Purification of active human vacuolar H+-ATPase in native lipid-containing nanodiscs

• Published in: The Journal of biological chemistry 2021-08, Vol.297 (2), p.100964, Article 100964
• Main Authors: Oot, Rebecca A., Yao, Yeqi, Manolson, Morris F., Wilkens, Stephan
• Format: Article
• Language: English
• Subjects: Accelerated Communication; ATP6V0a4; HEK293 Cells; human; Humans; Isoenzymes - chemistry; Isoenzymes - genetics; Isoenzymes - isolation & purification; Isoenzymes - metabolism; Lipids - chemistry; membrane protein; Nanostructures - chemistry; native lipid nanodisc; protein purification; Protein Subunits - chemistry; Protein Subunits - genetics; Protein Subunits - isolation & purification; Protein Subunits - metabolism; vacuolar H+-ATPase; Vacuolar Proton-Translocating ATPases - chemistry; Vacuolar Proton-Translocating ATPases - genetics; Vacuolar Proton-Translocating ATPases - isolation & purification; Vacuolar Proton-Translocating ATPases - metabolism
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1016/j.jbc.2021.100964

Vacuolar H+-ATPases (V-ATPases) are large, multisubunit proton pumps that acidify the lumen of organelles in virtually every eukaryotic cell and in specialized acid-secreting animal cells, the enzyme pumps protons into the extracellular space. In higher organisms, most of the subunits are expressed as multiple isoforms, with some enriched in specific compartments or tissues and others expressed ubiquitously. In mammals, subunit a is expressed as four isoforms (a1-4) that target the enzyme to distinct biological membranes. Mutations in a isoforms are known to give rise to tissue-specific disease, and some a isoforms are upregulated and mislocalized to the plasma membrane in invasive cancers. However, isoform complexity and low abundance greatly complicate purification of active human V-ATPase, a prerequisite for developing isoform-specific therapeutics. Here, we report the purification of an active human V-ATPase in native lipid nanodiscs from a cell line stably expressing affinity-tagged a isoform 4 (a4). We find that exogenous expression of this single subunit in HEK293F cells permits assembly of a functional V-ATPase by incorporation of endogenous subunits. The ATPase activity of the preparation is >95% sensitive to concanamycin A, indicating that the lipid nanodisc-reconstituted enzyme is functionally coupled. Moreover, this strategy permits purification of the enzyme’s isolated membrane subcomplex together with biosynthetic assembly factors coiled-coil domain–containing protein 115, transmembrane protein 199, and vacuolar H+-ATPase assembly integral membrane protein 21. Our work thus lays the groundwork for biochemical characterization of active human V-ATPase in an a subunit isoform-specific manner and establishes a platform for the study of the assembly and regulation of the human holoenzyme.