Coordinated conformational changes in the V1 complex during V-ATPase reversible dissociation

Abstract

Vacuolar-type ATPases (V-ATPases) are rotary enzymes that acidify intracellular compartments in eukaryotic cells. These multi-subunit complexes consist of a cytoplasmic V1 region that hydrolyzes ATP and a membrane-embedded VO region that transports protons. V-ATPase activity is regulated by reversible dissociation of the two regions, with the isolated V1 and VO complexes becoming autoinhibited upon disassembly and subunit C subsequently detaching from V1. In yeast, assembly of the V1 and VO regions is mediated by the RAVE complex through an unknown mechanism. We used cryoEM of yeast V-ATPase to determine structures of the intact enzyme, the dissociated but complete V1 complex, and the V1 complex lacking subunit C. Upon separation, V1 undergoes a dramatic conformational rearrangement, with its rotational state becoming incompatible for reassembly with VO. Loss of subunit C allows V1 to match the rotational state of VO, suggesting how RAVE could reassemble V1 and VO by recruiting subunit C.

Structure of V-ATPase from citrus fruit

“…However, 3D variability analysis of particle images (Punjani and Fleet, 2021) from both rotary states revealed that the C-terminal domain of subunit H could adopt two different conformations, with the consensus map missing the domain due to incoherent averaging. In one extreme of the principal component analysis, subunit H adopts the conformation seen in previous structures of intact V-ATPase (Benlekbir et al, 2012; Tan et al, 2021; Vasanthakumar et al, 2019, 2022; Wang et al, 2020a, 2020b; Zhao et al, 2015), with the C-terminal domain of H bound to the N-terminal domain of subunit a (Fig. 3B and C).…”

“…Previous structural studies of yeast V-ATPase have relied on purification of the detergent-solubilized endogenous enzyme following incorporation of sequence encoding an affinity tag into the yeast chromosomal DNA (Benlekbir et al, 2012; Mazhab-Jafari et al, 2016; Oot et al, 2016; Roh et al, 2018; Vasanthakumar et al, 2022; Zhao et al, 2015). V-ATPase from bovine brain, where the enzyme is highly enriched, was isolated for structural studies using a large mass of starting material and conventional chromatographic methods (Wang et al, 2020b).…”

“…Ubiquitous in eukaryotes, V-ATPase activity acidifies the lumen of various organelles, enabling pH-dependent processes and driving transmembrane transport (Forgac, 2007; Kane, 2007; Vasanthakumar and Rubinstein, 2020). Understanding of the structure-function relationship in V-ATPases has been driven by studies of the enzyme from yeast (Benlekbir et al, 2012; Mazhab-Jafari et al, 2016; Oot et al, 2016; Roh et al, 2018; Vasanthakumar et al, 2019, 2022; Zhao et al, 2015), mammals (Abbas et al, 2020; Tan et al, 2021; Wang et al, 2020a, 2020b), and insects (Muench et al, 2009). These studies revealed the overall architecture of the enzyme and the sequence of events that couples ATP hydrolysis to proton pumping.…”

“…V-ATPase activity in yeast (Kane, 1995), insects (Sumner et al, 1995), and mammals (Liberman et al, 2014; Sautin et al, 2005; Stransky and Forgac, 2015) is regulated by reversible separation of the V 1 region from the V O region. On separation, the V O complex adopts an inhibited conformation (Mazhab-Jafari et al, 2016) that is impermeable to protons (Ward and Sze, 1992; Zhang et al, 1992) and the V 1 complex undergoes a major conformational rearrangement that blocks ATP hydrolysis and prevents its premature reassembly with V O (Vasanthakumar et al, 2022). Inhibition of ATP hydrolysis in dissociated V 1 requires subunit H (Parra et al, 2000), which forms a new interaction with one of the peripheral stalks on dissociation to prevent the conformational changes needed for hydrolysis (Vasanthakumar et al, 2022).…”

“…On separation, the V O complex adopts an inhibited conformation (Mazhab-Jafari et al, 2016) that is impermeable to protons (Ward and Sze, 1992; Zhang et al, 1992) and the V 1 complex undergoes a major conformational rearrangement that blocks ATP hydrolysis and prevents its premature reassembly with V O (Vasanthakumar et al, 2022). Inhibition of ATP hydrolysis in dissociated V 1 requires subunit H (Parra et al, 2000), which forms a new interaction with one of the peripheral stalks on dissociation to prevent the conformational changes needed for hydrolysis (Vasanthakumar et al, 2022). Subunit C subsequently separates from V 1 (Gräf et al, 1996), with the RAVE complex (regulator of the A TPase of v acuoles and e ndosomes) required for reassembly of V 1 , V O , and subunit C to form a functional V-ATPase (Jaskolka et al, 2021; Smardon et al, 2002).…”