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