Molecular interactions of the Saccharomyces cerevisiae Atg1 complex provide insights into assembly and regulatory mechanisms |
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Authors: | Leon H Chew Shan Lu Xu Liu Franco Kk Li Angela Yh Yu Daniel J Klionsky Meng-Qiu Dong Calvin K Yip |
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Institution: | 1.Department of Biochemistry and Molecular Biology; University of British Columbia; Vancouver, BC Canada;2.National Institute of Biological Sciences; Beijing, China;3.Life Science Institute and the Department of Molecular; Cellular and Developmental Biology; University of Michigan; Ann Arbor, MI USA |
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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 (Atg1CTD]) and Atg13 (Atg13CTD]). 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 Atg1CTD] crosslinks exclusively to the Atg13CTD] and does not appear to make direct contact with the Atg17-Atg31-Atg29 scaffold. Finally, single-particle electron microscopy analysis revealed that both the Atg13CTD] and Atg1CTD] 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. |
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Keywords: | Atg1 autophagy crosslinking mass spectrometry electron microscopy macromolecular assembly Saccharomyces cerevisiae |
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