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Quaternary structures of Vac8 differentially regulate the Cvt and PMN pathways.

ABSTRACT: 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.

Abbreviations

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.

SUBMITTER: Park J

PROVIDER: S-EPMC7469494 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Quaternary structures of Vac8 differentially regulate the Cvt and PMN pathways.

Park Jumi J Kim Hye-In HI Jeong Hanbin H Lee Miriam M Jang Se Hwan SH Yoon So Young SY Kim Hyejin H Park Zee-Yong ZY Jun Youngsoo Y Lee Changwook C

Autophagy 20190912 6

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 t ...[more]

PMID: 31512555

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Project description:BackgroundDue to the recent European legislations posing a ban of animal tests for safety assessment within the cosmetic industry, development of in vitro alternatives for assessment of skin sensitization is highly prioritized. To date, proposed in vitro assays are mainly based on single biomarkers, which so far have not been able to classify and stratify chemicals into subgroups, related to risk or potency.MethodsRecently, we presented the Genomic Allergen Rapid Detection (GARD) assay for assessment of chemical sensitizers. In this paper, we show how the genome wide readout of GARD can be expanded and used to identify differentially regulated pathways relating to individual chemical sensitizers. In this study, we investigated the mechanisms of action of a range of skin sensitizers through pathway identification, pathway classification and transcription factor analysis and related this to the reactive mechanisms and potency of the sensitizing agents.ResultsBy transcriptional profiling of chemically stimulated MUTZ-3 cells, 33 canonical pathways intimately involved in sensitization to chemical substances were identified. The results showed that metabolic processes, cell cycling and oxidative stress responses are the key events activated during skin sensitization, and that these functions are engaged differently depending on the reactivity mechanisms of the sensitizing agent. Furthermore, the results indicate that the chemical reactivity groups seem to gradually engage more pathways and more molecules in each pathway with increasing sensitizing potency of the chemical used for stimulation. Also, a switch in gene regulation from up to down regulation, with increasing potency, was seen both in genes involved in metabolic functions and cell cycling. These observed pathway patterns were clearly reflected in the regulatory elements identified to drive these processes, where 33 regulatory elements have been proposed for further analysis.ConclusionsThis study demonstrates that functional analysis of biomarkers identified from our genomics study of human MUTZ-3 cells can be used to assess sensitizing potency of chemicals in vitro, by the identification of key cellular events, such as metabolic and cell cycling pathways.

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Quaternary structures of Vac8 differentially regulate the Cvt and PMN pathways.

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Quaternary structures of Vac8 differentially regulate the Cvt and PMN pathways.

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