Abstract
There has been no previous indication that vacuolar ATPases (V-ATPases) require sphingolipids for function. Here we show, by using Saccharomyces cerevisiae sur4Delta and fen1Delta cells, that sphingolipids with a C26 acyl group are required for generating V1 domains with ATPase activity. Sphingolipids in sur4Delta cells contain C22 and C24 acyl groups instead of C26 acyl groups whereas about 30% of the sphingolipids in fen1Delta cells have C26 acyl groups and the rest have C22 and C24 acyl groups. sur4Delta cells have several phenotypes (vacuolar membrane ATPase, Vma-) that indicate a defect in the V-ATPase, and vacuoles purified from sur4Delta cells have little to no ATPase activity. These phenotypes are less pronounced in fen1Delta 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 sur4Delta cells similar to wild-type cells. In vitro assembly studies show that V1 from sur4Delta cells associates with wild-type V0 but the complex lacks V-ATPase activity, indicating that V1 is defective. Reciprocal experiments with V0 from sur4Delta cells show that it is normal. We conclude that sphingolipids with a C26 acyl group are required for generating fully functional V1 domains.
Highlights
Vacuolar ATPases (V-ATPases)1 are found in all eukaryotes where they are required for receptor-mediated endocytosis, renal acidification, bone reabsoprtion, neurotransmitter accumulation, and activation of acid hydrolases
Sphingolipids in sur4⌬ cells contain C22 and C24 acyl groups instead of C26 acyl groups whereas about 30% of the sphingolipids in fen1⌬ cells have C26 acyl groups and the rest have C22 and C24 acyl groups. sur4⌬ cells have several phenotypes that indicate a defect in the V-ATPase, and vacuoles purified from sur4⌬ cells have little to no ATPase activity
The importance of the C26 acyl component of S. cerevisiae sphingolipids was demonstrated by the isolation of mutant strains that do not make sphingolipids [9], but instead make a set of novel sphingolipid mimics in which ceramide is replaced by diacylglycerol [10]
Summary
V-ATPase, vacuolar ATPase; DHS, dihydrosphingosine; LCB, long chain base; LCBP, long chain base phosphate; PHS, phytosphingosine; VLCFA, very long chain fatty acid; V1, peripheral domain of V-ATPase; Vma, vacuolar membrane ATPase; V0, membrane domain of V-ATPase; ER, endoplasmic reticulum; MES, 4-morpholineethanesulfonic acid; MOPS, 4-morpholinepropanesulfonic acid; HPLC, high pressure liquid chromatography; FITC, fluorescein isothiocyanate. S. cerevisiae V-ATPase contains two components or domains, V1 and V0, which associate to form an active V1V0 complex on the vacuolar membrane The V1 domain has ATPase activity and is composed of 8 different proteins It can exist free in the cytoplasm or complexed with V0 on the vacuolar membrane. The V1 and V0 domains are assembled and associate in the ER to form functional V1V0 complexes, which are transported from the Golgi to the vacuole Kohlwein et al [12] reported that sur4⌬ and fen1⌬ cells contain small vacuoles called fragmented vacuoles that fail to properly fuse to form larger vacuoles This observation suggested to us that sphingolipids with a C26 acyl group are needed for some vacuolar function(s). Our data are the first to implicate sphingolipids with a C26 acyl group in the generation of a fully functional V1 domain
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
