title: Molecular interactions of the Saccharomyces cerevisiae Atg1 complex provide insights into assembly and regulatory mechanisms.
abstract: The Atg1 complex, which contains 5 major subunits: Atg1, Atg13, Atg17, Atg29, and Atg31, regulates the induction of autophagy and autophagosome formation. To gain a better understanding of the overall architecture and assembly mechanism of this essential autophagy regulatory complex, we have reconstituted a core assembly of the Saccharomyces cerevisiae Atg1 complex composed of full-length Atg17, Atg29, and Atg31, along with the C-terminal domains of Atg1 (Atg1[CTD]) and Atg13 (Atg13[CTD]). Using chemical-crosslinking coupled with mass spectrometry (CXMS) analysis we systematically mapped the intersubunit interaction interfaces within this complex. Our data revealed that the intrinsically unstructured C-terminal domain of Atg29 interacts directly with Atg17, whereas Atg17 interacts with Atg13 in 2 distinct intrinsically unstructured regions, including a previously unknown motif that encompasses several putative phosphorylation sites. The Atg1[CTD] crosslinks exclusively to the Atg13[CTD] and does not appear to make direct contact with the Atg17-Atg31-Atg29 scaffold. Finally, single-particle electron microscopy analysis revealed that both the Atg13[CTD] and Atg1[CTD] localize to the tip regions of Atg17-Atg31-Atg29 and do not alter the distinct curvature of this scaffolding subcomplex. This work provides a comprehensive understanding of the subunit interactions in the fully assembled Atg1 core complex, and uncovers the potential role of intrinsically disordered regions in regulating complex integrity.
authors.affiliation: a Department of Biochemistry and Molecular Biology ; University of British Columbia ; Vancouver , BC Canada.
authors.forename: Leon H
authors.initials: LH
authors.lastname: Chew
authors.forename: Shan
authors.initials: S
authors.lastname: Lu
authors.forename: Xu
authors.initials: X
authors.lastname: Liu
authors.forename: Franco Kk
authors.initials: FK
authors.lastname: Li
authors.forename: Angela Yh
authors.initials: AY
authors.lastname: Yu
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Klionsky
authors.forename: Meng-Qiu
authors.initials: MQ
authors.lastname: Dong
authors.forename: Calvin K
authors.initials: CK
authors.lastname: Yip
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D001343:Autophagy / Q000502:physiology*; D000071183:Autophagy-Related Proteins; D002352:Carrier Proteins / Q000378:metabolism; D010588:Phagosomes / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000071183:Autophagy-Related Proteins; D002352:Carrier Proteins; D029701:Saccharomyces cerevisiae Proteins; D011494:Protein Kinases; C477823:ATG1 protein, S cerevisiae
keywords: Atg, autophagy related; Atg1; CTD, C-terminal domain; CXMS, chemical-crosslinking coupled with mass spectrometry; Cvt, cytoplasm-to-vacuole targeting; FDR, false-discovery rate; IDR, intrinsically disordered region; PAS, phagophore assembly site; PtdIns3K, phosphatidylinositol 3-kinase; Saccharomyces cerevisiae; autophagy; crosslinking mass spectrometry; electron microscopy; macromolecular assembly