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Structure and Function of Yeast Atg20, a Sorting Nexin That Facilitates Autophagy Induction

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Structure and Function of Yeast Atg20, a Sorting Nexin That Facilitates Autophagy Induction Structure and function of yeast Atg20, a sorting nexin 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 proteins 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 protein 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, Protein Data Bank (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
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