. Friedman SL
    636. Frigo DE
    637. Fu D
    638. Fuentes JM
    639. Fueyo J
    640. Fujitani Y
    641. Fujiwara Y
    642. Fujiya M
    643. Fukuda M
    644. Fulda S
    645. Fusco C
    646. Gabryel B
    647. Gaestel M
    648. Gailly P
    649. Gajewska M
    650. Galadari S
    651. Galili G
    652. Galindo I
    653. Galindo MF
    654. Galliciotti G
    655. Galluzzi L
    656. Galluzzi L
    657. Galy V
    658. Gammoh N
    659. Gandy S
    660. Ganesan AK
    661. Ganesan S
    662. Ganley IG
    663. Gannagé M
    664. Gao FB
    665. Gao F
    666. Gao JX
    667. García Nannig L
    668. García Véscovi E
    669. Garcia-Macía M
    670. Garcia-Ruiz C
    671. Garg AD
    672. Garg PK
    673. Gargini R
    674. Gassen NC
    675. Gatica D
    676. Gatti E
    677. Gavard J
    678. Gavathiotis E
    679. Ge L
    680. Ge P
    681. Ge S
    682. Gean PW
    683. Gelmetti V
    684. Genazzani AA
    685. Geng J
    686. Genschik P
    687. Gerner L
    688. Gestwicki JE
    689. Gewirtz DA
    690. Ghavami S
    691. Ghigo E
    692. Ghosh D
    693. Giammarioli AM
    694. Giampieri F
    695. Giampietri C
    696. Giatromanolaki A
    697. Gibbings DJ
    698. Gibellini L
    699. Gibson SB
    700. Ginet V
    701. Giordano A
    702. Giorgini F
    703. Giovannetti E
    704. Girardin SE
    705. Gispert S
    706. Giuliano S
    707. Gladson CL
    708. Glavic A
    709. Gleave M
    710. Godefroy N
    711. Gogal RM
    712. Gokulan K
    713. Goldman GH
    714. Goletti D
    715. Goligorsky MS
    716. Gomes AV
    717. Gomes LC
    718. Gomez H
    719. Gomez-Manzano C
    720. Gómez-Sánchez R
    721. Gonçalves DA
    722. Goncu E
    723. Gong Q
    724. Gongora C
    725. Gonzalez CB
    726. Gonzalez-Alegre P
    727. Gonzalez-Cabo P
    728. González-Polo RA
    729. Goping IS
    730. Gorbea C
    731. Gorbunov NV
    732. Goring DR
    733. Gorman AM
    734. Gorski SM
    735. Goruppi S
    736. Goto-Yamada S
    737. Gotor C
    738. Gottlieb RA
    739. Gozes I
    740. Gozuacik D
    741. Graba Y
    742. Graef M
    743. Granato GE
    744. Grant GD
    745. Grant S
    746. Gravina GL
    747. Green DR
    748. Greenhough A
    749. Greenwood MT
    750. Grimaldi B
    751. Gros F
    752. Grose C
    753. Groulx JF
    754. Gruber F
    755. Grumati P
    756. Grune T
    757. Guan JL
    758. Guan KL
    759. Guerra B
    760. Guillen C
    761. Gulshan K
    762. Gunst J
    763. Guo C
    764. Guo L
    765. Guo M
    766. Guo W
    767. Guo XG
    768. Gust AA
    769. Gustafsson ÅB
    770. Gutierrez E
    771. Gutierrez MG
    772. Gwak HS
    773. Haas A
    774. Haber JE
    775. Hadano S
    776. Hagedorn M
    777. Hahn DR
    778. Halayko AJ
    779. Hamacher-Brady A
    780. Hamada K
    781. Hamai A
    782. Hamann A
    783. Hamasaki M
    784. Hamer I
    785. Hamid Q
    786. Hammond EM
    787. Han F
    788. Han W
    789. Handa JT
    790. Hanover JA
    791. Hansen M
    792. Harada M
    793. Harhaji-Trajkovic L
    794. Harper JW
    795. Harrath AH
    796. Harris AL
    797. Harris J
    798. Hasler U
    799. Hasselblatt P
    800. Hasui K
    801. Hawley RG
    802. Hawley TS
    803. He C
    804. He CY
    805. He F
    806. He G
    807. He RR
    808. He XH
    809. He YW
    810. He YY
    811. Heath JK
    812. Hébert MJ
    813. Heinzen RA
    814. Helgason GV
    815. Hensel M
    816. Henske EP
    817. Her C
    818. Herman PK
    819. Hernández A
    820. Hernandez C
    821. Hernández-Tiedra S
    822. Hetz C
    823. Hiesinger PR
    824. Higaki K
    825. Hilfiker S
    826. Hill BG
    827. Hill JA
    828. Hill WD
    829. Hino K
    830. Hofius D
    831. Hofman P
    832. Höglinger GU
    833. Höhfeld J
    834. Holz MK
    835. Hong Y
    836. Hood DA
    837. Hoozemans JJ
    838. Hoppe T
    839. Hsu C
    840. Hsu CY
    841. Hsu LC
    842. Hu D
    843. Hu G
    844. Hu HM
    845. Hu H
    846. Hu MC
    847. Hu YC
    848. Hu ZW
    849. Hua F
    850. Hua Y
    851. Huang C
    852. Huang HL
    853. Huang KH
    854. Huang KY
    855. Huang S
    856. Huang S
    857. Huang WP
    858. Huang YR
    859. Huang Y
    860. Huang Y
    861. Huber TB
    862. Huebbe P
    863. Huh WK
    864. Hulmi JJ
    865. Hur GM
    866. Hurley JH
    867. Husak Z
    868. Hussain SN
    869. Hussain S
    870. Hwang JJ
    871. Hwang S
    872. Hwang TI
    873. Ichihara A
    874. Imai Y
    875. Imbriano C
    876. Inomata M
    877. Into T
    878. Iovane V
    879. Iovanna JL
    880. Iozzo RV
    881. Ip NY
    882. Irazoqui JE
    883. Iribarren P
    884. Isaka Y
    885. Isakovic AJ
    886. Ischiropoulos H
    887. Isenberg JS
    888. Ishaq M
    889. Ishida H
    890. Ishii I
    891. Ishmael JE
    892. Isidoro C
    893. Isobe K
    894. Isono E
    895. Issazadeh-Navikas S
    896. Itahana K
    897. Itakura E
    898. Ivanov AI
    899. Iyer AK
    900. Izquierdo JM
    901. Izumi Y
    902. Izzo V
    903. Jäättelä M
    904. Jaber N
    905. Jackson DJ
    906. Jackson WT
    907. Jacob TG
    908. Jacques TS
    909. Jagannath C
    910. Jain A
    911. Jana NR
    912. Jang BK
    913. Jani A
    914. Janji B
    915. Jannig PR
    916. Jansson PJ
    917. Jean S
    918. Jendrach M
    919. Jeon JH
    920. Jessen N
    921. Jeung EB
    922. Jia K
    923. Jia L
    924. Jiang H
    925. Jiang H
    926. Jiang L
    927. Jiang T
    928. Jiang X
    929. Jiang X
    930. Jiang X
    931. Jiang Y
    932. Jiang Y
    933. Jiménez A
    934. Jin C
    935. Jin H
    936. Jin L
    937. Jin M
    938. Jin S
    939. Jinwal UK
    940. Jo EK
    941. Johansen T
    942. Johnson DE
    943. Johnson GV
    944. Johnson JD
    945. Jonasch E
    946. Jones C
    947. Joosten LA
    948. Jordan J
    949. Joseph AM
    950. Joseph B
    951. Joubert AM
    952. Ju D
    953. Ju J
    954. Juan HF
    955. Juenemann K
    956. Juhász G
    957. Jung HS
    958. Jung JU
    959. Jung YK
    960. Jungbluth H
    961. Justice MJ
    962. Jutten B
    963. Kaakoush NO
    964. Kaarniranta K
    965. Kaasik A
    966. Kabuta T
    967. Kaeffer B
    968. Kågedal K
    969. Kahana A
    970. Kajimura S
    971. Kakhlon O
    972. Kalia M
    973. Kalvakolanu DV
    974. Kamada Y
    975. Kambas K
    976. Kaminskyy VO
    977. Kampinga HH
    978. Kandouz M
    979. Kang C
    980. Kang R
    981. Kang TC
    982. Kanki T
    983. Kanneganti TD
    984. Kanno H
    985. Kanthasamy AG
    986. Kantorow M
    987. Kaparakis-Liaskos M
    988. Kapuy O
    989. Karantza V
    990. Karim MR
    991. Karmakar P
    992. Kaser A
    993. Kaushik S
    994. Kawula T
    995. Kaynar AM
    996. Ke PY
    997. Ke ZJ
    998. Kehrl JH
    999. Keller KE
    1000. Kemper JK
    1001. Kenworthy AK
    1002. Kepp O
    1003. Kern A
    1004. Kesari S
    1005. Kessel D
    1006. Ketteler R
    1007. Kettelhut IC
    1008. Khambu B
    1009. Khan MM
    1010. Khandelwal VK
    1011. Khare S
    1012. Kiang JG
    1013. Kiger AA
    1014. Kihara A
    1015. Kim AL
    1016. Kim CH
    1017. Kim DR
    1018. Kim DH
    1019. Kim EK
    1020. Kim HY
    1021. Kim HR
    1022. Kim JS
    1023. Kim JH
    1024. Kim JC
    1025. Kim JH
    1026. Kim KW
    1027. Kim MD
    1028. Kim MM
    1029. Kim PK
    1030. Kim SW
    1031. Kim SY
    1032. Kim YS
    1033. Kim Y
    1034. Kimchi A
    1035. Kimmelman AC
    1036. Kimura T
    1037. King JS
    1038. Kirkegaard K
    1039. Kirkin V
    1040. Kirshenbaum LA
    1041. Kishi S
    1042. Kitajima Y
    1043. Kitamoto K
    1044. Kitaoka Y
    1045. Kitazato K
    1046. Kley RA
    1047. Klimecki WT
    1048. Klinkenberg M
    1049. Klucken J
    1050. Knævelsrud H
    1051. Knecht E
    1052. Knuppertz L
    1053. Ko JL
    1054. Kobayashi S
    1055. Koch JC
    1056. Koechlin-Ramonatxo C
    1057. Koenig U
    1058. Koh YH
    1059. Köhler K
    1060. Kohlwein SD
    1061. Koike M
    1062. Komatsu M
    1063. Kominami E
    1064. Kong D
    1065. Kong HJ
    1066. Konstantakou EG
    1067. Kopp BT
    1068. Korcsmaros T
    1069. Korhonen L
    1070. Korolchuk VI
    1071. Koshkina NV
    1072. Kou Y
    1073. Koukourakis MI
    1074. Koumenis C
    1075. Kovács AL
    1076. Kovács T
    1077. Kovacs WJ
    1078. Koya D
    1079. Kraft C
    1080. Krainc D
    1081. Kramer H
    1082. Kravic-Stevovic T
    1083. Krek W
    1084. Kretz-Remy C
    1085. Krick R
    1086. Krishnamurthy M
    1087. Kriston-Vizi J
    1088. Kroemer G
    1089. Kruer MC
    1090. Kruger R
    1091. Ktistakis NT
    1092. Kuchitsu K
    1093. Kuhn C
    1094. Kumar AP
    1095. Kumar A
    1096. Kumar A
    1097. Kumar D
    1098. Kumar D
    1099. Kumar R
    1100. Kumar S
    1101. Kundu M
    1102. Kung HJ
    1103. Kuno A
    1104. Kuo SH
    1105. Kuret J
    1106. Kurz T
    1107. Kwok T
    1108. Kwon TK
    1109. Kwon YT
    1110. Kyrmizi I
    1111. La Spada AR
    1112. Lafont F
    1113. Lahm T
    1114. Lakkaraju A
    1115. Lam T
    1116. Lamark T
    1117. Lancel S
    1118. Landowski TH
    1119. Lane DJ
    1120. Lane JD
    1121. Lanzi C
    1122. Lapaquette P
    1123. Lapierre LR
    1124. Laporte J
    1125. Laukkarinen J
    1126. Laurie GW
    1127. Lavandero S
    1128. Lavie L
    1129. LaVoie MJ
    1130. Law BY
    1131. Law HK
    1132. Law KB
    1133. Layfield R
    1134. Lazo PA
    1135. Le Cam L
    1136. Le Roch KG
    1137. Le Stunff H
    1138. Leardkamolkarn V
    1139. Lecuit M
    1140. Lee BH
    1141. Lee CH
    1142. Lee EF
    1143. Lee GM
    1144. Lee HJ
    1145. Lee H
    1146. Lee JK
    1147. Lee J
    1148. Lee JH
    1149. Lee JH
    1150. Lee M
    1151. Lee MS
    1152. Lee PJ
    1153. Lee SW
    1154. Lee SJ
    1155. Lee SJ
    1156. Lee SY
    1157. Lee SH
    1158. Lee SS
    1159. Lee SJ
    1160. Lee S
    1161. Lee YR
    1162. Lee YJ
    1163. Lee YH
    1164. Leeuwenburgh C
    1165. Lefort S
    1166. Legouis R
    1167. Lei J
    1168. Lei QY
    1169. Leib DA
    1170. Leibowitz G
    1171. Lekli I
    1172. Lemaire SD
    1173. Lemasters JJ
    1174. Lemberg MK
    1175. Lemoine A
    1176. Leng S
    1177. Lenz G
    1178. Lenzi P
    1179. Lerman LO
    1180. Lettieri Barbato D
    1181. Leu JI
    1182. Leung HY
    1183. Levine B
    1184. Lewis PA
    1185. Lezoualc'h F
    1186. Li C
    1187. Li F
    1188. Li FJ
    1189. Li J
    1190. Li K
    1191. Li L
    1192. Li M
    1193. Li M
    1194. Li Q
    1195. Li R
    1196. Li S
    1197. Li W
    1198. Li W
    1199. Li X
    1200. Li Y
    1201. Lian J
    1202. Liang C
    1203. Liang Q
    1204. Liao Y
    1205. Liberal J
    1206. Liberski PP
    1207. Lie P
    1208. Lieberman AP
    1209. Lim HJ
    1210. Lim KL
    1211. Lim K
    1212. Lima RT
    1213. Lin CS
    1214. Lin CF
    1215. Lin F
    1216. Lin F
    1217. Lin FC
    1218. Lin K
    1219. Lin KH
    1220. Lin PH
    1221. Lin T
    1222. Lin WW
    1223. Lin YS
    1224. Lin Y
    1225. Linden R
    1226. Lindholm D
    1227. Lindqvist LM
    1228. Lingor P
    1229. Linkermann A
    1230. Liotta LA
    1231. Lipinski MM
    1232. Lira VA
    1233. Lisanti MP
    1234. Liton PB
    1235. Liu B
    1236. Liu C
    1237. Liu CF
    1238. Liu F
    1239. Liu HJ
    1240. Liu J
    1241. Liu JJ
    1242. Liu JL
    1243. Liu K
    1244. Liu L
    1245. Liu L
    1246. Liu Q
    1247. Liu RY
    1248. Liu S
    1249. Liu S
    1250. Liu W
    1251. Liu XD
    1252. Liu X
    1253. Liu XH
    1254. Liu X
    1255. Liu X
    1256. Liu X
    1257. Liu Y
    1258. Liu Y
    1259. Liu Z
    1260. Liu Z
    1261. Liuzzi JP
    1262. Lizard G
    1263. Ljujic M
    1264. Lodhi IJ
    1265. Logue SE
    1266. Lokeshwar BL
    1267. Long YC
    1268. Lonial S
    1269. Loos B
    1270. López-Otín C
    1271. López-Vicario C
    1272. Lorente M
    1273. Lorenzi PL
    1274. Lõrincz P
    1275. Los M
    1276. Lotze MT
    1277. Lovat PE
    1278. Lu B
    1279. Lu B
    1280. Lu J
    1281. Lu Q
    1282. Lu SM
    1283. Lu S
    1284. Lu Y
    1285. Luciano F
    1286. Luckhart S
    1287. Lucocq JM
    1288. Ludovico P
    1289. Lugea A
    1290. Lukacs NW
    1291. Lum JJ
    1292. Lund AH
    1293. Luo H
    1294. Luo J
    1295. Luo S
    1296. Luparello C
    1297. Lyons T
    1298. Ma J
    1299. Ma Y
    1300. Ma Y
    1301. Ma Z
    1302. Machado J
    1303. Machado-Santelli GM
    1304. Macian F
    1305. MacIntosh GC
    1306. MacKeigan JP
    1307. Macleod KF
    1308. MacMicking JD
    1309. MacMillan-Crow LA
    1310. Madeo F
    1311. Madesh M
    1312. Madrigal-Matute J
    1313. Maeda A
    1314. Maeda T
    1315. Maegawa G
    1316. Maellaro E
    1317. Maes H
    1318. Magariños M
    1319. Maiese K
    1320. Maiti TK
    1321. Maiuri L
    1322. Maiuri MC
    1323. Maki CG
    1324. Malli R
    1325. Malorni W
    1326. Maloyan A
    1327. Mami-Chouaib F
    1328. Man N
    1329. Mancias JD
    1330. Mandelkow EM
    1331. Mandell MA
    1332. Manfredi AA
    1333. Manié SN
    1334. Manzoni C
    1335. Mao K
    1336. Mao Z
    1337. Mao ZW
    1338. Marambaud P
    1339. Marconi AM
    1340. Marelja Z
    1341. Marfe G
    1342. Margeta M
    1343. Margittai E
    1344. Mari M
    1345. Mariani FV
    1346. Marin C
    1347. Marinelli S
    1348. Mariño G
    1349. Markovic I
    1350. Marquez R
    1351. Martelli AM
    1352. Martens S
    1353. Martin KR
    1354. Martin SJ
    1355. Martin S
    1356. Martin-Acebes MA
    1357. Martín-Sanz P
    1358. Martinand-Mari C
    1359. Martinet W
    1360. Martinez J
    1361. Martinez-Lopez N
    1362. Martinez-Outschoorn U
    1363. Martínez-Velázquez M
    1364. Martinez-Vicente M
    1365. Martins WK
    1366. Mashima H
    1367. Mastrianni JA
    1368. Matarese G
    1369. Matarrese P
    1370. Mateo R
    1371. Matoba S
    1372. Matsumoto N
    1373. Matsushita T
    1374. Matsuura A
    1375. Matsuzawa T
    1376. Mattson MP
    1377. Matus S
    1378. Maugeri N
    1379. Mauvezin C
    1380. Mayer A
    1381. Maysinger D
    1382. Mazzolini GD
    1383. McBrayer MK
    1384. McCall K
    1385. McCormick C
    1386. McInerney GM
    1387. McIver SC
    1388. McKenna S
    1389. McMahon JJ
    1390. McNeish IA
    1391. Mechta-Grigoriou F
    1392. Medema JP
    1393. Medina DL
    1394. Megyeri K
    1395. Mehrpour M
    1396. Mehta JL
    1397. Mei Y
    1398. Meier UC
    1399. Meijer AJ
    1400. Meléndez A
    1401. Melino G
    1402. Melino S
    1403. de Melo EJ
    1404. Mena MA
    1405. Meneghini MD
    1406. Menendez JA
    1407. Menezes R
    1408. Meng L
    1409. Meng LH
    1410. Meng S
    1411. Menghini R
    1412. Menko AS
    1413. Menna-Barreto RF
    1414. Menon MB
    1415. Meraz-Ríos MA
    1416. Merla G
    1417. Merlini L
    1418. Merlot AM
    1419. Meryk A
    1420. Meschini S
    1421. Meyer JN
    1422. Mi MT
    1423. Miao CY
    1424. Micale L
    1425. Michaeli S
    1426. Michiels C
    1427. Migliaccio AR
    1428. Mihailidou AS
    1429. Mijaljica D
    1430. Mikoshiba K
    1431. Milan E
    1432. Miller-Fleming L
    1433. Mills GB
    1434. Mills IG
    1435. Minakaki G
    1436. Minassian BA
    1437. Ming XF
    1438. Minibayeva F
    1439. Minina EA
    1440. Mintern JD
    1441. Minucci S
    1442. Miranda-Vizuete A
    1443. Mitchell CH
    1444. Miyamoto S
    1445. Miyazawa K
    1446. Mizushima N
    1447. Mnich K
    1448. Mograbi B
    1449. Mohseni S
    1450. Moita LF
    1451. Molinari M
    1452. Molinari M
    1453. Møller AB
    1454. Mollereau B
    1455. Mollinedo F
    1456. Mongillo M
    1457. Monick MM
    1458. Montagnaro S
    1459. Montell C
    1460. Moore DJ
    1461. Moore MN
    1462. Mora-Rodriguez R
    1463. Moreira PI
    1464. Morel E
    1465. Morelli MB
    1466. Moreno S
    1467. Morgan MJ
    1468. Moris A
    1469. Moriyasu Y
    1470. Morrison JL
    1471. Morrison LA
    1472. Morselli E
    1473. Moscat J
    1474. Moseley PL
    1475. Mostowy S
    1476. Motori E
    1477. Mottet D
    1478. Mottram JC
    1479. Moussa CE
    1480. Mpakou VE
    1481. Mukhtar H
    1482. Mulcahy Levy JM
    1483. Muller S
    1484. Muñoz-Moreno R
    1485. Muñoz-Pinedo C
    1486. Münz C
    1487. Murphy ME
    1488. Murray JT
    1489. Murthy A
    1490. Mysorekar IU
    1491. Nabi IR
    1492. Nabissi M
    1493. Nader GA
    1494. Nagahara Y
    1495. Nagai Y
    1496. Nagata K
    1497. Nagelkerke A
    1498. Nagy P
    1499. Naidu SR
    1500. Nair S
    1501. Nakano H
    1502. Nakatogawa H
    1503. Nanjundan M
    1504. Napolitano G
    1505. Naqvi NI
    1506. Nardacci R
    1507. Narendra DP
    1508. Narita M
    1509. Nascimbeni AC
    1510. Natarajan R
    1511. Navegantes LC
    1512. Nawrocki ST
    1513. Nazarko TY
    1514. Nazarko VY
    1515. Neill T
    1516. Neri LM
    1517. Netea MG
    1518. Netea-Maier RT
    1519. Neves BM
    1520. Ney PA
    1521. Nezis IP
    1522. Nguyen HT
    1523. Nguyen HP
    1524. Nicot AS
    1525. Nilsen H
    1526. Nilsson P
    1527. Nishimura M
    1528. Nishino I
    1529. Niso-Santano M
    1530. Niu H
    1531. Nixon RA
    1532. Njar VC
    1533. Noda T
    1534. Noegel AA
    1535. Nolte EM
    1536. Norberg E
    1537. Norga KK
    1538. Noureini SK
    1539. Notomi S
    1540. Notterpek L
    1541. Nowikovsky K
    1542. Nukina N
    1543. Nürnberger T
    1544. O'Donnell VB
    1545. O'Donovan T
    1546. O'Dwyer PJ
    1547. Oehme I
    1548. Oeste CL
    1549. Ogawa M
    1550. Ogretmen B
    1551. Ogura Y
    1552. Oh YJ
    1553. Ohmuraya M
    1554. Ohshima T
    1555. Ojha R
    1556. Okamoto K
    1557. Okazaki T
    1558. Oliver FJ
    1559. Ollinger K
    1560. Olsson S
    1561. Orban DP
    1562. Ordonez P
    1563. Orhon I
    1564. Orosz L
    1565. O'Rourke EJ
    1566. Orozco H
    1567. Ortega AL
    1568. Ortona E
    1569. Osellame LD
    1570. Oshima J
    1571. Oshima S
    1572. Osiewacz HD
    1573. Otomo T
    1574. Otsu K
    1575. Ou JH
    1576. Outeiro TF
    1577. Ouyang DY
    1578. Ouyang H
    1579. Overholtzer M
    1580. Ozbun MA
    1581. Ozdinler PH
    1582. Ozpolat B
    1583. Pacelli C
    1584. Paganetti P
    1585. Page G
    1586. Pages G
    1587. Pagnini U
    1588. Pajak B
    1589. Pak SC
    1590. Pakos-Zebrucka K
    1591. Pakpour N
    1592. Palková Z
    1593. Palladino F
    1594. Pallauf K
    1595. Pallet N
    1596. Palmieri M
    1597. Paludan SR
    1598. Palumbo C
    1599. Palumbo S
    1600. Pampliega O
    1601. Pan H
    1602. Pan W
    1603. Panaretakis T
    1604. Pandey A
    1605. Pantazopoulou A
    1606. Papackova Z
    1607. Papademetrio DL
    1608. Papassideri I
    1609. Papini A
    1610. Parajuli N
    1611. Pardo J
    1612. Parekh VV
    1613. Parenti G
    1614. Park JI
    1615. Park J
    1616. Park OK
    1617. Parker R
    1618. Parlato R
    1619. Parys JB
    1620. Parzych KR
    1621. Pasquet JM
    1622. Pasquier B
    1623. Pasumarthi KB
    1624. Patschan D
    1625. Patterson C
    1626. Pattingre S
    1627. Pattison S
    1628. Pause A
    1629. Pavenstädt H
    1630. Pavone F
    1631. Pedrozo Z
    1632. Peña FJ
    1633. Peñalva MA
    1634. Pende M
    1635. Peng J
    1636. Penna F
    1637. Penninger JM
    1638. Pensalfini A
    1639. Pepe S
    1640. Pereira GJ
    1641. Pereira PC
    1642. Pérez-de la Cruz V
    1643. Pérez-Pérez ME
    1644. Pérez-Rodríguez D
    1645. Pérez-Sala D
    1646. Perier C
    1647. Perl A
    1648. Perlmutter DH
    1649. Perrotta I
    1650. Pervaiz S
    1651. Pesonen M
    1652. Pessin JE
    1653. Peters GJ
    1654. Petersen M
    1655. Petrache I
    1656. Petrof BJ
    1657. Petrovski G
    1658. Phang JM
    1659. Piacentini M
    1660. Pierdominici M
    1661. Pierre P
    1662. Pierrefite-Carle V
    1663. Pietrocola F
    1664. Pimentel-Muiños FX
    1665. Pinar M
    1666. Pineda B
    1667. Pinkas-Kramarski R
    1668. Pinti M
    1669. Pinton P
    1670. Piperdi B
    1671. Piret JM
    1672. Platanias LC
    1673. Platta HW
    1674. Plowey ED
    1675. Pöggeler S
    1676. Poirot M
    1677. Polčic P
    1678. Poletti A
    1679. Poon AH
    1680. Popelka H
    1681. Popova B
    1682. Poprawa I
    1683. Poulose SM
    1684. Poulton J
    1685. Powers SK
    1686. Powers T
    1687. Pozuelo-Rubio M
    1688. Prak K
    1689. Prange R
    1690. Prescott M
    1691. Priault M
    1692. Prince S
    1693. Proia RL
    1694. Proikas-Cezanne T
    1695. Prokisch H
    1696. Promponas VJ
    1697. Przyklenk K
    1698. Puertollano R
    1699. Pugazhenthi S
    1700. Puglielli L
    1701. Pujol A
    1702. Puyal J
    1703. Pyeon D
    1704. Qi X
    1705. Qian WB
    1706. Qin ZH
    1707. Qiu Y
    1708. Qu Z
    1709. Quadrilatero J
    1710. Quinn F
    1711. Raben N
    1712. Rabinowich H
    1713. Radogna F
    1714. Ragusa MJ
    1715. Rahmani M
    1716. Raina K
    1717. Ramanadham S
    1718. Ramesh R
    1719. Rami A
    1720. Randall-Demllo S
    1721. Randow F
    1722. Rao H
    1723. Rao VA
    1724. Rasmussen BB
    1725. Rasse TM
    1726. Ratovitski EA
    1727. Rautou PE
    1728. Ray SK
    1729. Razani B
    1730. Reed BH
    1731. Reggiori F
    1732. Rehm M
    1733. Reichert AS
    1734. Rein T
    1735. Reiner DJ
    1736. Reits E
    1737. Ren J
    1738. Ren X
    1739. Renna M
    1740. Reusch JE
    1741. Revuelta JL
    1742. Reyes L
    1743. Rezaie AR
    1744. Richards RI
    1745. Richardson DR
    1746. Richetta C
    1747. Riehle MA
    1748. Rihn BH
    1749. Rikihisa Y
    1750. Riley BE
    1751. Rimbach G
    1752. Rippo MR
    1753. Ritis K
    1754. Rizzi F
    1755. Rizzo E
    1756. Roach PJ
    1757. Robbins J
    1758. Roberge M
    1759. Roca G
    1760. Roccheri MC
    1761. Rocha S
    1762. Rodrigues CM
    1763. Rodríguez CI
    1764. de Cordoba SR
    1765. Rodriguez-Muela N
    1766. Roelofs J
    1767. Rogov VV
    1768. Rohn TT
    1769. Rohrer B
    1770. Romanelli D
    1771. Romani L
    1772. Romano PS
    1773. Roncero MI
    1774. Rosa JL
    1775. Rosello A
    1776. Rosen KV
    1777. Rosenstiel P
    1778. Rost-Roszkowska M
    1779. Roth KA
    1780. Roué G
    1781. Rouis M
    1782. Rouschop KM
    1783. Ruan DT
    1784. Ruano D
    1785. Rubinsztein DC
    1786. Rucker EB
    1787. Rudich A
    1788. Rudolf E
    1789. Rudolf R
    1790. Ruegg MA
    1791. Ruiz-Roldan C
    1792. Ruparelia AA
    1793. Rusmini P
    1794. Russ DW
    1795. Russo GL
    1796. Russo G
    1797. Russo R
    1798. Rusten TE
    1799. Ryabovol V
    1800. Ryan KM
    1801. Ryter SW
    1802. Sabatini DM
    1803. Sacher M
    1804. Sachse C
    1805. Sack MN
    1806. Sadoshima J
    1807. Saftig P
    1808. Sagi-Eisenberg R
    1809. Sahni S
    1810. Saikumar P
    1811. Saito T
    1812. Saitoh T
    1813. Sakakura K
    1814. Sakoh-Nakatogawa M
    1815. Sakuraba Y
    1816. Salazar-Roa M
    1817. Salomoni P
    1818. Saluja AK
    1819. Salvaterra PM
    1820. Salvioli R
    1821. Samali A
    1822. Sanchez AM
    1823. Sánchez-Alcázar JA
    1824. Sanchez-Prieto R
    1825. Sandri M
    1826. Sanjuan MA
    1827. Santaguida S
    1828. Santambrogio L
    1829. Santoni G
    1830. Dos Santos CN
    1831. Saran S
    1832. Sardiello M
    1833. Sargent G
    1834. Sarkar P
    1835. Sarkar S
    1836. Sarrias MR
    1837. Sarwal MM
    1838. Sasakawa C
    1839. Sasaki M
    1840. Sass M
    1841. Sato K
    1842. Sato M
    1843. Satriano J
    1844. Savaraj N
    1845. Saveljeva S
    1846. Schaefer L
    1847. Schaible UE
    1848. Scharl M
    1849. Schatzl HM
    1850. Schekman R
    1851. Scheper W
    1852. Schiavi A
    1853. Schipper HM
    1854. Schmeisser H
    1855. Schmidt J
    1856. Schmitz I
    1857. Schneider BE
    1858. Schneider EM
    1859. Schneider JL
    1860. Schon EA
    1861. Schönenberger MJ
    1862. Schönthal AH
    1863. Schorderet DF
    1864. Schröder B
    1865. Schuck S
    1866. Schulze RJ
    1867. Schwarten M
    1868. Schwarz TL
    1869. Sciarretta S
    1870. Scotto K
    1871. Scovassi AI
    1872. Screaton RA
    1873. Screen M
    1874. Seca H
    1875. Sedej S
    1876. Segatori L
    1877. Segev N
    1878. Seglen PO
    1879. Seguí-Simarro JM
    1880. Segura-Aguilar J
    1881. Seki E
    1882. Sell C
    1883. Seiliez I
    1884. Semenkovich CF
    1885. Semenza GL
    1886. Sen U
    1887. Serra AL
    1888. Serrano-Puebla A
    1889. Sesaki H
    1890. Setoguchi T
    1891. Settembre C
    1892. Shacka JJ
    1893. Shajahan-Haq AN
    1894. Shapiro IM
    1895. Sharma S
    1896. She H
    1897. Shen CK
    1898. Shen CC
    1899. Shen HM
    1900. Shen S
    1901. Shen W
    1902. Sheng R
    1903. Sheng X
    1904. Sheng ZH
    1905. Shepherd TG
    1906. Shi J
    1907. Shi Q
    1908. Shi Q
    1909. Shi Y
    1910. Shibutani S
    1911. Shibuya K
    1912. Shidoji Y
    1913. Shieh JJ
    1914. Shih CM
    1915. Shimada Y
    1916. Shimizu S
    1917. Shin DW
    1918. Shinohara ML
    1919. Shintani M
    1920. Shintani T
    1921. Shioi T
    1922. Shirabe K
    1923. Shiri-Sverdlov R
    1924. Shirihai O
    1925. Shore GC
    1926. Shu CW
    1927. Shukla D
    1928. Sibirny AA
    1929. Sica V
    1930. Sigurdson CJ
    1931. Sigurdsson EM
    1932. Sijwali PS
    1933. Sikorska B
    1934. Silveira WA
    1935. Silvente-Poirot S
    1936. Silverman GA
    1937. Simak J
    1938. Simmet T
    1939. Simon AK
    1940. Simon HU
    1941. Simone C
    1942. Simons M
    1943. Simonsen A
    1944. Singh R
    1945. Singh SV
    1946. Singh SK
    1947. Sinha D
    1948. Sinha S
    1949. Sinicrope FA
    1950. Sirko A
    1951. Sirohi K
    1952. Sishi BJ
    1953. Sittler A
    1954. Siu PM
    1955. Sivridis E
    1956. Skwarska A
    1957. Slack R
    1958. Slaninová I
    1959. Slavov N
    1960. Smaili SS
    1961. Smalley KS
    1962. Smith DR
    1963. Soenen SJ
    1964. Soleimanpour SA
    1965. Solhaug A
    1966. Somasundaram K
    1967. Son JH
    1968. Sonawane A
    1969. Song C
    1970. Song F
    1971. Song HK
    1972. Song JX
    1973. Song W
    1974. Soo KY
    1975. Sood AK
    1976. Soong TW
    1977. Soontornniyomkij V
    1978. Sorice M
    1979. Sotgia F
    1980. Soto-Pantoja DR
    1981. Sotthibundhu A
    1982. Sousa MJ
    1983. Spaink HP
    1984. Span PN
    1985. Spang A
    1986. Sparks JD
    1987. Speck PG
    1988. Spector SA
    1989. Spies CD
    1990. Springer W
    1991. Clair DS
    1992. Stacchiotti A
    1993. Staels B
    1994. Stang MT
    1995. Starczynowski DT
    1996. Starokadomskyy P
    1997. Steegborn C
    1998. Steele JW
    1999. Stefanis L
    2000. Steffan J
    2001. Stellrecht CM
    2002. Stenmark H
    2003. Stepkowski TM
    2004. Stern ST
    2005. Stevens C
    2006. Stockwell BR
    2007. Stoka V
    2008. Storchova Z
    2009. Stork B
    2010. Stratoulias V
    2011. Stravopodis DJ
    2012. Strnad P
    2013. Strohecker AM
    2014. Ström AL
    2015. Stromhaug P
    2016. Stulik J
    2017. Su YX
    2018. Su Z
    2019. Subauste CS
    2020. Subramaniam S
    2021. Sue CM
    2022. Suh SW
    2023. Sui X
    2024. Sukseree S
    2025. Sulzer D
    2026. Sun FL
    2027. Sun J
    2028. Sun J
    2029. Sun SY
    2030. Sun Y
    2031. Sun Y
    2032. Sun Y
    2033. Sundaramoorthy V
    2034. Sung J
    2035. Suzuki H
    2036. Suzuki K
    2037. Suzuki N
    2038. Suzuki T
    2039. Suzuki YJ
    2040. Swanson MS
    2041. Swanton C
    2042. Swärd K
    2043. Swarup G
    2044. Sweeney ST
    2045. Sylvester PW
    2046. Szatmari Z
    2047. Szegezdi E
    2048. Szlosarek PW
    2049. Taegtmeyer H
    2050. Tafani M
    2051. Taillebourg E
    2052. Tait SW
    2053. Takacs-Vellai K
    2054. Takahashi Y
    2055. Takáts S
    2056. Takemura G
    2057. Takigawa N
    2058. Talbot NJ
    2059. Tamagno E
    2060. Tamburini J
    2061. Tan CP
    2062. Tan L
    2063. Tan ML
    2064. Tan M
    2065. Tan YJ
    2066. Tanaka K
    2067. Tanaka M
    2068. Tang D
    2069. Tang D
    2070. Tang G
    2071. Tanida I
    2072. Tanji K
    2073. Tannous BA
    2074. Tapia JA
    2075. Tasset-Cuevas I
    2076. Tatar M
    2077. Tavassoly I
    2078. Tavernarakis N
    2079. Taylor A
    2080. Taylor GS
    2081. Taylor GA
    2082. Taylor JP
    2083. Taylor MJ
    2084. Tchetina EV
    2085. Tee AR
    2086. Teixeira-Clerc F
    2087. Telang S
    2088. Tencomnao T
    2089. Teng BB
    2090. Teng RJ
    2091. Terro F
    2092. Tettamanti G
    2093. Theiss AL
    2094. Theron AE
    2095. Thomas KJ
    2096. Thomé MP
    2097. Thomes PG
    2098. Thorburn A
    2099. Thorner J
    2100. Thum T
    2101. Thumm M
    2102. Thurston TL
    2103. Tian L
    2104. Till A
    2105. Ting JP
    2106. Titorenko VI
    2107. Toker L
    2108. Toldo S
    2109. Tooze SA
    2110. Topisirovic I
    2111. Torgersen ML
    2112. Torosantucci L
    2113. Torriglia A
    2114. Torrisi MR
    2115. Tournier C
    2116. Towns R
    2117. Trajkovic V
    2118. Travassos LH
    2119. Triola G
    2120. Tripathi DN
    2121. Trisciuoglio D
    2122. Troncoso R
    2123. Trougakos IP
    2124. Truttmann AC
    2125. Tsai KJ
    2126. Tschan MP
    2127. Tseng YH
    2128. Tsukuba T
    2129. Tsung A
    2130. Tsvetkov AS
    2131. Tu S
    2132. Tuan HY
    2133. Tucci M
    2134. Tumbarello DA
    2135. Turk B
    2136. Turk V
    2137. Turner RF
    2138. Tveita AA
    2139. Tyagi SC
    2140. Ubukata M
    2141. Uchiyama Y
    2142. Udelnow A
    2143. Ueno T
    2144. Umekawa M
    2145. Umemiya-Shirafuji R
    2146. Underwood BR
    2147. Ungermann C
    2148. Ureshino RP
    2149. Ushioda R
    2150. Uversky VN
    2151. Uzcátegui NL
    2152. Vaccari T
    2153. Vaccaro MI
    2154. Váchová L
    2155. Vakifahmetoglu-Norberg H
    2156. Valdor R
    2157. Valente EM
    2158. Vallette F
    2159. Valverde AM
    2160. Van den Berghe G
    2161. Van Den Bosch L
    2162. van den Brink GR
    2163. van der Goot FG
    2164. van der Klei IJ
    2165. van der Laan LJ
    2166. van Doorn WG
    2167. van Egmond M
    2168. van Golen KL
    2169. Van Kaer L
    2170. van Lookeren Campagne M
    2171. Vandenabeele P
    2172. Vandenberghe W
    2173. Vanhorebeek I
    2174. Varela-Nieto I
    2175. Vasconcelos MH
    2176. Vasko R
    2177. Vavvas DG
    2178. Vega-Naredo I
    2179. Velasco G
    2180. Velentzas AD
    2181. Velentzas PD
    2182. Vellai T
    2183. Vellenga E
    2184. Vendelbo MH
    2185. Venkatachalam K
    2186. Ventura N
    2187. Ventura S
    2188. Veras PS
    2189. Verdier M
    2190. Vertessy BG
    2191. Viale A
    2192. Vidal M
    2193. Vieira HL
    2194. Vierstra RD
    2195. Vigneswaran N
    2196. Vij N
    2197. Vila M
    2198. Villar M
    2199. Villar VH
    2200. Villarroya J
    2201. Vindis C
    2202. Viola G
    2203. Viscomi MT
    2204. Vitale G
    2205. Vogl DT
    2206. Voitsekhovskaja OV
    2207. von Haefen C
    2208. von Schwarzenberg K
    2209. Voth DE
    2210. Vouret-Craviari V
    2211. Vuori K
    2212. Vyas JM
    2213. Waeber C
    2214. Walker CL
    2215. Walker MJ
    2216. Walter J
    2217. Wan L
    2218. Wan X
    2219. Wang B
    2220. Wang C
    2221. Wang CY
    2222. Wang C
    2223. Wang C
    2224. Wang C
    2225. Wang D
    2226. Wang F
    2227. Wang F
    2228. Wang G
    2229. Wang HJ
    2230. Wang H
    2231. Wang HG
    2232. Wang H
    2233. Wang HD
    2234. Wang J
    2235. Wang J
    2236. Wang M
    2237. Wang MQ
    2238. Wang PY
    2239. Wang P
    2240. Wang RC
    2241. Wang S
    2242. Wang TF
    2243. Wang X
    2244. Wang XJ
    2245. Wang XW
    2246. Wang X
    2247. Wang X
    2248. Wang Y
    2249. Wang Y
    2250. Wang Y
    2251. Wang YJ
    2252. Wang Y
    2253. Wang Y
    2254. Wang YT
    2255. Wang Y
    2256. Wang ZN
    2257. Wappner P
    2258. Ward C
    2259. Ward DM
    2260. Warnes G
    2261. Watada H
    2262. Watanabe Y
    2263. Watase K
    2264. Weaver TE
    2265. Weekes CD
    2266. Wei J
    2267. Weide T
    2268. Weihl CC
    2269. Weindl G
    2270. Weis SN
    2271. Wen L
    2272. Wen X
    2273. Wen Y
    2274. Westermann B
    2275. Weyand CM
    2276. White AR
    2277. White E
    2278. Whitton JL
    2279. Whitworth AJ
    2280. Wiels J
    2281. Wild F
    2282. Wildenberg ME
    2283. Wileman T
    2284. Wilkinson DS
    2285. Wilkinson S
    2286. Willbold D
    2287. Williams C
    2288. Williams K
    2289. Williamson PR
    2290. Winklhofer KF
    2291. Witkin SS
    2292. Wohlgemuth SE
    2293. Wollert T
    2294. Wolvetang EJ
    2295. Wong E
    2296. Wong GW
    2297. Wong RW
    2298. Wong VK
    2299. Woodcock EA
    2300. Wright KL
    2301. Wu C
    2302. Wu D
    2303. Wu GS
    2304. Wu J
    2305. Wu J
    2306. Wu M
    2307. Wu M
    2308. Wu S
    2309. Wu WK
    2310. Wu Y
    2311. Wu Z
    2312. Xavier CP
    2313. Xavier RJ
    2314. Xia GX
    2315. Xia T
    2316. Xia W
    2317. Xia Y
    2318. Xiao H
    2319. Xiao J
    2320. Xiao S
    2321. Xiao W
    2322. Xie CM
    2323. Xie Z
    2324. Xie Z
    2325. Xilouri M
    2326. Xiong Y
    2327. Xu C
    2328. Xu C
    2329. Xu F
    2330. Xu H
    2331. Xu H
    2332. Xu J
    2333. Xu J
    2334. Xu J
    2335. Xu L
    2336. Xu X
    2337. Xu Y
    2338. Xu Y
    2339. Xu ZX
    2340. Xu Z
    2341. Xue Y
    2342. Yamada T
    2343. Yamamoto A
    2344. Yamanaka K
    2345. Yamashina S
    2346. Yamashiro S
    2347. Yan B
    2348. Yan B
    2349. Yan X
    2350. Yan Z
    2351. Yanagi Y
    2352. Yang DS
    2353. Yang JM
    2354. Yang L
    2355. Yang M
    2356. Yang PM
    2357. Yang P
    2358. Yang Q
    2359. Yang W
    2360. Yang WY
    2361. Yang X
    2362. Yang Y
    2363. Yang Y
    2364. Yang Z
    2365. Yang Z
    2366. Yao MC
    2367. Yao PJ
    2368. Yao X
    2369. Yao Z
    2370. Yao Z
    2371. Yasui LS
    2372. Ye M
    2373. Yedvobnick B
    2374. Yeganeh B
    2375. Yeh ES
    2376. Yeyati PL
    2377. Yi F
    2378. Yi L
    2379. Yin XM
    2380. Yip CK
    2381. Yoo YM
    2382. Yoo YH
    2383. Yoon SY
    2384. Yoshida K
    2385. Yoshimori T
    2386. Young KH
    2387. Yu H
    2388. Yu JJ
    2389. Yu JT
    2390. Yu J
    2391. Yu L
    2392. Yu WH
    2393. Yu XF
    2394. Yu Z
    2395. Yuan J
    2396. Yuan ZM
    2397. Yue BY
    2398. Yue J
    2399. Yue Z
    2400. Zacks DN
    2401. Zacksenhaus E
    2402. Zaffaroni N
    2403. Zaglia T
    2404. Zakeri Z
    2405. Zecchini V
    2406. Zeng J
    2407. Zeng M
    2408. Zeng Q
    2409. Zervos AS
    2410. Zhang DD
    2411. Zhang F
    2412. Zhang G
    2413. Zhang GC
    2414. Zhang H
    2415. Zhang H
    2416. Zhang H
    2417. Zhang H
    2418. Zhang J
    2419. Zhang J
    2420. Zhang J
    2421. Zhang J
    2422. Zhang JP
    2423. Zhang L
    2424. Zhang L
    2425. Zhang L
    2426. Zhang L
    2427. Zhang MY
    2428. Zhang X
    2429. Zhang XD
    2430. Zhang Y
    2431. Zhang Y
    2432. Zhang Y
    2433. Zhang Y
    2434. Zhang Y
    2435. Zhao M
    2436. Zhao WL
    2437. Zhao X
    2438. Zhao YG
    2439. Zhao Y
    2440. Zhao Y
    2441. Zhao YX
    2442. Zhao Z
    2443. Zhao ZJ
    2444. Zheng D
    2445. Zheng XL
    2446. Zheng X
    2447. Zhivotovsky B
    2448. Zhong Q
    2449. Zhou GZ
    2450. Zhou G
    2451. Zhou H
    2452. Zhou SF
    2453. Zhou XJ
    2454. Zhu H
    2455. Zhu H
    2456. Zhu WG
    2457. Zhu W
    2458. Zhu XF
    2459. Zhu Y
    2460. Zhuang SM
    2461. Zhuang X
    2462. Ziparo E
    2463. Zois CE
    2464. Zoladek T
    2465. Zong WX
    2466. Zorzano A
    2467. Zughaier SM
    (2016) Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)
    Autophagy 12:1–222.
    https://doi.org/10.1080/15548627.2015.1100356
    1. Li Y
    2. Li B
    3. Liu L
    4. Chen H
    5. Zhang H
    6. Zheng X
    7. Zhang Z
    (2015)
    FgMon1, a guanine nucleotide exchange factor of FgRab7, is important for vacuole fusion, autophagy and plant infection in Fusarium graminearum
    Scientific Reports 5:1–13.
    1. Nordmann M
    2. Ungermann C
    3. Cabrera M
    (2012)
    Rab GTPases and Membrane Trafficking
    132–143, Role of Rab7/Ypt7 in Organizing Membrane Trafficking at the Late Endosome, Rab GTPases and Membrane Trafficking.

Article and author information

Author details

  1. Jieqiong Gao

    Biochemistry Section, Department of Biology/Chemistry, University of Osnabrück, Osnabrück, Germany
    Contribution
    Conceptualization, Data curation, Formal analysis, Methodology
    Competing interests
    No competing interests declared
  2. Lars Langemeyer

    Biochemistry Section, Department of Biology/Chemistry, University of Osnabrück, Osnabrück, Germany
    Contribution
    Data curation, Formal analysis, Methodology
    Competing interests
    No competing interests declared
  3. Daniel Kümmel

    Structural Biology Section, Department of Biology/Chemistry, University of Osnabrück, Osnabrück, Germany
    Contribution
    Conceptualization, Investigation
    Competing interests
    No competing interests declared
  4. Fulvio Reggiori

    Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, Netherlands
    Contribution
    Conceptualization, Funding acquisition, Investigation, Writing—review and editing
    Competing interests
    No competing interests declared
  5. Christian Ungermann

    Biochemistry Section, Department of Biology/Chemistry, University of Osnabrück, Osnabrück, Germany
    Contribution
    Conceptualization, Data curation, Funding acquisition, Writing—original draft, Project administration
    For correspondence
    cu@uos.de
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-4331-8695

Funding

Deutsche Forschungsgemeinschaft (UN111/7-3)

  • Christian Ungermann

Deutsche Forschungsgemeinschaft (SFB 944)

  • Daniel Kuemmel
  • Christian Ungermann

ZonMw (VICI 016.130.606)

  • Fulvio Reggiori

European Commission (Marie Skłodowska-Curie ITN)

  • Fulvio Reggiori

Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (SNF Sinergia (CRSII3_154421)

  • Fulvio Reggiori

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

Acknowledgements

We thank Anna Lürick, Stephan Kiontke, and Claudio DeVirgilio for support and discussion, Sascha Martens and Ivan Dikic for constructs, and Kathrin Auffarth and Angela Perz for excellent technical assistance. DK is supported by the SFB944, Project P17. FR is supported by SNF Sinergia (CRSII3_154421), Marie Skłodowska-Curie ITN (765912), and ZonMW VICI (016.130.606) grants. JG received support by the SFB 944 graduate program. This work was funded by the DFG (UN111/7-3 and SFB 944, Project P11).

Version history

  1. Received: August 9, 2017
  2. Accepted: February 12, 2018
  3. Accepted Manuscript published: February 15, 2018 (version 1)
  4. Version of Record published: March 7, 2018 (version 2)

Copyright

© 2018, Gao 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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  1. Jieqiong Gao
  2. Lars Langemeyer
  3. Daniel Kümmel
  4. Fulvio Reggiori
  5. Christian Ungermann
(2018)
Molecular mechanism to target the endosomal Mon1-Ccz1 GEF complex to the pre-autophagosomal structure
eLife 7:e31145.
https://doi.org/10.7554/eLife.31145

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