期刊
EMBO JOURNAL
卷 41, 期 3, 页码 -出版社
WILEY
DOI: 10.15252/embj.2021109360
关键词
Cryo-electron microscopy; Oxr1p; reversible disassembly; TLDc domain; vacuolar ATPase
资金
- NIH [GM058600, GM141908, CA228340]
- Korean National Research Foundation [NRF-2019M3E5D6063871, 2019R1C1C1004598, 2020R1A5A1018081, 2021M3A9I4021220, 2020R1A6C101A183]
- SUHF foundation
- Creative-Pioneering Researchers Program of Seoul National UniversityS
- National Research Foundation of Korea [2019R1C1C1004598, 2019M3E5D6063871, 2020R1A5A1018081, 2020R1A6C101A183, 2021M3A9I4021220] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
The study presents cryo-EM structures of yeast V-ATPase assembled in vitro, revealing its active states and autoinhibition mechanism. The uneven spacing of proton-carrying glutamic acid residues in V-o helps alleviate symmetry mismatch between V-1 and V-o motors. Additionally, a factor involved in oxidative stress response, Oxr1, inhibits V-1-ATPase activity by causing disassembly of the holoenzyme.
The vacuolar ATPase (V-ATPase) is a rotary motor proton pump that is regulated by an assembly equilibrium between active holoenzyme and autoinhibited V-1-ATPase and V-o proton channel subcomplexes. Here, we report cryo-EM structures of yeast V-ATPase assembled in vitro from lipid nanodisc reconstituted V-o and mutant V-1. Our analysis identified holoenzymes in three active rotary states, indicating that binding of V-1 to V-o provides sufficient free energy to overcome V-o autoinhibition. Moreover, the structures suggest that the unequal spacing of V-o's proton-carrying glutamic acid residues serves to alleviate the symmetry mismatch between V-1 and V-o motors, a notion that is supported by mutagenesis experiments. We also uncover a structure of free V-1 bound to Oxr1, a conserved but poorly characterized factor involved in the oxidative stress response. Biochemical experiments show that Oxr1 inhibits V-1-ATPase and causes disassembly of the holoenzyme, suggesting that Oxr1 plays a direct role in V-ATPase regulation.
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