Early steps in assembly of the yeast vacuolar H+-ATPase

Abstract

Vacuolar proton-translocating ATPases are composed of a complex of integral membrane proteins, the V(o) 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 V(o) subunit associate within 3-5 min, followed by addition of other V(o) 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 V(o) sectors that form without any apparent V1V(o) 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 V(o) subunit during incubation at elevated temperature. This complex can assemble with the 17- kDa V(o) 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 V(o) subunits and an independent assembly pathway requiring full assembly of V1 and V(o) 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 V(o) subunits during or after exit from the endoplasmic reticulum.