Defining steps in RAVE-catalyzed V-ATPase assembly using purified RAVE and V-ATPase subcomplexes
| Autor: | Maureen Tarsio, Michael C. Jaskolka, Md. Murad Khan, Patricia M. Kane, Anne M. Smardon |
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| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
assembly Vacuolar Proton-Translocating ATPases Saccharomyces cerevisiae Proteins SEC size-exclusion chromatography Endosome MBP-C MBP-tagged subunit C Protein subunit Mutant MBP maltose-binding protein Vacuole Saccharomyces cerevisiae lysosomal acidification vacuolar ATPase yeast Biochemistry YEPD 03 medical and health sciences Maltose-binding protein chemistry.chemical_compound YEPD yeast extract peptone 2% dextrose V-ATPase BLI biolayer interferometry Molecular Biology RAVE TBSE Tris-buffered saline with EDTA 030102 biochemistry & molecular biology biology vacuole Chemistry RAVE Regulator of the H+-ATPase of Vacuoles and Endosomes regulation Cell Biology Yeast Cell biology 030104 developmental biology proton pump biology.protein YEP yeast extract–peptone medium without glucose Vph1NT N-terminal domain of Vo subunit Vph1 V-ATPase vacuolar H+-ATPase Research Article |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 1083-351X |
| Popis: | The vacuolar H+-ATPase (V-ATPase) is a highly conserved proton pump responsible for the acidification of intracellular organelles in virtually all eukaryotic cells. V-ATPases are regulated by the rapid and reversible disassembly of the peripheral V1 domain from the integral membrane Vo domain, accompanied by release of the V1 C subunit from both domains. Efficient reassembly of V-ATPases requires the Regulator of the H+-ATPase of Vacuoles and Endosomes (RAVE) complex in yeast. Although a number of pairwise interactions between RAVE and V-ATPase subunits have been mapped, the low endogenous levels of the RAVE complex and lethality of constitutive RAV1 overexpression have hindered biochemical characterization of the intact RAVE complex. We describe a novel inducible overexpression system that allows purification of native RAVE and RAVE–V1 complexes. Both purified RAVE and RAVE–V1 contain substoichiometric levels of subunit C. RAVE–V1 binds tightly to expressed subunit C in vitro, but binding of subunit C to RAVE alone is weak. Neither RAVE nor RAVE–V1 interacts with the N-terminal domain of Vo subunit Vph1 in vitro. RAVE–V1 complexes, like isolated V1, have no MgATPase activity, suggesting that RAVE cannot reverse V1 inhibition generated by rotation of subunit H and entrapment of MgADP that occur upon disassembly. However, purified RAVE can accelerate reassembly of V1 carrying a mutant subunit H incapable of inhibition with Vo complexes reconstituted into lipid nanodiscs, consistent with its catalytic activity in vivo. These results provide new insights into the possible order of events in V-ATPase reassembly and the roles of the RAVE complex in each event. |
| Databáze: | OpenAIRE |
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