Structure and function of yeast Atg20, a that facilitates autophagy induction

Hana Popelkaa, Alejandro Damasiob, Jenny E. Hinshawc, Daniel J. Klionskya,d,1, and Michael J. Ragusab,1

aLife Sciences Institute, University of Michigan, Ann Arbor, MI 48109; bDepartment of Chemistry, Dartmouth College, Hanover, NH 03755; cLaboratory of Cell and Molecular Biology, National Institutes of Health, Bethesda, MD 20892; and dDepartment of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, MI 48109

Edited by Jennifer Lippincott-Schwartz, Howard Hughes Medical Institute, and approved October 16, 2017 (received for review May 19, 2017) The Atg20 and Snx4/Atg24 have been identified in a screen Snx4 is involved in retrieval of late-Golgi SNAREs and has a for mutants defective in a type of selective macroautophagy/auto- mammalian homolog, SNX4, thought to function in endocytosis phagy. Both proteins are connected to the Atg1 kinase complex, which and intracellular trafficking (8). Homologs of Atg20 exist in fungi is involved in autophagy initiation, and bind phosphatidylinositol-3- from Saccharomyces cerevisiae to Schizosaccharomyces pombe; phosphate. Atg20 and Snx4 contain putative BAR domains, suggesting however, a human sequence-based homolog of Atg20 has not a possible role in membrane deformation, but they have been been identified, although optineurin (OPTN) has been proposed relatively uncharacterized. Here we demonstrate that, in addition as a functional counterpart (9) and SNX30 has been proposed as a to its function in selective autophagy, Atg20 plays a critical role in mammalian equivalent based on its dimerization pattern and the efficient induction of nonselective autophagy. Atg20 is a phylogenetic tree (10–12). The function of Atg20 is unknown, dynamic posttranslationally modified that engages both except that it is important for an efficient Cvt pathway, for the structurally stable (PX and BAR) and intrinsically disordered domains degradation of the peroxisomal thiolase enzyme Pot1/Fox3 during for its function. In addition to its PX and BAR domains, Atg20 uses a pexophagy (6), and for clearance of accumulated mitochondria third membrane-binding module, a membrane-inducible amphi- during mitophagy (13). pathic helix present in a previously undescribed location in Atg20 In the present study, we probed the structure and function of within the putative BAR domain. Taken together, these findings the least explored subunit of the Atg1 complex, Atg20. We dem- yield insights into the molecular mechanism of the autophagy onstrate a facilitating role for this protein in autophagy induction, machinery. which requires a hybrid native conformation composed of struc- tured domains mixed with intrinsically disordered regions. We also autophagy | vacuole | yeast demonstrate that Atg20 forms a heterodimer with Snx4 in vitro, and characterize this complex using analytical ultracentrifugation ealthy cells maintain homeostasis via a vital self-cleaning (AUC) and small-angle X-ray scattering (SAXS). These results Hmechanism, macroautophagy (hereinafter autophagy), that show how Atg20 uses distinct regions, including a unique gapped is conserved from yeast to mammals. Autophagy involves the BAR domain, for optimal function. sequestration of cargo by a double-membrane compartment, the phagophore, which expands and seals to form an autophago- Results some. Cargo selection distinguishes nonselective from selective Atg20 Facilitates Autophagy Induction. Previous studies concluded autophagy. Nonselective autophagy engulfs random cytoplasm that Atg20 is not required for nonselective autophagy, because the Δ during starvation, whereas selective autophagy targets specific budding yeast, S. cerevisiae, atg20 strain showed only a minor cargo (e.g., mitochondria, peroxisomes, vacuolar hydrolases) for transport to the vacuole/lysosome (1–3). Each of these processes Significance involves what has been termed the “core” autophagy machinery, proteins that are required for both nonselective and selective Autophagy is a cellular process that results in the capture of autophagy. Many of the core autophagy machinery proteins have cytosolic material in double-membrane vesicles, which sub- conserved homologs from yeast to mammals. sequently fuse with lysosomes to degrade the captured con- Forty-one autophagy-related (Atg) proteins have been identi- tents. Autophagy is essential to maintain cellular homeostasis, fied in fungi, many of which have been categorized into functional respond to cellular stress, and prevent the accumulation of groups (3, 4). The first functional group of Atg proteins assembles material that could damage the cell. The initiation of auto- into the induction complex, also termed the Atg1 complex, to phagy is carried out by the Atg1 complex. Whereas recent initiate autophagy. In budding yeast, this complex for the non- work has provided functional and mechanistic insight into selective autophagy pathway is composed of Atg1, Atg13, Atg17, many components of the Atg1 complex, one member of this Atg29, and Atg31. In mammals, the homologous ULK1 complex complex—Atg20—has remained relatively uncharacterized. is composed of ULK1 (or its homolog ULK2), ATG13, RB1CC1/ Here we report a detailed investigation into the structure and FIP200, and ATG101 (5). Although the stable Atg17-Atg31-Atg29 function of Atg20, including the identification of an amphi- subcomplex is required for efficient nonselective autophagy, it is pathic helix in Atg20 that is required for efficient autophagy not needed for selective autophagic processes. Instead, this sub- and membrane tubulation. complex is replaced by Atg11, which also binds Atg1 and assem- Author contributions: H.P., J.E.H., D.J.K., and M.J.R. designed research; H.P., A.D., and bles along with Snx4 (also termed Atg24) and Atg20. The latter J.E.H. performed research; H.P., A.D., J.E.H., D.J.K., and M.J.R. analyzed data; and H.P., two proteins are sorting nexins containing a PX domain that binds A.D., J.E.H., D.J.K., and M.J.R. wrote the paper. membranes enriched in phosphatidylinositol-3-phosphate (PtdIns3P), The authors declare no conflict of interest. thereby providing a functional connection between the Atg1 complex This article is a PNAS Direct Submission. and the PtdIns 3-kinase complex that also plays a critical role in Published under the PNAS license. autophagy induction (6, 7). 1To whom correspondence may be addressed. Email: [email protected] or michael.j. Recent structural studies have partially clarified the role of [email protected]. some of the subunits of the Atg1/ULK1 complex; however, Snx4 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. and Atg20 and their role in the Atg1 complex remain unexplored. 1073/pnas.1708367114/-/DCSupplemental.

E10112–E10121 | PNAS | Published online November 7, 2017 www.pnas.org/cgi/doi/10.1073/pnas.1708367114 Downloaded by guest on September 27, 2021 decrease in autophagy activity after 4 h of nitrogen starvation (6). N terminus of Atg20 is disordered. The Atg20[FR] is followed PNAS PLUS Atg20 binds the Atg11 scaffold (2), and a recent report revealed a by the PX domain (residues 160–297), which is connected to a new facilitating function of Atg11 in the autophagy induction BAR domain through a region denoted here as the linker complex (14). This finding led us to revisit the question of whether (residues 298–358). This BAR domain (residues 359–636) has a Atg20 can also function as a facilitator in nonselective autophagy gap in the consensus sequence, which we refer to here as the initiation. To answer this question, we first used the Pho8Δ60 BAR-GAP (residues 487–574) (Fig. 2A). The BAR-GAP is pre- assay to monitor autophagy (15). This assay relies on the cytosolic dicted to be partially disordered, and no similar regions have Pho8Δ60 zymogen, which must be targeted to the vacuole by been observed in any BAR domain structures deposited in the nonselective autophagy to become active. In wild-type (WT) cells, (PDB). Together, the results of the sequence- a substantial increase in Pho8Δ60 activity is observed during ni- analyzing algorithms show that the native Atg20 protein is a trogen starvation (Fig. 1A). In comparison, the SEY6210 atg20Δ member of the PX-BAR domain family of sorting nexins (21), strain showed an ∼30% decrease in Pho8Δ60 activity relative to and is enriched in functionally unassigned intrinsically dis- the WT strain (Fig. 1A), a result that may in fact correspond to the ordered protein regions (IDPRs) at the N terminus and in decrease in activity seen previously (6). To check whether this the BAR-GAP. defect is background-dependent, we carried out the Pho8Δ60 as- IDPRs have unique physiochemical properties that render them say in the W303 background and did not detect a clear defect unable to adopt a well-defined 3-dimensional structure. The bi- upon ATG20 deletion (Fig. 1 and SI Appendix,Fig.S1A). ological function of IDPRs relies on various functional elements The Pho8Δ60 assay relies on colorimetric detection of a cleaved including binding modules, which undergo a disorder-to-order substrate and is typically analyzed at least 3–4 h postinduction. transition upon binding proteins, nucleic acids, or lipids (22–25). Accordingly, a defect manifested early in the process, such as at Depending on how these functional elements are identified from a the stage of autophagy initiation, may be difficult to detect with protein amino acid sequence, the binding modules are termed this assay. Therefore, we analyzed the WT and atg20Δ cells with molecular recognition features (MoRFs) or ANCHOR-based dis- the GFP-Atg8 processing assay. In brief, Atg8 (or GFP-Atg8) lines ordered binding regions (BRs). The bioinformatics tool MoRFPred both sides of the phagophore, and the population on the concave (biomine-ws.ece.ualberta.ca/MoRFpred/index.html)(26)predicts side becomes enclosed within the completed autophagosome. MoRFs, and ANCHOR (anchor.enzim.hu/) (27, 28) predicts Following vacuolar delivery, Atg8 is readily degraded, whereas ANCHOR-based disordered BRs. We applied these tools to the GFP is relatively stable and can be monitored as a measure of S. cerevisiae Atg20 sequence and found 6 MoRFs and 10 predicted autophagy flux (16). In both the SEY6210 and W303 backgrounds, BRs, some of which overlap in the Atg20 protein (Fig. 2A and SI autophagy induction was significantly delayed in atg20Δ cells, with Appendix,Fig.S2). MoRF1/BR1, MoRF2/BR2, and BR3-6 are a clear difference detectable after 1–3 h of nitrogen starvation located at the flexible N terminus; MoRF3, MoRF4, and BR7 map (Fig. 1B and SI Appendix,Fig.S1B). on mobile segments of the PX domain; and BR8 and BR9 flank the To further test whether Atg20 is required for nonselective first segment of the BAR domain; therefore, they exhibit relatively autophagy, we probed the transport of the precursor form of high (SI Appendix,Fig.S2). MoRF5/BR10 aminopeptidase I (prApe1) to the vacuole during nitrogen localizes to the BAR-GAP, and MoRF6isneartheveryCterminus starvation in cells where the Cvt pathway was blocked by VAC8 of Atg20. Of all the MoRFs and BRs located in disordered, poorly deletion (17). As expected, the SEY6210 vac8Δ mutant was conserved segments of Atg20, BR3 is the most highly conserved BR completely blocked for prApe1 maturation before autophagy in- likely involved in protein binding, presumably via formation of a duction, whereas processing to the mature enzyme could be conserved secondary structure element (α-helix or β-sheet). In- readily detected within 0.5 h after shifting to starvation conditions terestingly, BR6 contains several conserved serine or threonine (Fig. 1C). In comparison, the SEY6210 atg20Δ vac8Δ strain residues, and could be a target for an unknown kinase. The exhibited a significant delay (∼80% decrease) in prApe1 matu- Eukaryotic Linear Motif (ELM) database (elm.eu.org) reports the ration after 0.5 h of nitrogen starvation. This defect gradually RTSLS sequence in BR6 as a canonical arginine-containing phos- disappeared with the progression of autophagy. A similar result pho motif mediating a strong interaction with YWHA/14–3-3 was found in the W303 background, with a clear lag in prApe1 proteins. maturation in the W303 atg20Δ vac8Δ cells relative to the W303 vac8Δ strain, although the onset of the starvation-induced trans- Mapping Atg20 Domains Interacting with Atg11 and Snx4. To probe port of prApe1 to the vacuole was delayed compared with the how Atg20 uses its hybrid architecture, we constructed three deletion SEY6210 background (Fig. 1C and SI Appendix, Fig. S1C). Taken mutants—Atg20[ΔFR], Atg20[Δ380–480], and Atg20[Δ533–632]— together, these data reveal that Atg20 is required for the efficient and one site-directed mutant, Atg20[Aroma]. Atg20[ΔFR] induction of nonselective autophagy (Fig. 1 B and C and SI Ap- lacks the FR and exhibits an ∼30% defect in the Cvt pathway, as pendix, Fig. S1 B and C). determined by the prApe1 processing assay (Fig. 2C), but no defect in nonselective autophagy induced by nitrogen starvation Native Atg20 Is Predicted to Have Large Disordered Regions. Atg20 (SI Appendix, Fig. S3 A–C). The Atg20[Δ380–480] mutant lacks contains a PX domain, which recruits the protein to PtdIns3P- the majority of the first consensus sequence for the BAR do- enriched lipid membranes (6); no structure-function information is main, whereas the Atg20[Δ533–632] mutant lacks a part of the available regarding other domains in Atg20. To gain insight into the BAR-GAP, including MoRF5/BR10 and the second BAR domain functional conformation of Atg20, we first applied a multialgorithm consensus sequence. These deletions produce dysfunctional pro- bioinformatics approach that analyzed the amino acid sequence of teins in the Cvt pathway (Fig. 2C), as well as in nonselective

S. cerevisiae Atg20. Protein-protein BLAST (https://blast.ncbi.nlm. autophagy (SI Appendix, Fig. S3 A–C). CELL BIOLOGY nih.gov/Blast.cgi) and two metapredictors of intrinsically disor- To evaluate the importance of flexibility in the FR of Atg20, dered regions (PONDR-FIT and MetaDisorderMD2) (18–20) we constructed Atg20[Aroma] by replacing nine Glu or Lys revealed that Atg20 has a long, disordered N terminus, denoted residues with the aromatic residues of Tyr, Phe, or Trp: K47W, here as the flexible region (FR; residues 1–160) (Fig. 2A). In K52Y, E108W, E123W, E143W, E148F, E149W, K156F, and agreement with this prediction, circular dichroism of the K157W (black asterisks in SI Appendix,Fig.S2). This Atg20[Aroma] recombinant purified Atg20[FR] (Fig. 2B, Left) shows a nega- mutant has a lower propensity for disorder in its FR (Fig. 2D) tive peak at 200 nm, which is typical of disordered regions. The and has a partial defect in the Cvt pathway (Fig. 2C) compared 1D 1H NMR spectra of Atg20[FR] (Fig. 2B, Right) also exhibits with the WT, but no defect in nonselective autophagy (SI Ap- poor dispersion in the amide region, further confirming that the pendix, Fig. S3A). Together, the mutants demonstrate a functional

Popelka et al. PNAS | Published online November 7, 2017 | E10113 Downloaded by guest on September 27, 2021 role of the Atg20 FR in the Cvt pathway and an indispensable A 120 +N –N (4 h) function of the Atg20 BAR in both the Cvt pathway and 100 nonselective autophagy. 80 *** We next investigated how Atg20 binds its partner subunits in the Atg11-Atg20-Snx4 trimer. To map Atg11 and Snx4 BRs on Atg20, 60 we carried out a coimmunoprecipitation (co-IP) analysis with 40 three aforementioned deletion mutants. Atg20 was tagged on the 20 N terminus with protein A (PA), and was used for affinity isolation of HA-tagged Atg11. We found that the Atg20 FR was required 0 Pho8∆60 activity (%) WT atg20∆ for efficient binding to Atg11 (Fig. 3A). The weak interaction Strain: between Atg20[ΔFR] and Atg11 was completely lost when the HA SEY6210 tag on Atg11 was replaced with the larger GFP tag (SI Appendix, Fig. S3D). Atg20 lacking residues 380–480 also nearly failed to B Strain: WT atg20∆ bind HA-tagged Atg11, revealing a second binding site for Atg11 (Fig. 3B), whereas deletion of the C-terminal amino acids 533– –N (h): 01230123 632 had no detectable effect. Taken together, these results suggest GFP-Atg8 that Atg20 encompasses two binding sites for the Atg11 scaffold protein, the Atg20 FR domain and the 380–480 region, each of which is necessary for efficient interaction of the two proteins. GFP Snx4 is also a member of the BAR domain family of proteins, Pgk1 based on the analysis of its amino acid sequence by protein-protein BLAST. Probing the same deletion mutants of Atg20 for binding with Snx4 by co-IP identified the C-terminal region 533–632 as the 100 WT *** atg20∆ *** single binding site for Snx4 (Fig. 3 C and D). In agreement with this 80 finding, an Atg20 peptide encompassing residues 533–640 showed a strong interaction with Snx4 (Fig. 3E). Thus, the region of 60 *** Atg20 encompassed by amino acids 533–640 is both necessary and 40 sufficient for binding Snx4. – 20 Residues 533 632 of Atg20 contain the highly conserved motif 626NLExW (SI Appendix, Figs. S2 and S4). We analyzed the role 0 of this motif in the interaction with Snx4 by mutating L627 and

Free GFP:Total GFP (%) 0123 W630 to alanine (LW/AA). This mutation has no effect on the Nitrogen starvation (h) interaction of Atg20 and Snx4 (SI Appendix, Fig. S5A), but does affect the function of Atg20 in terms of prApe1 maturation (SI Appendix, Fig. S5B). Thus, the 626NLExW motif in Atg20 ap- C pears to play a structural role, perhaps in proper folding of the atg20∆ BAR domain, rather than serving as a binding site for Snx4. Strain: vac8∆ atg11∆ vac8∆ Taken together, the data presented in Fig. 3 A–E reveal a model –N (h): 0 0.5 1 1.5 0 0 0.5 1 1.5 kDa: for the Atg11-Atg20-Snx4 trimer (Fig. 3F). prApe1 55 Ape1 Atg20 Is a Posttranslationally Modified Protein. Protein function, stability, or folding efficiency can be affected by posttranslational Pgk1 modifications (PTMs) (29). To determine whether this mechanism fine-tunes Atg20 function, we applied LC-MS/MS analysis on the partially purified Atg20 protein and searched for PTMs (Fig. 4A). 3 vac8∆ We detected 10 acetylated lysines in Atg20, specifically at residues 22, 75, 218, 226, 277, 372, 502, 532, 590, and 613. These acetyla-

atg20∆ vac8∆ tion sites, confirmed by b and/or y ions (SI Appendix, Figs. S6 and 2 *** S7), are distributed evenly throughout the protein. Of the identi- fied acetylation sites, lysine 218, located in the PX domain, is the most conserved, with 11 of the 12 yeast sequences that are aligned 1 *** containing a lysine in this position (SI Appendix, Figs. S2 and S4). We detected phosphorylation of Atg20 exclusively on IDPRs, in

Ape1:prApe1 ratio agreement with a previous study showing correlation of phos- 0 phorylation with disordered regions (30). Atg20 is phosphorylated 0 0.5 1 1.5 on Ser139, Thr144, Ser145, Ser307, Ser342, Ser343, and Thr517, Nitrogen starvation (h) all of which are previously unreported phosphorylation sites me- diated by unknown kinases. In addition to these sites, our MS Fig. 1. Atg20 is essential for the efficient initiation of bulk autophagy. (A) WT (WLY176) and atg20Δ (HPY063) cells expressing Pho8Δ60 were shifted from nutrient-rich conditions to SD-N medium for 4 h. (B) Autophagy as measured by the GFP-Atg8 processing assay in WT (SEY6210) and atg20Δ vac8Δ (CWY230) strain. The atg11Δ (SEY6210) strain served as a negative (D3Y009) cells. Cells transformed with the plasmid (pRS426) carrying a GFP- control, and Pgk1 served as a loading control. Quantitative evaluation of Atg8 construct were grown in rich selective medium and then starved for 1, western blot data was done with three to four independent experiments 2, and 3 h. The free GFP:total GFP ratio was measured and normalized to carried out in nutrient-rich and nitrogen starvation conditions. Error bars that of WT after 3 h of starvation (100%). (C) Kinetics of prApe1 maturation represent the SD from three to four independent experiments. Statistical in nutrient-rich medium and after the shift to SD-N medium for the in- significance was tested using the unpaired two-tailed Student’s t test: **P < dicated times. The vac8Δ atg20Δ (HPY079) strain was compared with the 0.01; ***P < 0.005.

E10114 | www.pnas.org/cgi/doi/10.1073/pnas.1708367114 Popelka et al. Downloaded by guest on September 27, 2021 Δ PNAS PLUS A BR1 (1-14) BR4 (83-101) BR7 (176-184) MoRF1 (7-12) MoRF4 (250-252) prApe1 processing assay under growing conditions in the atg20 BR2 (29-43) BR5 (115-124) BR8 (376-383) MoRF2 (35, 37-38) MoRF5 (539-545) strain showed that phosphorylation and acetylation of Atg20 are BR3 (63-81) BR6 (128-138) BR9 (468-475) MoRF3 (157-165) MoRF6 (632-636) BR10 (534-550) necessary for an efficient Cvt pathway (Fig. 4B).

1 262912061 792 359 486 57536 6 046 To test the importance of Atg20 PTMs in nonselective auto- BAR- phagy, we examined these plasmid-driven Atg20 variants using the FR PX Linker BAR GAP BAR prApe1 maturation assay in the atg20Δ vac8Δ background. We MoRF1 MoRF2 MoRF3 MoRF4 MoRF5 MoRF6 BR1-2 BR3 BR4-6 BR7 BR8 BR9 BR10 found that the Atg20[10STA] mutant exhibited no significant de- MetaDisorderMD2 PONDR-FIT fect in the induction of autophagy, whereas the Atg20[4KR] mu- 1.0 0.8 tant displayed only a 10% (albeit consistent) defect in this pathway 0.6 relative to the WT (Fig. 4C). Taken together, the data in Fig. 4 0.4 suggest that phosphorylation and acetylation modulate the optimal 0.2 Disorder score 0 ordered disordered conformation of Atg20, which is essential for an efficient Cvt 0 1500200 300 400 00006 pathway, but not for nonselective autophagy induction. A reason for Residue number the differing results, which were also obtained with the Atg20[ΔFR]

) 10,000 – B -1 and Atg20[Aroma] mutants (Fig. 2C and SI Appendix,S3A C), 5,000 could be that PTMs finely tune two binding sites—one of them within 0

x dmol — 2 -5,000 the Atg20 FR (Fig. 3) for interaction with Atg11, which is critical -10,000 for the Cvt pathway, but not for nonselective autophagy. -15,000 Molar ellipticity Intensity (relative) (deg x cm -20,000 Structural Characterization of Atg20. Some sorting nexins both 200 210 220 230 240 250 1086420 Wavelength (nm) 1H (ppm) homodimerize and heterodimerize, whereas others prefer only heterodimers. The Atg20 mammalian equivalent, SNX30, does atg20∆ atg11∆ C ∆380- ∆533- D not form homodimers, as judged by co-IP from HEK-293T cells GFP-Atg20: – WT ∆FR 480 632 Aroma – (11). In agreement with these findings (33), we were able to GFP-Atg20 1.2 produce recombinant Atg20 only in a stable heterodimeric com- prApe1 Atg20[Aroma] 1.0 Atg20 Ape1 plex with Snx4, but not as a homodimer (Fig. 5 and SI Appendix, 0.8 Fig. S11). Heterodimer formation was not dependent on the FR Pgk1 0.6 of Atg20, as Atg20 – -Snx4 – was also able to form a stable 1.0 0.4 156 640 21 423 ** 0.8 *** 0.2 heterodimer. Dynamic light scattering on Atg20156–640-Snx421–423

0.6 PONDR-FIT score 0 revealed a molecular mass of 127.6 kDa, demonstrating that the 0.4 0200400 600 Residue number 0.2 *** *** Ape1:prApe1 *** 0 GFP-Atg20: – WT ∆FR ∆380- ∆533-Aroma 480 632 Lysate IP: PA-Atg20 IP: PA-Atg20 Lysate A HA-Atg11: +++ ++ + B HA-Atg11: Fig. 2. Bioinformatics and biochemical analysis of structured and intrinsically PA-Atg20: – WT ∆FR – WT ∆FR kDa: + +++ ++++ HA-Atg11 250 disordered domains in Atg20. (A) Domain representation of Atg20 that in- 130 PA-Atg20: – WT ∆380-480∆533-632 – WT ∆380-480∆533-632kDa: corporates the results of the protein amino acid sequence analyses by 95 PA-Atg20 HA-Atg11 the PONDR-FIT, MetaDisorderMD2, protein-protein BLAST, MoRFPred, and 72 130

ANCHOR algorithms. (B, Left) Far UV circular dichroism spectrum of the PA-Atg20 95 recombinant purified Atg20[FR]. (B, Right) One-dimensional 1HNMRspectrum C IP: PA-Snx4 Lysate D IP: PA-Snx4 Lysate of Atg20[FR]. (C) Empty vector, the plasmids pCuGFP-Atg20(426), pCuGFP- PA-Snx4: +++ +++ PA-Snx4: GFP-Atg20: – WT ∆FR – WT ∆FR + +++ + +++ Atg20[ΔFR](426), pCuGFP-Atg20[Δ380–480](426), pCuGFP-Atg20[Δ533–632] kDa: GFP-Atg20 Δ 95 (426), and pCuGFP-Atg20[Aroma](426) were transformed into atg20 (D3Y009) GFP-Atg20: – WT ∆380-480∆533-632– WT ∆380-480∆533-632kDa: Δ 130 cells and examined for prApe1 processing in rich selective medium. The atg11 72 GFP-Atg20 PA-Snx4 95 (SEY6210) strain served as a negative control. Quantification of the Ape: PA-Snx4 prApe1 ratio was determined from three independent experiments. Error bars 55 represent SDs. Statistical significance was tested using the unpaired two-tailed E IP: PA-Snx4 Lysate ’ < < PA-Snx4: + +++ + +++ Student s t test: **P 0.05; ***P 0.005. (D) Comparison of PONDR-FIT scores F Atg11 for WT (black) and Atg20[Aroma] (magenta). N GFP-Atg20: – WT ∆533-632533-640 – WT ∆533-632533-640kDa: C 130 GFP-Atg20 Snx4/Atg24 BAR 95 Atg20 72 PX analysis also detected phosphorylation on Ser45 and Ser49, which PA-Snx4 BAR

are targeted by Cdc28/Cdk1 (31), and on Ser363 and Thr365, sites GFP-Atg20 [533-640] 36 recognized by casein kinase 2 (CK2) (32), in agreement with PtdIns3P previous high-throughput studies. The third site reportedly mod- PX ified by CK2, Ser361, was confirmed by peptide mass only (SI Fig. 3. Mapping of the Atg11 and Snx4 binding sites on Atg20. (A and B) Appendix, Figs. S8 and S9). The most conserved phosphorylation Coprecipitation of HA-Atg11 by PA-Atg20. The plasmids pCuPA(424), pCuPA- site is Ser307, with either a serine or a threonine present in this Atg20(424), pCuPA-Atg20[ΔFR](424), pCuPA-Atg20[Δ380–480](424), and position in 10 of the 12 yeast sequences that were aligned (SI pCuPA-Atg20[Δ533–632](424) were transformed into MKO (YCY123) cells

Appendix, Figs. S2 and S4). Ser307 is located in the linker between and coexpressed with a plasmid encoding HA-Atg11 (pCuHA-Atg11; 416) CELL BIOLOGY the PX and BAR domains. under the CUP1 promotor. (C–E) Coprecipitation of GFP-Atg20 by PA- To investigate whether acetylation and phosphorylation of Snx4. The plasmids pCuGFP(426), pCuGFP-Atg20(426), pCuGFP-Atg20[ΔFR] Atg20 are important for its function in vivo, we generated a (426), pCuGFP-Atg20[Δ380–480](426), pCuGFP-Atg20[Δ533–632](426), and multiple-nonphosphorylatable and multiple-nonacetylatable mu- pCuGFP-Atg20[533-640](426) were transformed into MKO (YCY123) cells tant by replacing 10 serines or threonines (Ser45, Ser49, Ser139, and coexpressed under the CUP1 promotor with a plasmid encoding PA-Snx4 (pCuPA-Snx4; 424). For A–E, cells were cultured in SMD, and cell lysates were Thr144, Ser145, Ser342, Ser343, Ser361, Ser363, and Thr365) with prepared and incubated with IgG-Sepharose for affinity purification. The alanine (Atg20[10STA]) and replacing four lysines (Lys226, proteins were separated by SDS/PAGE and detected with the indicated an- Lys277, Lys372, and Lys532) with arginine (Atg20[4KR]), re- tibody. (F) Schematic representation depicting the Atg11-Atg20-Snx4 trimer, spectively (SI Appendix,Fig.S10). Probing these mutants using the based on the results presented in the figure.

Popelka et al. PNAS | Published online November 7, 2017 | E10115 Downloaded by guest on September 27, 2021 S342/S343 Phosphate To generate a model for the Atg20-Snx4 heterodimer, we used A S45/S49 S139/T144/S145 S361/S363/T365 T517 S307 Acetyl SWISS-MODEL to create a homology model for Snx4 based on 1 160 219 262 297 359 486 575 640636 the most similar template, human SNX9. The BAR domains FR PX Linker BAR BAR-GAP BAR from the Snx4 and Atg20 homology models were aligned to the human SNX1 BAR domain dimer (Fig. 5F); the Atg20 PX do-

K226 K502 K613 main model was connected to the Atg20 BAR domain model by K22 K75 K372 K590 K218 K277 K532 a flexible linker. The Atg20-Snx4 homology model was used as a B atg20∆ C atg20∆ vac8∆ atg11∆ starting point for molecular dynamics (MD)-based fitting of +N +N –N (0.5 h) the SAXS data by BilboMD (37). During the MD analysis, the linkers between the end of the PX domains and the start of the :02gtA-PFG – WT 10STA 4KR :02gtA-PFG – WT 10STA4KR – WT 10STA4KR – GFP-Atg20 GFP-Atg20 BAR domains were defined as flexible. The overall best fit to χ = prApe1 prApe1 the experimental data ( 4.88) resulted from an ensemble of Ape1 Ape1 three dimeric structures (Fig. 5 B and G). Ape1:prApe1 0 1 0.5 0.5 Ape1:prApe1 0 0000.51 1 0.9 0 Pgk1 Pgk1 Membrane-Inducible Amphipathic Helix in Atg20. The Atg20 BAR- GAP is a specific sequence separating the BAR domain into two 1.0 N+ N– 1.0 *** segments. The BAR-GAP is predicted to be disordered by two 0.8 0.8 *** *** metapredictors (18, 20) (Fig. 2A) and three unrelated, recently 0.6 *** 0.6 0.4 0.4 0.2 0.2

Ape1:prApe1 ratio *** Ape1:prApe1 ratio 0 0 GFP-Atg20: –WT 10STA 4KR GFP-Atg20: –WT 10STA 4KR A 0.5 B 1000 4.1 0.45 4.0 0.4 100 3.9 Fig. 4. Atg20 is a posttranslationally modified protein. (A) Schematic domain 3.8 0.35 ln I 3.7 representation of Atg20 with all PTM sites that were experimentally detected 0.3 10 3.6 by LC-MS/MS analysis. (B) Functionality of Atg20 variants in the Cvt pathway 0.25 3.5 C(s) 0.0001 0.0003 0.0005 analyzed by the prApe1 maturation assay. The SEY6210 atg20Δ strain was 0.2 1 q2 (Å-2) 0.15 transformed with the plasmids pCuGFP(426), pCuGFP-Atg20(426), pCuGFP- 0.1 Intensity (relative units) 0.1 Atg20[10STA](426), and pCuGFP-Atg20[4KR](426). Cells were cultured in rich 0.05 selective medium. (C) Autophagy induction examined for WT and PTM mutants 0 0.01 02468101214161820 0 0.05 0.1 0.15 0.2 0.25 0.3 of Atg20 using the prApe1 maturation assay. The SEY6210 atg20Δ vac8Δ strain Sedimentation Coefficient (S) q (Å-1) was transformed with the same plasmids as indicated in B. Cells were cultured C0.06 D in rich selective medium and then shifted to SD-N medium for 0.5 h. The atg11Δ 0.05 Y177 BR7 MoRF4 (SEY6210) strain served as a negative control, and Pgk1 served as a loading C 0.04 control. Quantification of the Ape:prApe1 ratio was determined from three α2 independent experiments. Error bars represent SDs. Statistical significance was 0.03 Y180 ’ < P(r) tested using the unpaired two-tailed Student s t test: ***P 0.005. 0.02 N MoRF3 0.01

0 recombinant complex is indeed a heterodimer, as its expec- 0 50 100 150 ted mass is 121.5 kDa. r (Å) We next used AUC and SAXS to investigate the overall shape E BAR-GAP F

of the heterodimer. Both the AUC (Fig. 5A) and SAXS data N Y469 (Fig. 5 B and C) demonstrated an elongated architecture for the I382 L472 F542 F539α5 BR9 MoRF5/BR10 heterodimer, as expected for a PX-BAR dimer. Y385 We attempted to crystalize the purified Atg20-Snx4 hetero- C BR8 dimer, but the dynamic of this complex rendered this G attempt unsuccessful. To obtain an approximate image of the structured domains of Atg20, we used the SWISS-MODEL workspace (swissmodel.expasy.org/interactive) (34–36) and cre- o ated homology models of the Atg20 PX and BAR domains based 90 on the most similar template, human SNX1. SNX1 contains a long, disordered N terminus, as does Atg20, but the PX and BAR domains of SNX1 are more structured, with no BAR-GAP Fig. 5. Structural characterization of Atg20. (A) Sedimentation velocity AUC recorded on the full-length Atg20-Snx4 heterodimer. (B) In-line SEC with SAXS in the consensus sequence (based on the PONDR-FIT profile data recorded on the Atg20156–640-Snx421–423 heterodimer. (Inset) The Guinier and protein BLAST), in contrast to Atg20. The overlap of data region for these data. SAXS data calculated from the Atg20-Snx4 ensemble in Fig. 2A with the homology model of the Atg20 PX domain homology model in F are shown in red. (C) Pair distance distribution function (I161-N297) (Fig. 5D) based on the SNX1 template (PDB ID calculated from the SEC-SAXS data in B.(D and E) Homology modeling of code 2I4K) shows that the second half of MoRF3 and the short structurally stable domains in Atg20. (D) Structural alignment of the crystal structure of the PX domain of SNX1 (gray) with the homology model of the PX MoRF4 map onto flexible loops, and that BR7 maps onto the β domain of Atg20 (blue). (E) Structural alignment of the crystal structure of the beginning of the 2 sheet of the PX domain. The same model BAR domain of SNX1 (gray) with the homology model of the BAR domain of also indicates that the Atg20 PX might have an additional helix Atg20 (blue). The amino acid residues of Atg20 mutated to Glu (Tyr177, (α2) compared with the SNX1 PX, which carries a flexible loop Tyr180, Ile382, Tyr385, Tyr469, Leu472, Phe539, and Phe542) are shown (black). in the homologous position. The homology model of the Atg20 Phe539 and Phe542 are within MoRF5/BR10 that maps on the putative α5helix BAR domain (R360-E632) (Fig. 5E) based on the SNX1 tem- of the BAR-GAP. (F) Atg20-Snx4 homology model of the BAR domain dimer. α Atg20 is shown in blue; Snx4, in red. (G) Atg20-Snx4 ensemble model gener- plate (PDB ID code 4FZS) shows -helical rods that are typical ated using BilboMD. The BAR domains of the different ensemble models are for a BAR domain and that overlap with BAR rods of SNX1; superimposed. The PX domain and linker from each model are shown in a BR8 and BR9 map onto these rods (Fig. 2A). different shade of blue (Atg20) or red (Snx4).

E10116 | www.pnas.org/cgi/doi/10.1073/pnas.1708367114 Popelka et al. Downloaded by guest on September 27, 2021 reported individual predictors (SI Appendix, Fig. S12). In the Amph/ (SI Appendix, Fig. S13B), and human SNX9 PNAS PLUS homology model, the MoRF5/BR10 element within the BAR- (42). In all three of these proteins, the AH is located in an IDPR, GAP maps onto a putative helix, specifically on the α5 helix (Fig. outside the BAR domain consensus sequence, and is detected by 5E). A helical wheel representation (Fig. 6A) indicates that this at least one of the algorithms designed to search for disordered helix may be amphipathic. In fact, out of all predicted MoRFs foldable elements (MoRFs or ANCHOR BRs). The putative AH and BRs in Atg20, MoRF5/BR10 is the only element with a in S. cerevisiae Atg20 exhibits all these features in the compar- predicted amphipathicity. Moreover, the amino acid sequences ative bioinformatics analysis (SI Appendix, Fig. S14), indicating a homologous to S. cerevisiae MoRF5/BR10 in other organisms similar molecular mechanism of function. that express an Atg20 homolog (Kluyveromyces lactis and Can- A typical test for the functionality of a foldable element that dida glabrata) are also predicted to form amphipathic helices forms a membrane-induced AH is to replace one or two residues on its hydrophobic face—which normally becomes buried on AH (AHs) (SI Appendix, Fig. S13A). In general, binding-coupled — AHs can mediate protein–protein interaction (38), but are insertion into the lipid bilayer with negatively charged residue(s). common in BAR proteins (39), where they function as sensors of We hypothesized that if disordered MoRF5/BR10 folds into a functional membrane-sensing AH in vivo, then a mutagenic membrane curvature (40) or promote membrane fission (41). disruption in its hydrophobic face should create a dysfunctional The most well-studied and experimentally well-proven AHs in Atg20 protein. In contrast, such a mutation should have no sig- BAR proteins are those of rat SH3GL2/endophilin A1, fruit fly nificant effect if MoRF5/BR10 does not fold in vivo, and would retain mutagenically added negatively charged residues exposed to a hydrophilic environment. To test our hypothesis, we mutated Phe539 and Phe542 si- A MoRF5 (Atg20): D Atg20: WT F539,542E BR10 (Atg20): Microsomes: –+–+ SPP SPSPS multaneously to glutamic acid. For comparison, we produced 537 kDa: K three double mutants as negative controls, including one in the PX K M Atg20 72 D GA 55 domain (Y177E Y180E) and two in the BAR domain (I382E N S His-Snx4 S L Y385E and Y469E L472E) of Atg20 (Fig. 5 D and E), reasoning 537K-554K Dpm1 28 554 K L that disruption of the PX or BAR domain would yield functionally N V E WT defective proteins, because a structural defect induced by a mu- K F F A V tation in one of these membrane-binding modules might prevent proper binding of the protein to the lipid membrane. The prApe1 B atg20∆ vac8∆ atg11∆ –N (0.5 h) +N processing assay (SI Appendix,Fig.S15A) showed that the Atg20 Y177E I382E Y469E F539E protein mutated in MoRF5/BR10 (F539E F542E) was signifi- GFP-Atg20: – WT Y180E Y385E L472E F542E – GFP-Atg20 cantly defective in the Cvt pathway, as were the mutants disrupting prApe1 the PX and BAR domains. Ape1 To probe the importance of the putative AH in facilitating F539,542E Pgk1 induction of autophagy by Atg20 (Fig. 2), we carried out the prApe1 processing assay in starved atg20Δ vac8Δ cells over- 1.0 expressing plasmid-driven WT Atg20 or the PX, BAR, or AH 0.8 ** 0.6 mutant. Again, all these mutants were largely dysfunctional rel- 0.4 *** ative to the WT (Fig. 6B). 0.2 *** *** Ape1:prApe1 *** We used co-IP to test whether this dysfunction of the 0 F539,542E GFP-Atg20: – WT Y177E I382E Y469E F539E Atg20 mutant in the Cvt pathway (SI Appendix, Fig. Y180E Y385E L472E F542E S15A) and autophagy induction (Fig. 6B) are due to a weakened C No lipid MLVs 1 μm0.4 μm 0.1 μm 1.2 *** or lost interaction with Snx4. Our findings show that neither the PS SPP SPSPS 1.0 F539,542E Y177,180E Atg20 0.8 Atg20 mutant nor the negative control (Atg20 ) 0.6 Snx4/Atg24 was defective in binding to Snx4 (SI Appendix, Fig. S15B), sug- 0.4 SPP SPSPSPS Normalized 0.2 gesting that the Snx4 binding site does not overlap with the pu- Atg20F539,542E liposome tube length 0 tative AH on Atg20, and can be narrowed down to the 546–625 Snx4/Atg24 Atg20-Snx4: WT F539,542E region (between MoRF5 and the 626NLExW motif). Fig. 6. The membrane-induced AH in Atg20. (A) Helical wheel representation To strengthen our bioinformatics analysis and in vivo experi- of the amino acid sequence in Atg20 that corresponds to MoRF5/BR10. Black ments suggesting the existence of the membrane-inducible AH in arrows indicate the double mutation F539E, F542E. Yellow indicates hydro- Atg20, we conducted liposome sedimentation assays with the phobic amino acid residues; green, polar; red, negatively charged; blue, posi- recombinant Atg20-Snx4 or Atg20F539,F542E-Snx4 heterodimer tively charged. (B)Thevac8Δ atg20Δ (HPY079) cells transformed with the against a range of Folch liposome sizes, including multilamellar plasmids {pCuGFP(426), pCuGFP-Atg20(426), pCuGFP-Atg20[Y177E Y180/E] vesicles and unilamellar vesicles with diameters of 1,000, 400, and (426), pCuGFP-Atg20[I382E Y385E](426), pCuGFP-Atg20[Y469E L472E](426), 100 nm. Recombinant protein and liposomes were mixed, in- and pCuGFP-Atg20[F539E F542E](426)} were cultured in rich selective medium and then nitrogen-starved for 0.5 h. Error bars represent SD from three in- cubated for 30 min at room temperature, and subjected to cen- dependent experiments. (C) Liposome sedimentation assay for the recombi- trifugation. The recombinant heterodimers did not pellet in the nant Atg20-Snx4 heterodimer, in which Atg20 was either the WT or F539E absence of lipid; therefore, the presence of protein in the pellet F542E mutant, with Folch liposomes of varying diameter. (D) In vitro re- fractions is a result of lipid binding. The Atg20-Snx4 heterodimer

constitution of yeast microsomes, isolated from SEY6210 atg20Δ snx4Δ cells, bound preferentially to larger vesicles, whereas the heterodimer CELL BIOLOGY with the recombinant purified heterodimer Atg20-Snx4, in which Atg20 was mutated in the Atg20 AH exhibited lower lipid binding than the either WT or mutant including the F539E F542E mutation. Supernatant (S) and WT, as manifested by a weaker band in the pellet fraction (Fig. 6C). pellet (P) were obtained by ultracentrifugation and analyzed by western blot To further confirm a membrane-binding defect of the μ analysis. (E, Upper) Representative negative stain EM images of 1.0- m vesicles Atg20F539,542E mutant relative to the WT, we also carried out in incubated with WT Atg20-Snx4 or mutant Atg20-Snx4 [F539E F542E] hetero- Δ μ vitro reconstitution of yeast microsomes, isolated from atg20 dimer. (Scale bars: 0.2 m.) (E, Lower) Quantification of lipid tube length from Δ grid squares, which are 484 μm2. Data from each trial were normalized to the cells, with the purified full-length Atg20-Snx4 heterodimer tube length from WT. Error bars represent the SD from three independent (Fig. 6D and SI Appendix, Fig. S16). western blot analysis qual- experiments. Statistical significance was tested using the unpaired two-tailed itatively showed that the Atg20-Snx4 heterodimer harboring the Student’s t test: **P < 0.01; ***P < 0.005. mutation in the Atg20 AH failed to efficiently bind to yeast

Popelka et al. PNAS | Published online November 7, 2017 | E10117 Downloaded by guest on September 27, 2021 membranes (i.e., with a physiological composition of lipids) Atg20 are two membrane-binding modules that function in compared with the WT heterodimer. remodeling and sensing of PtdIns3P-enriched membranes (6, 21, Finally, we examined the effect of the AH mutation on remod- 49). The BAR domain of Atg20 is unique in that it is separated into eling of membranes. In many BAR proteins, AHs are important for two segments by an intrinsically disordered region, the BAR-GAP membrane remodeling, specifically for tubulation (11). Protein and (Fig. 2A). Here we present bioinformatics and experimental evi- vesicles were mixed, incubated for 1 h, and applied to EM grids. dence showing that a portion of the BAR-GAP folds into a func- The length of membrane tubes on the grids was measured and tional, membrane-inducible AH (Fig. 6 and SI Appendix,Figs.S12– normalized to the liposome tube length measured from the WT S15). This helix is analogous to AHs found in other BAR proteins Atg20-Snx4 sample. Our negative-stain EM images of membrane (11, 40, 42). Our data reveal a unique position for this type of F539,542E tubulation by the Atg20-Snx4 or Atg20 -Snx4 heterodimer membrane-binding module occurring among sorting nexins. showed that the mutant was far less efficient in membrane remod- Together, the membrane-binding modules of Atg20 (PX, BAR, eling than the WT (Fig. 6E). and AH) have dual functions, operating both in the Cvt pathway – Taken together, our data in Fig. 6 and SI Appendix,Figs.S12 and during the induction of nonselective autophagy (Fig. 7 and SI S15 reveal that Atg20 encompasses a membrane-inducible AH that Appendix,Fig.S15). Based on this duality, it is tempting to spec- arises from a disordered region and is localized within the protein ulate that a membrane source recruited by Atg20 to the Cvt ves- at an as-yet unknown position among sorting nexins, the BAR- icles is also used, at least in part, for the rapid formation of the GAP domain. Along with the PX and BAR domains, this AH in phagophore. This would explain why autophagy induction is Atg20 is critical for protein function in the Cvt pathway and non- delayed in the absence of Atg20 (Fig. 1). It is also possible that the selective autophagy induction. Atg20-Snx4 BAR heterodimer stabilizes a certain curvature of the Discussion growing phagophore, which is why it preferentially binds in vitro to larger vesicles (Fig. 6C). Here we show that Atg20 is a dynamic protein with a hybrid ar- The data presented in this study yield a model of the Atg11- chitecture that combines structurally stable and dynamic domains Atg20-Snx4 heterotrimer interacting with a PtdIns3P-enriched to facilitate the efficiency of nonselective and selective autophagy membrane (Fig. 7). Snx4 dimerizes with Atg20 via binding to the (6), (Fig. 1). This hybrid conformation is modulated by phos- Atg20 BAR domain near the very C terminus (in the region 546– phorylation and acetylation on numerous serines or threonines, as well as lysines. These residues form substrate motifs for mostly 625), in analogy to other BAR heterodimers (50), whereas unknown kinases and acetyltransferases. The exception is the Atg11 has two binding sites on Atg20, the BAR domain in the region 380–480 and the disordered N terminus. Each site is nec- known substrate motif 42GVGSPKK and 46PKKSPKK of the – Cdc28/Cdk1 protein kinase, which phosphorylates Ser45 and essary for an efficient Atg11 Atg20 interaction. The overall func- tion of Atg20 is fine-tuned by phosphorylation and acetylation, and Ser49 (31), and the 362KSFTNIE motif of CK2, which phos- phorylates Ser363 and Thr365 (32). The multiplicity of phos- phorylation and acetylation observed in Atg20 is not unusual, and has been found in other proteins (43, 44) targeted by multisite Atg11 modifications. A proposed reason for the multiplicity of mod- ifications is a robust response to above-threshold stimulatory signals (45). This would indicate that Atg20 might function as a sensor mediating a response to signals aimed at the Atg11- Atg20-Snx4 trimer. N Although the exact purpose and interplay of phosphorylated BAR sites in Atg20 are difficult to elucidate, several aspects can be C C proposed or eliminated. First, an earlier study showed that phos- AH Atg20 phorylation can induce or stabilize α-helices in cases where p-Ser PX or p-Thr at position i in the sequence are preceded or followed by BAR lysines at positions i + 4 or i − 4, respectively (46). Since no p-Ser Snx4/Atg24 or p-Thr in Atg20 is preceded/followed by a lysine at positions i + 4 or i − 4, we can exclude the possibility that Atg20 is phos- phorylated to induce/stabilize an α helix via this mechanism. Phosphate PX Acetyl Second, another study showed that p-Ser and p-Thr at N-terminal N PtdIns3P helix positions stabilize those α-helices (47). Phosphorylation on S363 and T365 in Atg20 maps on the N terminus of the BAR domain consensus sequence; thus, these residues could be phos- phorylated to stabilize the N-terminal α-helical rod in the BAR domain of Atg20. Finally, phosphorylation and acetylation change Fig. 7. Proposed molecular mechanism of Atg20 function depicted by a schematic model of the Atg11-Atg20-Snx4 heterotrimer interacting with the the net charge in proteins; phosphate groups add a negative PtdIns3P-enriched membrane in yeast S. cerevisiae. The PX domain of charge, whereas acetyl groups neutralize existing positive charges Atg20 acts as a lipid-selective module that interacts with PtdIns3P. The BAR on lysines. Accordingly, these two modifications on Atg20 can domain and AH of Atg20 are membrane-sensing and -remodeling modules modulate its overall charge and thereby a degree of compaction, that detect and stabilize a membrane curvature. The lipid-binding modules which increases with decreased net charge (48). Therefore, a are the main targets of acetylation, whereas phosphorylation is located possible purpose of acetylation of lysines in Atg20 could be to gain predominantly in IDPRs of Atg20. Together, these two PTMs modulate the compaction, especially in the Atg20 PX and BAR domains. Be- optimal Atg20 conformation that uses the disordered N terminus (FR do- cause BAR domains bind negatively charged membranes elec- main) and the first segment of the BAR domain (region 380–480) to interact trostatically using lysine and arginine residues (39), acetylated with Atg11. Fine-tuning of Atg20 architecture by PTMs is important for the Cvt pathway, in which Atg11 is a critical component. Snx4 forms the heter- lysines in Atg20 presumably are not the lysines involved in odimer with Atg20 via binding to its second segment of the BAR domain membrane binding. near the very C terminus. The three lipid-binding modules of Atg20 along Atg20 exists only in a heterodimer with Snx4, where both these with the PX and BAR domain of Snx4 recruit the PtdIns3P-enriched mem- sorting nexins adopt an elongated overall conformation, as revealed branes. The rate of this process is critical for the efficient initiation of bulk by our AUC and SAXS data (Fig. 5). The PX and BAR domains in autophagy.

E10118 | www.pnas.org/cgi/doi/10.1073/pnas.1708367114 Popelka et al. Downloaded by guest on September 27, 2021 uses membrane-binding modules (PX, BAR, and AH) to recruit (www.matrixsciences.com) and X! Tandem (www.proteomesoftware.com) PNAS PLUS PtdIns3P-enriched membranes into the forming Cvt vesicles and search engines. The results from the gel segments of each sample were com- phagophore. Thus, a possible purpose of Atg20 in autophagy in- bined and displayed using Scaffold software (www.proteomesoftware.com). duction could be to enhance the efficiency of recruitment of PTMs of interest were specified in the database searches and displayed in the Scaffold file. PtdIns3P-enriched membranes and thereby speed buildup of the phagophore during autophagy initiation and/or stabilization of the Overexpression, Purification, and Characterization of the Recombinant Atg20[FR]. curvature of the growing phagophore. S. cerevisiae Atg20 comprising residues 1–160 was subcloned into pHis2 to Our model of the Atg11-Atg20-Snx4 heterotrimer (Fig. 7) generate Atg20[FR] pHis2. Escherichia coli BL21 (DE3) Star cells (Invitrogen), proposes a molecular mechanism of Atg20 function in budding transformed with Atg20[FR] pHis2, were grown in LB medium at 37 °C to an yeast. At the same time, it raises questions in the field of mam- OD600 of 0.6, induced with 1 mM isopropyl-beta-D-thiogalactopyranoside, and malian autophagy of whether there is an analogous mechanism in grown for 18 h at 18 °C. Cells were pelleted and stored at −80 °C. Cell pellets human cells that facilitates autophagy induction via a membrane- were thawed and resuspended in 50 mM Tris, pH 8.0, 500 mM NaCl, 0.1% μ binding protein and, if so, whether this analogous mechanism is Triton X-100 containing 0.1 mM 4-(2-aminoethyl)benzenesulfonyl and 1 g/mL embedded in the function of the fairly unexplored RB1CC1/ leupeptin. Cells were lysed with three passes through a French press (Thermo Electron). Lysates were cleared by centrifugation and added to TALON resin FIP200 protein. (Clontech). The resin was washed with 50 mM Tris, pH 8.0, 500 mM NaCl, 2.5 mM imidazole, and protein was eluted using 50 mM Tris, pH 8.0, 500 mM Materials and Methods NaCl, 200 mM imidazole. Strains, Media, and Growth Conditions. Deletion strains created for this work Elutions were subjected to ion exchange chromatography using a HiTrap Q were produced as described previously (51). Strains used in this study are listed column (GE Healthcare) and eluted with a gradient from 50 mM NaCl to 1 M in SI Appendix,TableS1. Yeast cells were grown in nutrient-rich medium [YPD; NaCl. Fractions containing protein were pooled and further purified using a 1% (wt/vol) yeast extract, 2% (wt/vol) peptone, 2% (wt/vol) glucose] or syn- HiLoad 16/600 Superdex 75-pg column (GE Healthcare) equilibrated in 20 mM thetic minimal medium [SMD; 0.67% yeast nitrogen base, 2% (wt/vol) glucose, sodium phosphate, pH 6.5, 100 mM NaCl, 0.2 mM Tris(2-carboxyethyl)phosphine and auxotrophic amino acids and vitamins]. Autophagy was induced by (TCEP). For circular dichroism, protein was concentrated to 25 μM. Spectra shifting the cells to nitrogen starvation medium [SD-N; 0.17% nitrogen base were acquired with a 0.1-cm cuvette from 198 to 250 nm at 20 °C using a Jasco without ammonium sulfate or amino acids and 2% (wt/vol) glucose]. J-185 spectrometer. For NMR spectroscopy, protein was concentrated to 1 324 μM, D2O was added to a final concentration of 10%, and 1D Hspectra Plasmids. pCuPA-Atg20(424), pCuGFP-Atg20(426), pCu-PA-Snx4(424) (6), and were recorded on a Bruker UltraShield Plus 600-MHz magnet. pCuHA-Atg11(416) (14, 52) have been reported previously. Other plasmids were generated from pCuPA-Atg20(424) or pCuGFP-Atg20(426) by site- Overexpression and Purification of the Recombinant Full-Length Atg20-Snx4 directed mutagenesis as described previously (53). To generate the plasmid or Atg20F539E, F542E-Snx4 Heterodimer. Full-length S. cerevisiae Snx4 and Snx4 pCuGFP-Atg20[Aroma], the DNA fragment of Atg20[Aroma] that was syn- comprising residues 21–423 were each subcloned into the pET His6 tobacco etch thesized by GeneArt (Life Technologies) was inserted into the ClaI and BglII virus (TEV) ligation-independent cloning vector (1B), a gift from Scott Gradia sites of pCuGFP-Atg20. All mutations were verified by DNA sequencing. (29653; Addgene), to generate Snx4 1B and Snx421–423 1B. His6-TEV-Snx4 from Snx4 1B and full-length S. cerevisiae Atg20 were subcloned into the Precursor Ape1 Processing, GFP Processing, and Enzymatic Assays for Autophagy. polycistronic vector pST39 to generate Atg20-Snx4 pST39 (55). Atg20F539E, The prApe1 processing assay was carried out by culturing the cells from 0.05 F542E-Snx4 pST39 was generated from Atg20-Snx4 pST39 using QuikChange

OD600 units to 0.55 OD600 units in nutrient-rich medium. This allows for mutagenesis. His6-TEV-Snx421–423 from Snx421–423 1B and S. cerevisiae monitoring the Cvt pathway in mid-log phase cells that are actively trans- Atg20 comprising residues 156–640 were subcloned into pST39 to gener-

porting prApe1 to the vacuole. Cells (1 OD600 unit) were harvested, washed ate Atg20156–640-Snx421–423 pST39. E. coli BL21 (DE3) Star cells, transformed with 1 mL of H2O, and analyzed by western blot analysis. For the GFP pro- with the appropriate plasmid, were grown in LB, and protein was expressed cessing assay, cells expressing the GFP-Atg8 construct under the CUP1 pro- using the same protocol as for Atg20[FR] pHis2. Cell pellets were thawed and moter on the plasmid (pRS426) were cultured from 0.25 OD600 units to 1.00 resuspended in 50 mM Tris, pH 8.0, 500 mM NaCl, 0.1% Triton X-100 con- OD600 units in selective nutrient-rich medium, and then washed in 1 mL of H2O taining one Mini Complete EDTA-Free Protease Inhibitor tablet (Roche). Cells and shifted to SD-N medium for 1, 2, or 3 h. The harvested cells were washed were lysed with three passes through a French press (Thermo Electron). Ly- with 1 mL of H2O and subjected to western blot analysis as described sates were cleared by centrifugation and added to TALON resin (Clontech). previously (54). For phosphatase assays, autophagy was induced by Protein was eluted using 50 mM Tris, pH 8.0, 500 mM NaCl, 200 mM imidazole. shifting the cells from nutrient-rich medium to SD-N medium. The har- For Atg20-Snx4 and Atg20F539E, F542E-Snx4, elutions were further purified vested cells were washed in 0.85% NaCl and stored at −80 °C until the using a Hiload 16/60 Superdex 200 prep grade column (GE Healthcare) enzymatic assay. equilibrated in 20 mM Tris, pH 8.0, 200 mM NaCl, 0.2 mM TCEP. For

Atg20156–640-Snx421–423, TEV protease was added to the elutions, the protein Phosphatase Activity. The phosphatase activity of Pho8Δ60 or mitoPho8Δ60 was was cleaved overnight at 4 °C and subjected to ion exchange chromatog- assayed as described previously (15). raphy using a HiTrap SP column (GE Healthcare). Fractions containing

Atg20156–640-Snx421–423 were further purified using a Hiload 16/60 Superdex

Immunoprecipitation. Cells (50 OD600 units) were lysed in 4 mL of lysis buffer 200 prep grade column (GE Healthcare) equilibrated in 20 mM Tris, pH 8.0, [1× PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4), 200 mM NaCl, 0.2 mM TCEP. 0.2 M sorbitol, 1 mM MgCl2, 0.1% Tween-20, 1 mM PMSF inhibitor, Com- plete EDTA-Free Protease Inhibitor (Roche)] with glass beads. After centri- Liposome Sedimentation Assay. Folch fraction type I lipids isolated from bovine fugation at 1,750 × g for 5 min, the resulting supernatant fraction was brain (Sigma-Aldrich) were dried under a nitrogen stream for 1 h and then dried incubated with IgG Sepharose 6 Fast Flow (GE Healthcare) for 2 h at 4 °C. overnight in a vacuum oven. Dried lipids were resuspended in 20 mM, Tris pH After five washes with lysis buffer, the bound proteins were eluted by in- 8.0, 200 mM NaCl, 0.2 mM TCEP to a final concentration of 2.5 mg/mL. Lipids cubating the Sepharose at 55 °C with SDS/PAGE buffer, followed by western were subjected to freeze-thaw using dry ice and then extruded using an Avanti blot analysis with the appropriate antibody. Mini Extruder with the appropriate size membrane. Then 25 μLof3μM purified F539E, F542E μ Atg20-Snx4 or Atg20 -Snx4 was mixed with 25 Lof2.5mg/mLli- CELL BIOLOGY GeLC-Mass Spectrometry. PA affinity-purified Atg20 was resolved by SDS/PAGE, posomes. The mixture was incubated at room temperature for 30 min, fol- and the entire gel lane of the sample was divided into equal segments and lowed by centrifugation at 45,000 rpm for 50 min at 4 °C using a TLA45 rotor. excised. The gel bands were then subjected to in-gel tryptic digestion, followed by LC-MS/MS analysis with detection via collision-induced dissociation. This Negative Stain Electron Microscopy of Membrane Tubulation. For the tubula- allowed for sequencing of the tryptic peptides while keeping the side chain tion assay, 25 μL of protein (WT and F539,542E), at a concentration of 6 μM, modifications intact for straightforward identification and localization of was incubated with 1 μm extruded liposomes of Folch lipid for 60 min at the modification. The analysis was performed on an Orbitrap Velos in- room temperature. The sample was then applied to carbon-coated 400- strument (Thermo Fisher Scientific) coupled to a Waters NanoAcquity liquid mesh Cu/Rh grids for 1 min, followed by two washings and then staining chromatography system. The proteins present in the samples were identi- with 2% uranyl acetate for 30 s. Images were obtained on a T12 transmission

fied by database searches using the SWISS-PROT database as well as Mascot electron microscope (FEI) using low-dose conditions at 120 kV with a LaB6

Popelka et al. PNAS | Published online November 7, 2017 | E10119 Downloaded by guest on September 27, 2021 filament. Images were recorded using a Gatan 2k × 2k CCD camera. Tubu- Microsome Isolation and in Vitro Binding Experiment. To isolate microsomes, 2 lation was quantified by imaging at least four grid squares (484 μm /grid atg20Δ snx4Δ SEY6210 cells (25 OD600 units) were resuspended in resuspension square) for each sample, WT and F539,542E. Samples were normalized to the buffer [50 mM Tris, pH 7.5, 1 mM EDTA, 1 mM PMSF, 1 mM β−mercaptoe- total WT tube length produced. thanol, and Complete EDTA-Free Protease Inhibitor (Roche)], mixed with glass beads, and vortexed four times for 1 min with intervals on ice between vor- × AUC. Atg20156–640-Snx421–423 (50 μM) was subjected to sedimentation velocity texing. The lysate was centrifuged at 5,900 g for 10 min at 4 °C. Supernatant AUC using a Beckman Coulter ProteomeLab XL-A. Centrifugation was per- was split into two ultracentrifuge tubes and ultracentrifuged at 55,000 rpm by formed at 30,000 rpm for 22 h at 20 °C using an AN 60 Ti rotor. SEDNTERP using a TLA 100.4 rotor (Beckman) for 15 min at 4 °C. The pellet was washed in (computer program written by D. B. Hayes, J. P. Philo, and T. M. Laue, 1994) resuspension buffer and ultracentrifuged again at 55,000 rpm for 15 min was used to determine density, viscosity, and specific volume. SEDFIT was used at 4 °C. to analyze raw AUC data and calculate sedimentation coefficient and For in vitro reconstitution of yeast microsomes with the recombinant frictional coefficient. purified Atg20-Snx4 heterodimer, tubes without or with a microsomal pellet

(from cells at 12.5 OD600 units) were incubated for 15 min at room tem- Dynamic Light Scattering. Dynamic light scattering was performed on protein perature with the purified heterodimer (2.5 μM) in the presence of buffer μ at 1 M or liposomes at 2.5 mg/mL at 20 °C using DynaPro NanoStar (Wyatt (50 mM Tris, pH 7.5, 150 mM NaCl, 2.5 mM PMSF, 200 mM MgSO4). Technology). Light scattering data were analyzed using Dynamics v7.1.8 Reconstitution samples were ultracentrifuged at 55,000 rpm for 15 min at (Wyatt Technology). 4 °C. Supernatant and pellet were analyzed by western blot analysis using rabbit anti-Atg20 polyclonal antibody to detect Atg20 and mouse anti- In-Line Size Exclusion Chromatography with SAXS. In-line size exclusion polyhistidine monoclonal antibody (H1029; Sigma-Aldrich) to detect the chromatography (SEC) with SAXS was performed at National Synchrotron His-tag at the N terminus of Snx4. Dpm1 was detected by mouse anti-

Light Source II beamline 16-ID. Atg20156–640-Snx421–423 (3.5 mg/mL) was in- Dpm1 monoclonal antibody (A6429; Life Technologies). jected onto a Superdex 200 Increase 5/150 GL column (GE Healthcare) equilibrated with 20 mM Tris, pH 8.0, 200 mM NaCl, 0.2 mM TCEP. PRIMUS ACKNOWLEDGMENTS. We thank Maria Pellegrini for NMR assistance. This was used to plot scattering data and determine the radius of gyration (Rg) work was supported by National Institutes of Health Grant GM053396 (to 2 2/3 using the Guinier approximation, I(q) = I(0)exp(−q Rg ), where a plot of I(q) D.J.K.) and the Protein Folding Diseases FastForward Initiative, University 2 of Michigan. M.J.R. is supported by National Institutes of Health Grant and q is linear for q < 1.3/Rg (56). The linearity of the Guinier plot was used to confirm that no aggregation was present in the sample. GNOM was used GM113132. J.E.H. is supported by National Institute of Diabetes and Digestive and Kidney Diseases Intramural Research Program. SAXS data were collected to determine the pair distribution function, P(r), and maximum particle di- at the life science X-ray scattering (LiX) beamline at National Synchrotron Light mension, D (57). A starting model of Atg20 – -Snx4 – for MD was max 156 640 21 423 Source II (NSLSII). LiX operates under Department of Energy (DOE) Biological generated using SWISS-MODEL. BilboMD was used to generate 9,500 dif- and Environmental Research Contract DE-SC0012704 and is supported by ferent conformations of Atg20156–640-Snx421–423, with the PX and BAR do- National Institutes of Health–National Institute of General Medical Sciences mains defined as rigid bodies, while regions between the PX and BAR Grant P41GM111244. NSLSII is operated under DOE Basic Energy Sciences domains (37) were defined as flexible. Contract DE-SC0012704.

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