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

Rebecca A Oot,Yeqi Yao,Morris F Manolson,Stephan Wilkens

doi:10.1016/j.jbc.2021.100964

Rebecca A Oot, Yeqi Yao + Show 2 more

Open Access

https://doi.org/10.1016/j.jbc.2021.100964

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Abstract

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.

Highlights

Including Wnt, Notch, and mTOR [1, 2]
The V-ATPase can be found on the plasma membrane of dedicated acidsecreting cells such as osteoclasts and renal α-intercalated cells where the enzyme is required for bone demineralization and urinary acidification, respectively [3, 4]
These studies showed that the exogenously expressed a isoforms were properly posttranslationally modified and assembled with endogenous subunits to form intact V-ATPase complexes [78]. We used this plasmid for stable expression of C-terminally FLAGtagged isoform a4 in suspension HEK293 (FreeStyle 293F) cells. This cell line was chosen solely for the purpose of accumulation of sufficient cell mass for purification of the low-abundance V-ATPase and not based on possible endogenous isoform content (evidence suggests that while HEK cells are derived from fetal kidney tissue, the origin of this cell line is most likely adrenal [80, 81])

Summary

Introduction

Including Wnt, Notch, and mTOR [1, 2]. The V-ATPase can be found on the plasma membrane of dedicated acidsecreting cells such as osteoclasts and renal α-intercalated cells where the enzyme is required for bone demineralization and urinary acidification, respectively [3, 4]. Only subunit a is expressed as two isoforms, presenting an ideal system for study of different isoforms containing V-ATPases [46, 47]. Active human V-ATPase of a defined a subunit isoform composition would allow analyses of possible intrinsic differences in biochemical properties of enzyme subpopulations, as seen for the yeast enzyme.

Results

Conclusion