Early steps in assembly of the yeast vacuolar H+-ATPase.
Vacuolar proton-translocating ATPases are composed of a complex of integral membrane proteins, the Vo sector, attached to a complex of peripheral membrane proteins, the V1 sector. We have examined the early steps in biosynthesis of the yeast vacuolar ATPase by biosynthetically labeling wild-type and mutant cells for varied pulse and chase times and immunoprecipitating fully and partially assembled complexes under nondenaturing conditions. In wild-type cells, several V1 subunits and the 100-kDa Vo subunit associate within 3-5 min, followed by addition of other Vo subunits with time. Deletion mutants lacking single subunits of the enzyme show a variety of partial complexes, including both complexes that resemble intermediates in the assembly pathway of wild-type cells and independent V1 and Vo sectors that form without any apparent V1Vo subunit interaction. Two yeast sec mutants that show a temperature-conditional block in export from the endoplasmic reticulum accumulate a complex containing several V1 subunits and the 100-kDa Vo subunit during incubation at elevated temperature. This complex can assemble with the 17-kDa Vo subunit when the temperature block is reversed. We propose that assembly of the yeast V-ATPase can occur by two different pathways: a concerted assembly pathway involving early interactions between V1 and Vo subunits and an independent assembly pathway requiring full assembly of V1 and Vo sectors before combination of the two sectors. The data suggest that in wild-type cells, assembly occurs predominantly by the concerted assembly pathway, and V-ATPase complexes acquire the full complement of Vo subunits during or after exit from the endoplasmic reticulum.
Kane
P M
PM
Department of Biochemistry and Molecular Biology, State University of New York Health Science Center, Syracuse, New York 13210, USA. kanepm@hscsyr.edu
Tarsio
M
M
Liu
J
J
D001692:Biological Transport; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005656:Fungal Proteins / Q000096:biosynthesis; D008954:Models, Biological; D009154:Mutation; D011485:Protein Binding; D017494:Proton Pumps / Q000096:biosynthesis* / Q000235:genetics; D006180:Proton-Translocating ATPases / Q000096:biosynthesis* / Q000235:genetics; D025262:Vacuolar Proton-Translocating ATPases; D014617:Vacuoles / Q000201:enzymology*; D015003:Yeasts / Q000235:genetics / Q000378:metabolism
D005656:Fungal Proteins; D017494:Proton Pumps; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases