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Reference: Ediger B, et al. (2009) 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. J Biol Chem 284(29):19522-32

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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 membrane-bound 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 Vo subunit d was harmed. Subunit d did not co-immunoprecipitate with subunit a and the c-ring. Cells contained pools of V1 and Vo(-d) that failed to form V1Vo, 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 V1Vo assembled after yeast cells were co-transformed with VMA6 and mutant VPH1. Tether-less V1Vo was delivered to the vacuole and active. It retained 63-71% of the wild-type activity and was responsive to glucose. Tether-less V1Vo disassembled and reassembled after brief glucose depletion and readdition. N-terminus retained binding to V1 subunits and C-terminus to phoshofructokinase. Thus, no major structural damage was generated at the N- and C-terminus of subunit a. We concluded that early steps of Vo assembly and trafficking were likely impaired by shorter tethers and rescued by VMA6.

Reference Type
Journal Article
Authors
Ediger B, Melman SD, Pappas DL, Finch M, Applen J, Parra KJ
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