title: Phosphorylation of Atg31 is required for autophagy.
abstract: Autophagy is an evolutionarily conserved cellular process which degrades intracellular contents. The Atg17-Atg31-Atg29 complex plays a key role in autophagy induction by various stimuli. In yeast, autophagy occurs with autophagosome formation at a special site near the vacuole named the pre-autophagosomal structure (PAS). The Atg17-Atg31-Atg29 complex forms a scaffold for PAS organization, and recruits other autophagy-related (Atg) proteins to the PAS. Here, we show that Atg31 is a phosphorylated protein. The phosphorylation sites on Atg31 were identified by mass spectrometry. Analysis of mutants in which the phosphorylated amino acids were replaced by alanine, either individually or in various combinations, identified S174 as the functional phosphorylation site. An S174A mutant showed a similar degree of autophagy impairment as an Atg31 deletion mutant. S174 phosphorylation is required for autophagy induced by various autophagy stimuli such as nitrogen starvation and rapamycin treatment. Mass spectrometry analysis showed that S174 is phosphorylated constitutively, and expression of a phosphorylation-mimic mutant (S174D) in the Atg31 deletion strain restores autophagy. In the S174A mutant, Atg9-positive vesicles accumulate at the PAS. Thus, S174 phosphorylation is required for formation of autophagosomes, possibly by facilitating the recycling of Atg9 from the PAS. Our data demonstrate the role of phosphorylation of Atg31 in autophagy.
authors.affiliation: PTN Program, College of Life Science, Peking University, Beijing, 100871, China.
authors.forename: Wenzhi
authors.initials: W
authors.lastname: Feng
authors.forename: Tong
authors.initials: T
authors.lastname: Wu
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Dan
authors.forename: Yuling
authors.initials: Y
authors.lastname: Chen
authors.forename: Lin
authors.initials: L
authors.lastname: Li
authors.forename: She
authors.initials: S
authors.lastname: Chen
authors.forename: Di
authors.initials: D
authors.lastname: Miao
authors.forename: Haiteng
authors.initials: H
authors.lastname: Deng
authors.forename: Xinqi
authors.initials: X
authors.lastname: Gong
authors.forename: Li
authors.initials: L
authors.lastname: Yu
mesh_terms: D000409:Alanine / Q000737:chemistry / Q000378:metabolism; D020816:Amino Acid Motifs; D001224:Aspartic Acid / Q000737:chemistry / Q000378:metabolism; D001343:Autophagy / Q000235:genetics*; D000071183:Autophagy-Related Proteins; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000172:deficiency; D010588:Phagosomes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D010766:Phosphorylation; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction; D020123:Sirolimus / Q000494:pharmacology
chemical_list: C506770:ATG29 protein, S cerevisiae; C434065:ATG9 protein, S cerevisiae; C505602:Atg17 protein, S cerevisiae; C520043:Atg31 protein, S cerevisiae; D000071183:Autophagy-Related Proteins; D002352:Carrier Proteins; D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D001224:Aspartic Acid; D012694:Serine; D009584:Nitrogen; D000409:Alanine; D020123:Sirolimus