The Tether Connecting Cytosolic (N Terminus) and Membrane (C Terminus) Domains of Yeast V-ATPase Subunit a (Vph1) Is Required for Assembly of V0 Subunit d

Abstract

V-ATPases are molecular motors that reversibly disassemble in vivo. Anchored in the membrane is subunit a. Subunit a has a movable N terminus that switches positions during disassembly and reassembly. Deletions were made at residues securing the N terminus of subunit a (yeast isoform Vph1) to its membranebound C-terminal domain in order to understand the role of this conserved region for V-ATPase function. Shrinking of the tether made cells pH-sensitive (vma phenotype) because assembly of V 0 subunit d was harmed. Subunit d did not co-immunoprecipitate with subunit a and the c-ring. Cells contained pools of V 1 and V 0 (؊d) that failed to form V 1 V 0 , and very low levels of V-ATPase subunits were found at the membrane. Although subunit d expression was stable and at wild-type levels, growth defects were rescued by exogenous VMA6 (subunit d). Stable V 1 V 0 assembled after yeast cells were co-transformed with VMA6 and mutant VPH1. Tether-less V 1 V 0 was delivered to the vacuole and active. It retained 63-71% of the wild-type activity and was responsive to glucose. Tether-less V 1 V 0 disassembled and reassembled after brief glucose depletion and readdition. The N terminus retained binding to V 1 subunits and the C terminus to phosphofructokinase. Thus, no major structural change was generated at the N and C termini of subunit a. We concluded that early steps of V 0 assembly and trafficking were likely impaired by shorter tethers and rescued by VMA6. V-ATPase4 proton pumps are highly conserved proteins fundamental for pH homeostasis (for review, See Refs. 1-6). Located in the endomembrane system, V-ATPases establish and maintain the low pH essential for endocytic and exocytic vesicular transport, zymogen activation, and protein sorting (for review, see Refs. 1-3). Cells specialized for active proton secretion, like kidney epithelial cells and osteoclasts, also express V-ATPases at the plasma membrane, where they transfer protons from the cytosol to the extracellular milieu (4, 5). In the kidney, plasma membrane V-ATPases of the intercalated cells are critical for regulation of the systemic acid-base balance (5, 6). Mutations in human kidney V-ATPase cause distal-renal tubular acidosis (6). V-ATPases at the plasma membrane of osteoclasts are essential for bone resorption, and mutations result in osteopetrosis, a disease characterized by thickening of the bones (1, 4, 7). Complete loss of V-ATPase activity is lethal in eukaryotes other than fungi (3).V-ATPases are multisubunit complexes that consist of two domains, V 1 (peripheral) and V 0 (membrane-bound) (1, 2). Each of the subunits in the V-ATPase complex is critical for function and V 1 V 0 assembly (8). Deletion of a peripheral V 1 subunit leads to disruption of the entire V 1 domain in yeast. Loss of a V 0 subunit does not affect V 1 assembly but disrupts the entire V 0 domain, which also prevents V 1 from associating with the membrane. An exception is subunit a for which two functional isoforms (Vph1, Stv1) exist in yeast (9). Disruption of subunit a requi...

Inhibition of Osteoclast Bone Resorption by Disrupting Vacuolar H+-ATPase a3-B2 Subunit Interaction

“…The "beam" that extends between the fingers is shown to cradle the H subunit and contact the d subunit, although this latter interaction is likely transient or manifest only when V 1 and V 0 are dissociated. These interactions have been demonstrated elsewhere (25,59), but the precise assembly is speculative. Both ends of the beam, which traverses ϳ120°around the rotor axis, are thought to support one each of three EG heterodimer peripheral stalks, and one end contacts the C subunit, which supports the third EG heterodimer at its distal end (see also C).…”

Inhibitors of V-ATPase Proton Transport Reveal Uncoupling Functions of Tether Linking Cytosolic and Membrane Domains of V0 Subunit a (Vph1p)

“…Western Blot-Vacuolar membrane protein was separated by SDS-PAGE in 8% polyacrylamide gels and transferred to a nitrocellulose membrane, and V-ATPase subunits a, A, and B were visualized with the monoclonal antibodies 10D7, 8B1, and 13D11 as described previously (35).…”

“…Yeast vph1⌬stv1⌬ does not express endogenous subunit a, because the gene VPH1 and its functional homolog, STV1, are deleted (34). Previously, we showed that biosynthetic assembly of tether-less V 1 V 0 (vph1-362-407⌬) complexes is complete when cells modestly overexpress V 0 subunit d (Vma6p) and that the assembled vph1-362-407⌬ V 1 V 0 fully retains ATP hydrolytic activity (35). Thus, we simultaneously co-transformed the vph1⌬stv1⌬ cells with VPH1-362-407⌬ and subunit d (VMA6), each of them expressed from a CEN plasmid (pRS316) under control of their natural promoters as described previously (35).…”

“…The yeast S. cerevisiae strains referred to throughout the in vitro studies involving vacuolar membranes and phenotype analyses are SF838-1D␣ (MAT␣ ade6 leu2-3,112 ura3-52 pep4 -3 his4 -519 gal2), the vph1⌬stv1⌬ strain, MM112 (MAT␣ his3-⌬200 leu2 lys2 ⌬stvl::LYS2 ura3-52 ⌬vphl::LEU2) (34), and the vph1⌬stv1⌬ strain transformed with the CEN plasmid pRS316 carrying either the wild-type VPH1 gene or the tether-less VPH1 gene (vph1-362-407⌬) (35). MM112 was a gift from Dr. Morris Manolson (University of Toronto, Toronto, Ontario, Canada), and pRS316-HA-VPH1 was a gift from Michael Forgac (Tufts University, Boston).…”

N‐terminal domain of the V‐ATPase a2‐subunit displays integral membrane protein properties

“…Crosslinking, two-hybrid screening, coimmunoprecipitation, and electron microscopy showed that the a-subunit isoforms directly interact with A-, E-, H-, C-, and G-subunits of the V 1 sector [9][10][11][12][13] as well as with c-and d-subunits of the V o sector. 2,[14][15][16] Recently, it has also been shown that the V-ATPase a-subunit directly assembles with proteins that are not part of the V-ATPase complex including: (i) aldolase B, 17,18 (ii) phosphofrucktokinase-1, 19,20 (iii) calmodulin, 21 and (iv) t-SNARES syntaxin and SNAP-25. 22 However, the mechanisms and molecular details of interactions with these proteins remain poorly understood, mainly due to the lack of a high-resolution structure of the a-subunit and the absence of studies designed for mapping interaction sites on both the a-subunit and its associated proteins.…”