PI 3P binding by Atg21 organises Atg8 lipidation
2015; Springer Nature; Volume: 34; Issue: 7 Linguagem: Inglês
10.15252/embj.201488957
ISSN1460-2075
AutoresLisa Juris, Marco Montino, Peter Rube, Petra Schlotterhose, Michael Thumm, Roswitha Krick,
Tópico(s)Biochemical and Molecular Research
ResumoArticle17 February 2015free access PI3P binding by Atg21 organises Atg8 lipidation Lisa Juris Lisa Juris Georg-August-University, University Medicine, Institute of Cellular Biochemistry, Goettingen, Germany Search for more papers by this author Marco Montino Marco Montino Georg-August-University, University Medicine, Institute of Cellular Biochemistry, Goettingen, Germany Search for more papers by this author Peter Rube Peter Rube Georg-August-University, University Medicine, Institute of Cellular Biochemistry, Goettingen, Germany Search for more papers by this author Petra Schlotterhose Petra Schlotterhose Georg-August-University, University Medicine, Institute of Cellular Biochemistry, Goettingen, Germany Search for more papers by this author Michael Thumm Corresponding Author Michael Thumm Georg-August-University, University Medicine, Institute of Cellular Biochemistry, Goettingen, Germany Search for more papers by this author Roswitha Krick Roswitha Krick Georg-August-University, University Medicine, Institute of Cellular Biochemistry, Goettingen, Germany Search for more papers by this author Lisa Juris Lisa Juris Georg-August-University, University Medicine, Institute of Cellular Biochemistry, Goettingen, Germany Search for more papers by this author Marco Montino Marco Montino Georg-August-University, University Medicine, Institute of Cellular Biochemistry, Goettingen, Germany Search for more papers by this author Peter Rube Peter Rube Georg-August-University, University Medicine, Institute of Cellular Biochemistry, Goettingen, Germany Search for more papers by this author Petra Schlotterhose Petra Schlotterhose Georg-August-University, University Medicine, Institute of Cellular Biochemistry, Goettingen, Germany Search for more papers by this author Michael Thumm Corresponding Author Michael Thumm Georg-August-University, University Medicine, Institute of Cellular Biochemistry, Goettingen, Germany Search for more papers by this author Roswitha Krick Roswitha Krick Georg-August-University, University Medicine, Institute of Cellular Biochemistry, Goettingen, Germany Search for more papers by this author Author Information Lisa Juris1, Marco Montino1, Peter Rube1, Petra Schlotterhose1, Michael Thumm 1 and Roswitha Krick1 1Georg-August-University, University Medicine, Institute of Cellular Biochemistry, Goettingen, Germany *Corresponding author. Tel: +49 551 39 5947; E-mail: [email protected] The EMBO Journal (2015)34:955-973https://doi.org/10.15252/embj.201488957 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Autophagosome biogenesis requires two ubiquitin-like conjugation systems. One couples ubiquitin-like Atg8 to phosphatidylethanolamine, and the other couples ubiquitin-like Atg12 to Atg5. Atg12~Atg5 then forms a heterodimer with Atg16. Membrane recruitment of the Atg12~Atg5/Atg16 complex defines the Atg8 lipidation site. Lipidation requires a PI3P-containing precursor. How PI3P is sensed and used to coordinate the conjugation systems remained unclear. Here, we show that Atg21, a WD40 β-propeller, binds via PI3P to the preautophagosomal structure (PAS). Atg21 directly interacts with the coiled-coil domain of Atg16 and with Atg8. This latter interaction requires the conserved F5K6-motif in the N-terminal helical domain of Atg8, but not its AIM-binding site. Accordingly, the Atg8 AIM-binding site remains free to mediate interaction with its E2 enzyme Atg3. Atg21 thus defines PI3P-dependently the lipidation site by linking and organising the E3 ligase complex and Atg8 at the PAS. Synopsis Atg21 binds to PI3P at the preautophagosomal structure, recruits ubiquitin-like Atg8 and the E3 ligase complex Atg12~Atg5/Atg16 and arranges them for efficient lipidation of Atg8. Atg21 binds via PI3P to the preautophagosomal structure (PAS). Atg21 interacts with the coiled-coil domain of Atg16 and thus recruits the E3 ligase complex Atg12˜Atg5/Atg16 to the PAS. Atg21 interacts with ubiquitin-like Atg8. Atg21 arranges the E3 ligase complex and Atg8 at the PAS for efficient lipidation of Atg8. Introduction Macroautophagy (hereafter autophagy) is an evolutionarily conserved process where cytosol and damaged or superfluous organelles are enclosed into double-membraned autophagosomes. After fusion of autophagosomes with the lysosome, their content is degraded and recycled (Farré et al, 2009; Backues & Klionsky, 2012; Hamasaki et al, 2013; Reggiori & Klionsky, 2013; Zavodszky et al, 2013). Autophagy affects ageing, cell death, removal of intracellular bacteria and antigen presentation. It is involved in cancer, Parkinson's, Huntington's and Crohn's disease (Choi et al, 2013). In S. cerevisiae, most of the autophagy (Atg) proteins colocalise at least transiently at the preautophagosomal structure (PAS), where the isolation membrane (phagophore) is assembled, elongated and sealed to an autophagosome. Autophagy subtypes selectively remove bulky cargos such as organelles or aggregates. The yeast cytoplasm-to-vacuole (Cvt) pathway, which targets proaminopeptidase I (pApeI) under vegetative conditions to the vacuole, is a prototype of selective autophagy. Crucial PAS components are the regulatory serine/threonine protein kinase Atg1, the transmembrane protein Atg9, which is proposed to deliver membranes (Mari et al, 2010; Yamamoto et al, 2012) and a phosphatidylinositol 3-kinase complex containing Atg14. Autophagosome biogenesis further requires two ubiquitin-like conjugation systems. First, ubiquitin-like Atg8 is processed by the cysteine proteinase Atg4, then activated by the E1 enzyme Atg7, transferred to the E2 Atg3 and finally coupled to phosphatidylethanolamine (PE). Atg8-PE might act as tether during phagophore expansion (Nakatogawa et al, 2007; Xie et al, 2008; Weidberg et al, 2010, 2011; Nair et al, 2011). Secondly, ubiquitin-like Atg12 is activated by Atg7, transferred to the E2 Atg10 and covalently coupled to Atg5. The Atg12~Atg5 conjugate then binds Atg16, which dimerises via its coiled-coil domain. Binding of dimeric Atg12~Atg5/Atg16 to the PAS activates Atg3 analogous to an E3 ubiquitin-ligase complex (Hanada et al, 2007; Fujioka et al, 2010; Romanov et al, 2012; Noda et al, 2013; Sakoh-Nakatogawa et al, 2013). Atg16 is the key determinant of the lipidation site (Hanada et al, 2007; Fujita et al, 2008). Although no phosphoinositide-binding sites have been detected, yeast Atg5 and Atg16 require PI3P for PAS recruitment (Suzuki et al, 2007) and the PI3P-kinase inhibitor wortmannin affects mammalian Atg5 membrane association (Mizushima et al, 2001). Autophagosome biogenesis requires PI3P at the PAS. Its presence on autophagosomal membranes is deciphered by members of the PROPPIN family. These β-propellers that bind phosphoinositides are a WD40 protein family conserved from yeast to human (Lemmon, 2008; Moravcevic et al, 2012). PROPPINs fold as seven-bladed β-propellers, with each blade containing four antiparallel β-strands (A to D, from in- to outside) (Fig 4A). We and others identified based on the crystal structure two lipid-binding sites at the circumference of the propeller; they act jointly and can bind both PI3P and PI(3,5)P(2) (Dove et al, 2004; Baskaran et al, 2012; Krick et al, 2012; Watanabe et al, 2012). The S. cerevisiae PROPPINs Atg18, Atg21 and Hsv2 are homologous, but differently affect autophagy subtypes. Atg18 binds PI3P-dependently to the PAS, where it mediates with Atg2 the retrieval of Atg9 from mature autophagosomes (Reggiori et al, 2004; Obara et al, 2008). Loops 2AB, 2CD and 2D3A in Atg18, localised opposite to the PIP-binding sites, are involved in Atg2 binding (Watanabe et al, 2012; Rieter et al, 2013). Interestingly, for Atg18 in P. pastoris, modulation of phosphoinositide binding by phosphorylation was reported (Tamura et al, 2013). Hsv2 partially affects piecemeal microautophagy of the nucleus, where non-essential parts of the nucleus are removed by autophagy (Krick et al, 2008a,b). Atg21 is essential for selective autophagy as the Cvt pathway or mitophagy; in its absence, unselective autophagy is retarded (Meiling-Wesse et al, 2004; Obara et al, 2008; Nair et al, 2010; Reggiori & Klionsky, 2013). We here dissect the molecular function of the yeast PROPPIN Atg21. Results Atg21 localises PI3P-dependently to the PAS Atg21 was so far only detected at endosomes. Depletion of Atg21 from endosomes did not affect autophagy, leaving its role during autophagy elusive (Krick et al, 2008a; Obara et al, 2008). With a sensitive fluorescence microscope, we now detected in growing cells colocalisation of the PAS marker pApeI-RFP with one of the multiple Atg21-YFP punctae (Fig 1A and B). Atg14 is crucial for the PAS PtdIns 3-kinase complex (Obara & Ohsumi, 2011). Diminished colocalisation in atg14∆ cells showed that Atg21 PAS binding depends on PI3P (Fig 1A and B). Fluorescence microscopy (Fig 5A) further showed for the PI3P-binding-deficient cherry-Atg21FTTG only 0.0035 puncta/cell (1 punctum in 420 cells), while cherry-Atg21 formed 3.5 ± 0.21 puncta/cell. Atg21 PAS localisation was confirmed by reduced colocalisation in cells lacking the pApeI receptor (atg19∆) and in cells with PAS assembly defects (atg9∆) (Fig 1A and B). Also, pApeI-YFP and cherry-Atg21 colocalised to 46% in atg21∆ and to 23% in atg14∆ cells. Cherry-Atg21 also colocalised in atg8∆ cells (Fig 6F and G) with the PAS markers Atg16-GFP (65%) and Atg5-YFP (78%). Figure 1. Part of Atg21 localises to the PAS Cells expressing Atg21-YFP and the PAS marker pApeI-RFP were grown to log-phase and visualised in fluorescence microscopy. Images were deconvoluted with SoftWoRx (Applied Precision). To dissect the requirement of other Atg proteins for Atg21 PAS localisation, the colocalisation rate as the percentage of perivacuolar pApeI-RFP PAS dots overlapping with Atg21-YFP was determined. Images from at least three cultures were taken, and for each image, puncta/cells were counted. Error bars are SEM. Statistical relevance was determined by unpaired two-tailed t-test. ns, not significant P > 0.05; *P < 0.05; **P < 0.01. Download figure Download PowerPoint Atg21 recruits Atg8 to the PAS by direct interaction with the Atg8 FK-motif In growing atg21∆ cells, Atg8 PAS recruitment is impaired (Meiling-Wesse et al, 2004; Stromhaug et al, 2004), and we thus speculated that Atg21 might interact with Atg8. Since cell lysis often affects Atg21 membrane association, we used the split-ubiquitin system in intact cells. The split-ubiquitin system is similar to the 2-hybrid system but better suited for membrane-associated proteins. Briefly, the amino terminal half of ubiquitin (Nub) is fused to the bait and its carboxy-terminal domain (Cub) to the prey (Müller & Johnsson, 2008). Protein interaction restores ubiquitin leading to degradation of a proteolytically sensitive Ura3 variant at the carboxy-terminal ubiquitin domain. Interaction thus decreases growth without uracil and allows growth with 5-fluoroorotic acid (FOA). The split-ubiquitin system indeed indicated interaction of Atg21 with Atg8 comparable to the known interaction between Ste14 and Ubc6 (Fig 2A). The interaction was unaffected in atg4∆ and atg5∆ cells, suggesting that Atg21 can interact with unprocessed and unlipidated Atg8 (Fig 2A). To further confirm this interaction, E. coli-expressed GST-Atg8 on glutathione beads was incubated with extracts of S. cerevisiae cells expressing Atg21-TAP. Atg21-TAP effectively bound GST-Atg8, but not GST (Fig 2B and C). Which Atg8 region mediates interaction with Atg21? Leucine 50 in the Atg8 carboxy-terminal ubiquitin-like domain is part of the AIM (Atg8 interacting motif)-binding site crucial for interaction with the WxxL-like motif (AIM) in proteins such as Atg1, Atg3 and Atg19 (Noda et al, 2010; Alemu et al, 2012; Nakatogawa et al, 2012; Shaid et al, 2013). The additional N-terminal helical domain (NHD) of Atg8 is flexible in solution (Schwarten et al, 2010), and a conserved FK-motif (residues 5 and 6) mediates further interactions (Krick et al, 2010). Interestingly, replacement of the FK-motif with glycine almost blocked Atg21-TAP binding from yeast extracts to GST-Atg8 beads (Fig 2B and C). While mutagenesis of the non-conserved NHD residues 3 and 4 (ST) of Atg8 did not affect Atg21-TAP binding, GST-Atg8L50A showed reduced binding. We further coexpressed GFP-Atg8 and Atg21-TAP in atg1∆ pep4∆ cells and immunoprecipitated GFP-Atg8 with a GFP-binding protein on beads. GFP-Atg8F5G/K6G and GFP-Atg8L50A coprecipitated less Atg21-TAP than GFP-Atg8 (Fig 2D and E). Figure 2. Atg21 interacts with Atg8 In the split-ubiquitin system, the N-terminal ubiquitin domain (Nub) is fused to the bait and its C-terminal part (Cub) to the prey. Protein interaction restores ubiquitin and induces degradation of a destabilised Ura3 variant, which was attached to the Cub. Cells were diluted in tenfold steps and spotted on CM –Trp –His (growth control), CM –Trp –His +FOA (growth implies protein interaction) and CM –Trp –His –Ura (growth indicates no interaction). Images were taken after three days at 30°C. Ste14-Cub/Nub-Ubc6: positive control; Ste14-Cub/pRS314: negative control. GST-Atg8 variants on beads were incubated with extracts from atg21∆ pep4∆ cells expressing Atg21-TAP. Bound fraction (PD), lysate (L) and supernatant (S) corresponding to 4% of the PD were included. Immunoblots were probed with TAP (top) or GST antibodies (bottom). GST samples were diluted 1:10. Molecular weight markers are in kDa. Experiments from (B) were quantified as detailed in 4. The ratio of bound Atg21-TAP and related lysate was calculated. The ratio for GST-Atg8 was set to 100%. SEM is from three experiments. ns, P > 0.05; ****P < 0.0001 (unpaired two-tailed t-test). Extracts of atg1∆ pep4∆ cells expressing GFP-Atg8 and Atg21-TAP were incubated with GFP-binding protein on beads. The co-immunoprecipitates were immunobloted with TAP (top), pApeI (middle) and GFP antibodies (lower row). Quantification of (D). The bound fraction for GFP-Atg8 was set to 100%. SEM is from four experiments. ns, P > 0.05; ****P < 0.0001 (unpaired two-tailed t-test). Download figure Download PowerPoint Since crude extracts were used, the Atg21–Atg8 interaction could depend on further components. As shown in Fig 3A and B, the Atg12~Atg5/Atg16 complex, the Atg21 homologue Atg18 and the cargo receptor Atg19 do not mediate interaction of Atg21 with Atg8. To prove a direct interaction, we used recombinant E. coli proteins. We generated a SUMO-Atg21 fusion, which was isolated with Ni2+ beads via a His6-tag (Supplementary Fig S1). Its ability to pull down pApeI and cherry-Atg19 from yeast extracts showed its functionality (Supplementary Fig S2). Binding of E. coli-expressed GST-Atg8 to SUMO-Atg21 carrying beads (Fig 3C and D) and of SUMO-Atg21 to GST-Atg8 on beads (Supplementary Fig S3) confirmed direct interaction. Interaction between the recombinant proteins strongly required the FK-motif, but not the ST-motif in the Atg8-NHD. In contrast to the experiments with yeast extracts, GST-Atg8L50A bound almost normally to SUMO-Atg21 (Fig 3C and D). This suggests that direct interaction between Atg21 and Atg8 mainly depends on the FK-motif of the Atg8-NHD, but not the AIM-binding site of Atg8 via leucine 50. According to the binding of Atg19 and pApeI (Supplementary Fig S2), we expect that from yeast extracts, additional components stabilize the interaction of Atg21 with Atg8 via Atg8 leucine 50. Figure 3. Atg21 directly interacts with the FK-motif of Atg8, but not with its AIM-binding site A. To test whether the interaction of Atg21 with Atg8 depends on other proteins, GST-Atg8 on beads was incubated with extracts from BY4741 deletion strains. PD: bound fraction; lysate (L) and supernatant (S). Immunoblots were with TAP (top) or GST antibodies (lower row). B. Quantification of (A). Again, the ratio of bound Atg21-TAP and related lysate was normalised to wild-type. C. Sepharose-bound SUMO-Atg21 was incubated with recombinant GST-Atg8 variants. Samples of the load (L), supernatant (S), wash (W) and bound fraction (PD) are immunobloted with GST (top) or His-tag antibodies (bottom). D. Quantification of (C). Normalised ratios of bound and lysates are shown. ns, P > 0.05; ****P < 0.0001 (unpaired two-tailed t-test). E, F. Logarithmic (OD600 1.5) atg8Δ atg1∆ cells expressing GFP-Atg8F5G/K6G showed significantly reduced PAS puncta/cell. Fluorescence microscopy (E) and quantification (F) are shown. ns, P > 0.05; ****P < 0.0001 (unpaired two-tailed t-test). G, H. Expression of Atg8F5G/K6G affected pApeI maturation. Cell extracts are immunoblotted with anti-pApeI (top) and anti-Pgk1 (3-phosphoglycerate kinase). Percentage of mature ApeI was calculated and set to 100% for Atg8. I, J. atg8∆ cells expressing Atg8 variants and the mitochondrial marker mito-GFP were grown on lactate medium, and mitophagy was induced with rapamycin. Samples were taken and after immunoblotting with GFP antibodies the level of free GFP after 6 h was quantified (J). The GFP level corresponds to the mitophagy rate and was set to 100% for Atg8. Molecular weights are in kDa. Data information: Error bars are SEM. Statistical relevance was determined by unpaired two-tailed t-test. ns, not significant P > 0.05; *P < 0.05; ***P < 0.001; ****P < 0.0001. Download figure Download PowerPoint Disturbed interaction of Atg8 with Atg21 should affect PAS recruitment of Atg8. Indeed, in growing cells, the number of GFP-Atg8F5G/K6G puncta per cell was significantly reduced (Fig 3E and F). This reduction is comparable to that of GFP-Atg8L50A, which has defects in interactions with WxxL-containing Atg proteins (Noda et al, 2010). In line with the normal PAS recruitment of Atg21 in atg8∆ cells (Fig 1B), the Atg8 variants had little effect on Atg21 localisation (Atg8: 3.45 ± 0.14 Atg21 puncta/cell; Atg8F5G/K6G: 3.51 ± 0.13; Atg8S3A/T4A: 3.69 ± 0.17; Atg8L50A: 3.9 ± 0.16). Expression of Atg8F5G/K6G affected pApeI maturation slightly but significantly (Fig 3G and H). Atg21 is required for selective autophagic subtypes. Indeed, the effect of Atg8F5G/K6G on selective rapamycin-induced mitophagy was severe (Fig 3I and J). To analyse the importance of PI3P binding by Atg21, we included PI3P-binding-defective Atg21-FTTG (Fig 5A and B). Cherry-Atg21FTTG almost completely failed to form puncta confirming that Atg21 PAS recruitment depends on PI3P. Compared to Atg21, cherry-Atg21FTTG significantly reduced the number of Atg8 puncta/cell similar to atg21∆ cells. This shows that the PI3P binding of Atg21 is required for normal formation of Atg8 puncta. Previous studies suggested an almost complete lack of Atg8 puncta in atg21∆ cells. The detection of residual Atg8 puncta in atg21∆ cells is due to our sensitive microscope. Additionally, we determined colocalisation of GFP-Atg8 with pApeI-RFP (Supplementary Fig S4) in cells expressing Atg21 or Atg21FTTG. The significant (P < 0.001) difference further confirms requirement of Atg21 PI3P binding for efficient PAS recruitment of Atg8. As expected, Atg21FTTG expression caused defects in the Cvt pathway and Atg8 lipidation (Fig 5C–E). Loop 2D to 3A on the top side of Atg21 contributes to the interaction with Atg8 Yeast PROPPINs are non-velcro closed β-propellers. The four antiparallel β-sheets of each blade are denoted A to D, from the in- to the outside (Fig 4A–C). The propeller top is typically smaller than the bottom and exposes loops connecting D with A strands of the next blade (Stirnimann et al, 2010; Krick et al, 2012). Interestingly, a functionally diverse set of WD40 β-propellers, among them components of ubiquitin E3 ligases, binds ubiquitin via their top region (Fig 4D) (Pashkova et al, 2010). To identify the Atg21 domain involved in interaction with ubiquitin-like Atg8, we used synthetic 15 amino acid peptides corresponding to the Atg21 loops of the top. For longer loops, peptides overlapping for five amino acids were synthesised. SUMO-Atg21 on beads was incubated with GST-Atg8 and these peptides. The peptide CEIVFPHEIVDVVMN (Atg21 amino acids 136–150, connecting strand 2D with 3A), but no other peptide caused a significant reduction in GST-Atg8 binding to SUMO-Atg21 (Fig 4F and G). Interestingly, in Atg18, the beginning of this loop has been identified to mediate interaction with Atg2 (Fig 4H), (Rieter et al, 2013). Sequence alignment showed that D146 of this loop is conserved among Atg21, but not Atg18 orthologues from different yeasts (Fig 4H). D146 is at the beginning of blade A at the entry of the central β-propeller channel, a site often involved in protein interactions (Stirnimann et al, 2010), (Fig 4A–C). We next replaced D146 in peptides by alanine or lysine. The D146K peptide showed a significantly reduced competition in GST-Atg8 binding (Fig 4I and J). In parallel, other residues of the peptide were consecutively replaced by alanine. Due to problems with synthesis, the P141A peptide was omitted. Only replacement of the non-conserved E143 caused some loss of competition (Fig 4I and J). To verify the relevance of these residues, we coexpressed cherry-Atg21 variants and GFP-Atg8 in atg8∆ atg21∆ atg1∆ cells. Lack of the serine/threonine kinase Atg1 leads to Atg8 accumulation at the PAS. Cells expressing Atg21 had 1.29 Atg8 puncta/cell, Atg21D146K reduced this to 0.845 (P < 0.0001), Atg21FTTG yielded 0.54, and atg21∆ yielded 0.497. Additional replacement of E143 with K had no significant effect (Fig 5A and B). We conclude that D146K of Atg21 is significantly involved in recruiting Atg8 to the PAS, but that additional interactions must exist. Maturation of pApeI (Fig 5C and D) and Atg8 lipidation (Fig 5E) was not obviously reduced by Atg21D146K. Most likely, the residual PAS recruitment of Atg8 is sufficient for these processes. Figure 4. Interaction of Atg21 with Atg8 depends on D146 on the top of the Atg21 β-propeller A–C. Cartoons based on the homologous K. lactis Hsv2 structure. The two phosphoinositide-binding sites are in green. They indicate the orientation upon membrane binding. In the top view (A), the position equivalent to D146 of Atg21 is marked red. (B) Side view, the membrane plane would be at the bottom. (C) The propeller top is typically smaller than the bottom. D. Interaction of ubiquitin with the WD40 β-propeller of Doa1 (Pashkova et al, 2010). E. Atg3 and the cargo receptor Atg19 contain a WxxL-like AIM-motif (Atg8 interacting motif), which mediates interaction with Atg8 (Noda et al, 2008). Orientation of Atg8 similar to ubiquitin in (D) indicates that the AIM of Atg19 (red) would be opposed to the Atg8 region interacting with the propeller. Part of the AIM-binding site of Atg8 is in yellow. Pictures were made with PyMOL (Schrodinger, 2010). F, G. In competition experiments, GST-Atg8 was incubated with peptides and added to Sepharose-bound His6-SUMO-Atg21. After bead sedimentation, supernatant (S) and bound (PD) fractions were immunoblotted with GST (top) or His-tag antibodies (bottom). Samples of the GST-Atg8 peptide mixture (L) were included. (L) and (S) correspond to 8% of the (PD). Quantification of three experiments is shown in (G). Ratios of bound and lysates were normalised to the mock-treated sample. Peptide_10 corresponding to loop 2D to 3A affected binding. H–J. Sequence alignment of loop 2D to 3A of Atg18 and Atg21 orthologues with Jalview (Waterhouse et al, 2009). Conserved residues are coloured; the binding site of Atg18 for Atg2 is indicated on top as open box. As in (F), effects of amino acid substitutions in peptide_10 on competition were analysed by immunoblots (I) and quantified (J). Molecular weight is in kDa. Data information: Error bars are SEM. Statistical relevance was determined by unpaired two-tailed t-test. ns, not significant P > 0.05; *P < 0.05; ****P < 0.0001. Download figure Download PowerPoint Figure 5. Relevance of Atg21 mutants A, B. Fluorescence microscopy of growing (OD600 1.5) atg1∆ atg8Δ atg21∆ cells expressing cherry-Atg21 and GFP-Atg8. Cherry-Atg21D146K significantly reduced the number of Atg8 puncta/cell (B). The PI3P-binding-deficient Atg21FTTG reduced the Atg8 puncta/cell comparable to the absence of Atg21. C–E. pApeI maturation (C, D) and Atg8 lipidation (E) are affected by Atg21FTTG. Percentages of mApeI were calculated and those for Atg21 set to 100%. Data information: Error bars are SEM. Statistical relevance was determined by unpaired two-tailed t-test. ns, not significant P > 0.05; *P < 0.05; ****P < 0.0001. Download figure Download PowerPoint Atg21 interacts with Atg16 In atg8∆ cells, the otherwise faint PAS pool of Atg5 and Atg16 is enhanced (Stromhaug et al, 2004; Suzuki et al, 2007). We analysed Atg5-YFP and Atg16-GFP in atg8∆ and atg8∆ atg21∆ cells. As published, in growing atg21∆ atg8∆ cells, colocalisation of Atg5-YFP with pApeI-RFP was reduced (Fig 6A and C), while Atg12~Atg5 conjugation was unaffected (Stromhaug et al, 2004). We now found that also PAS recruitment of Atg16-GFP requires Atg21 (Fig 6B and C). Does Atg21 recruit Atg16 or the Atg12-Atg5 conjugate? In growing atg8∆ atg21∆ cells of our background, the number of Atg16-GFP puncta/cell (Fig 6D and E) and the colocalisation rate of Atg16-GFP with cherry-Atg21 (Fig 6D and F) did not significantly change in the absence of Atg5. In contrast, ATG16 deletion decreased the number of Atg5 puncta/cell drastically (Fig 6G and H). Since Atg12~Atg5 conjugation occurs in atg16∆ cells (Mizushima et al, 1999), our data suggest that Atg21 specifically recruits Atg16. Compared to Atg21, the number of Atg16-GFP puncta/cell was significantly reduced with the PI3P-binding-deficient mutant Atg21FTTG (Fig 6I). This shows that PAS recruitment of Atg16 depends on the ability of Atg21 to bind PI3P. Figure 6. Atg21 affects PAS recruitment of Atg5 and Atg16 in growing cells A–C. Fluorescence microscopy of growing cells expressing the PAS marker pApeI-RFP and Atg5-YFP (A) or Atg16-GFP (B). The percentage of pApeI-RFP-positive cells showing a perivacuolar Atg5-YFP or Atg16-GFP puncta was determined (C). D–F. Fluorescence microscopy of growing atg8∆ atg21∆ and atg5∆ atg8∆ atg21∆ cells expressing Atg16-GFP and cherry-Atg21. Quantification of (D) showed that lack of Atg5 did not affect the number of Atg16-GFP puncta/cell (E) and the colocalisation rate of Atg16-GFP and cherry-Atg21 (F). G, H. Fluorescence microscopy of atg8∆ atg21∆ and atg8∆ atg21∆ atg16∆ cells expressing Atg5-YFP and cherry-Atg21 showed that lack of Atg16 significantly reduced the number of Atg5-YFP puncta/cell (H). I. Quantification of fluorescence microscopy of atg8∆ atg16∆ atg2∆ cells expressing Atg16-GFP and Atg21-TAP variants. Expression of PI3P-binding-deficient Atg21FTTG-TAP reduced Atg16 puncta/cell similar to lack of Atg21. This shows the importance of PI3P binding of Atg21 in recruiting Atg16 to the PAS. Data information: Error bars are SEM. Statistical relevance was determined by unpaired two-tailed t-test. ns, not significant P > 0.05; *P < 0.05; ***P < 0.001; ****P < 0.0001. Download figure Download PowerPoint Atg16 interacts with the Atg12-Atg5 conjugate via Atg5, and this transfers Atg12 in an open conformation able to bind and activate Atg3 (Romanov et al, 2012; Noda et al, 2013). Accordingly, 2-hybrid analyses indicated interaction of Atg16 with Atg12 in wild-type, but not in atg5∆ cells (Mizushima et al, 1999). Indeed, with the split-ubiquitin system, we found interaction between Atg16 and Atg5 (Fig 7A, row 10,14). The interaction between Atg16 and Atg12 was weaker (Fig 7A, row 11) and absent in atg5∆ cells (row 15). Nub-Atg21 showed strong interaction with Atg16-Cub (row 12), which was only slightly reduced in atg5∆ cells (row 16), suggesting interaction of Atg21 with Atg16 without Atg12~Atg5. Also, Atg21-Cub showed some interaction with Atg16 (row 6), but not with Atg5 (row 4) or Atg12 (row 5). Figure 7. Atg21 interacts with Atg16 A. In a split-ubiquitin assay, tenfold serial dilutions of cells expressing Cub (bait) and Nub (prey) constructs were spotted on plates with CM –Trp –His (growth control), CM –Trp –His +FOA (growth indicates interaction) and CM –Trp –His –Ura (growth indicates no interaction). Ste14-Cub/Nub-Ubc6: positive control, Ste14-Cub/pRS314: negative control. B, C. Recombinant His6-SUMO-Atg21 and as control His6-SUMO were purified on Ni2+-NTA beads and incubated with extract of growing cells chromosomally expressing Atg16-HA. Samples from the E. coli lysates (E. coli L), cleared lysate (E. coli S), yeast lysates (yeast L), unbound fraction (yeast S) and the purified proteins (PD) were immunoblotted with antibodies against HA and His6. Quantification of bound Atg16 is shown in (C). Error bars are SEM. **P < 0.01(unpaired two-tailed t-test). D. Lysates of growing cells expressing Atg16-HA from the chromosome and GFP-Atg21 or GFP from plasmids were immunoprecipitated with beads carrying GFP-binding protein. Samples from the cell lysate (L), the supernatant (S) and the purified proteins (PD) were analysed by immunoblotting using HA and GFP antibodies. Download figure Download PowerPoint To biochemically confirm the interaction of Atg21 with Atg16, we incubated recombinant SUMO-Atg21 on beads with extracts from yeast cells chromosomally expressing Atg16-HA from its endogenous promoter. Atg16-HA bound to SUMO-Atg21, but not to SUMO (Fig 7B, lane 3, 6, and C). We further used extracts from yeast cells
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