article: 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.
  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). 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.
  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). 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. 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. 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.
  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.
  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 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. 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). 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). 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 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. 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.
summary: 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.
keywords: biochemistry and chemical biology
  cell biology
title: Molecular mechanism to target the endosomal Mon1-Ccz1 GEF complex to the pre-autophagosomal structure