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Quaternary structures of Vac8 differentially regulate the Cvt and PMN pathways

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journal contribution
posted on 12.09.2019 by Jumi Park, Hye-In Kim, Hanbin Jeong, Miriam Lee, Se Hwan Jang, So Young Yoon, Hyejin Kim, Zee-Yong Park, Youngsoo Jun, Changwook Lee

Armadillo (ARM) repeat proteins constitute a large protein family with diverse and fundamental functions in all organisms, and armadillo repeat domains share high structural similarity. However, exactly how these structurally similar proteins can mediate diverse functions remains a long-standing question. Vac8 (vacuole related 8) is a multifunctional protein that plays pivotal roles in various autophagic pathways, including piecemeal microautophagy of the nucleus (PMN) and cytoplasm-to-vacuole targeting (Cvt) pathways in the budding yeast Saccharomyces cerevisiae. Vac8 comprises an H1 helix at the N terminus, followed by 12 armadillo repeats. Herein, we report the crystal structure of Vac8 bound to Atg13, a key component of autophagic machinery. The 70-Å extended loop of Atg13 binds to the ARM domain of Vac8 in an antiparallel manner. Structural, biochemical, and in vivo experiments demonstrated that the H1 helix of Vac8 intramolecularly associates with the first ARM and regulates its self-association, which is crucial for Cvt and PMN pathways. The structure of H1 helix-deleted Vac8 complexed with Atg13 reveals that Vac8[Δ19–33]-Atg13 forms a heterotetramer and adopts an extended superhelical structure exclusively employed in the Cvt pathway. Most importantly, comparison of Vac8-Nvj1 and Vac8-Atg13 provides a molecular understanding of how a single ARM domain protein adopts different quaternary structures depending on its associated proteins to differentially regulate 2 closely related but distinct cellular pathways.

Ape1: aminopeptidase I; ARM: armadillo repeat; Atg: autophagy-related; AUC: analytical ultracentrifugation; Cvt: cytoplasm-to-vacuole targeting; DIC: differential interference contrast; GFP: green fluorescent protein; GST: glutathione-S-transferase; ITC: isothermal titration calorimetry; NVJ: nucleus-vacuole junction; PDB: protein data bank; PMN: piecemeal microautophagy of the nucleus; prApe1: precursor Ape1; RMSD: root-mean-square deviation; SAXS: small-angle X-ray scattering; SD-N: nitrogen starvation medium; SEC: size-exclusion chromatography; tAtg13: Atg13 construct comprising residues 567–695; tNvj1: Nvj1 construct comprising residues 229–321; tVac8: Vac8 construct comprising residues 10–515; Vac8: vacuole related 8


This research was supported by the Cell Logistics Research Center (grant number 2016R1A5A1007318) funded by the National Research Foundation of Korea. This research was also supported by the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number HI18C1395), the U-K Brand Research Fund (1.180064.01) of UNIST, and the GIST Research Institute (GRI) at GIST.