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.
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 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.
Ediger
Benjamin
B
Department of Biochemistry and Molecular Biology, University of New Mexico School of Medicine, Albuquerque, New Mexico 87131, USA.
Melman
Sandra D
SD
Pappas
Donald L
DL
Finch
Mark
M
Applen
Jeremy
J
Parra
Karlett J
KJ
D001483:Base Sequence; D015153:Blotting, Western; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D006863:Hydrogen-Ion Concentration; D047468:Immunoprecipitation; D009154:Mutation; D010641:Phenotype; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D014170:Transformation, Genetic; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D015003:Yeasts / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development
D005656:Fungal Proteins; D021122:Protein Subunits; D025262:Vacuolar Proton-Translocating ATPases