VMA13 ENCODES A 54-KDA VACUOLAR H-ATPASE SUBUNIT REQUIRED FOR ACTIVITY BUT NOT ASSEMBLY OF THE ENZYME COMPLEX IN SACCHAROMYCES-CEREVISIAE()

Previous purifications and characterizations of the Saccharomyces cere visiae vacuolar proton-translocating ATPase (V-ATPase) have indicated that this enzyme is a multisubunit complex composed of at least eight subunits of 100-, 69-, 60-, 42-, 36-, 32-, 27-, and 17-kDa (Kane, P. M ., Yamashiro, C. T., and Stevens, T. H. (1989) J. Biol. Chem. 264, 192 36-19244). We report the cloning and characterization of an additional V-ATPase subunit, the 54-kDa subunit, which is encoded by the VMA13 g ene. VMA13 was isolated by complementation of the growth phenotypes as sociated with the vma13 mutation, which was originally described as cl s11 (Ohya, Y., Umemoto, N., Tanida, I., Ohta, A., Iida, H., and Anraku , Y. (1991) J. Biol. Chem. 266, 13971-13977). The nucleotide sequence of the VMA13 gene predicted a hydrophilic polypeptide with a calculate d molecular mass of 54,415 daltons. The VMA13 54-kDa gene product resi des on the vacuolar membrane and co-purified with the active V-ATPase complex. Characterization of a null vma13 mutant (DELTAvma13) revealed that the Vma13 polypeptide is essential for V-ATPase activity. Howeve r, the Vma13 polypeptide is not required for targeting of the other V- ATPase subunits (100-, 69-, 60-, 42-, 27-, or 17-kDa subunits) to the vacuolar membrane as shown by the association of these subunits with v acuolar membranes isolated from DELTAvma13 cells. The nature of the V- ATPase ''complex'' in DELTAvma13 mutant is, nevertheless, fundamentall y different from the wild-type enzyme. This is evidenced by the fact t hat the inactive V-ATPase complex from DELTAvma13 cells is less stable than the wild-type enzyme. Taken together, these results indicate tha t DELTAvma13 encodes the 54-kDa subunit of the V-ATPase and that this subunit is essential for activity, but not assembly, of the enzyme com plex.