Abstract
Previous purifications and characterizations of the Saccharomyces cerevisiae 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, 19236-19244). We report the cloning and characterization of an additional V-ATPase subunit, the 54-kDa subunit, which is encoded by the VMA13 gene. VMA13 was isolated by complementation of the growth phenotypes associated with the vma13 mutation, which was originally described as cls11 (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 calculated molecular mass of 54,415 daltons. The VMA13 54-kDa gene product resides on the vacuolar membrane and co-purified with the active V-ATPase complex. Characterization of a null vma13 mutant (delta vma13) revealed that the Vma13 polypeptide is essential for V-ATPase activity. However, 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 vacuolar membranes isolated from delta vma13 cells. The nature of the V-ATPase "complex" in delta vma13 mutant is, nevertheless, fundamentally different from the wild-type enzyme. This is evidenced by the fact that the inactive V-ATPase complex from delta vma13 cells is less stable than the wild-type enzyme. Taken together, these results indicate that VMA13 encodes the 54-kDa subunit of the V-ATPase and that this subunit is essential for activity, but not assembly, of the enzyme complex.
Highlights
Deletion of the VMA1.3 gene in loss of A T P a~ctiv~ity~from the vacuolar membrane. These results demonstrate that the Vma13 protein (Vma13p) is a subunit Of the yeast Vmal3P is a subunit of the enzyme, this Polypeptide is not required for the assembly of the remaining V-ATPase subunits onto thevacuolar membrane
The VMAll gene encodes a homologue of the VMA3-encoded 17-kDa subunit, which is essential for the assembly of the V-ATPase and may be a component of the enzyme complex (Umemoto et al, 1991)
Adetailed molecular characterization of the VMAl2 gene indicates that the Vmalp2rotein is required for assembly of the yeast V-ATPase, yet it is not a subunitof the active enzyme complex (Hirata et al, 1993).In thispaper, we report on the VMA13 gene and its requirement for yeast VATPase function
Summary
Materials-Enzymes for recombinant DNA methods were purchased from Takara Shuzo (Kyoto). Plasmids and RecombinantDNA Methods-Yeast single-copyplasmid pRS315 (LEU2) (Sikorski and Hieter, 1989) was used for subcloning and complementation analysis of the VMAZ3 gene. Cloning, Sequencing, and Disruption of the VMA13Gene-A umal mutant strain,NUY34 (MATa leu2) was transformed with a yeast genomic DNA library carried on the yeast multi-copy vector YEpl (Yoshihisa and Anraku, 1989) by the lithium acetate method. The resulting plasmid was digested with BamHI and X b I , and the mutant allele of the VMAl3 gene introduced into YPH500 to yield yeast strain RH302. SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis-Protein extracts of whole cells and vacuolar membrane vesicles were prepared as described by Kane et al (1992).SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed by the method of Laemmli (1970).Immunoblots were prepared and probed as described. V-ATPase, a-mannosidaasned, dipeptidyl aminopeptidase B (DPAP-B) activities wereassayed as previously described (Uchida et al, 1985;Kane et al, 1989)
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