Sphingolipid Requirement for Generation of a Functional V1 Component of the Vacuolar ATPase

Abstract There has been no previous indication that vacuolar ATPases (V-ATPases) require sphingolipids for function. Here we show, by using Saccharomyces cerevisiae sur4Δ and fen1Δ cells, that sphingolipids with a C26 acyl group are required for generating V1 domains with ATPase activity. Sphingolipids in sur4Δ cells contain C22 and C24 acyl groups instead of C26 acyl groups whereas about 30% of the sphingolipids in fen1Δ cells have C26 acyl groups and the rest have C22 and C24 acyl groups. sur4Δ cells have several phenotypes (vacuolar membrane ATPase, Vma–) that indicate a defect in the V-ATPase, and vacuoles purified from sur4Δ cells have little to no ATPase activity. These phenotypes are less pronounced in fen1Δ cells, consistent with the idea that the C26 acyl group in sphingolipids is necessary for V-ATPase activity. Other results show that the two V-ATPase domains, V1 and V0, are assembled and delivered to the vacuolar membrane in sur4Δ cells similar to wild-type cells. In vitro assembly studies show that V1 from sur4Δ cells associates with wild-type V0 but the complex lacks V-ATPase activity, indicating that V1 is defective. Reciprocal experiments with V0 from sur4Δ cells show that it is normal. We conclude that sphingolipids with a C26 acyl group are required for generating fully functional V1 domains.