Expression and localization of the mouse homologue of the yeast V-ATPase 21-kDa Subunit c" (Vma16p).
We have identified a cDNA encoding the mouse homologue of the yeast V-ATPase 21-kDa subunit c" (Vma16p). The encoded protein contains 205 amino acid residues with five putative membrane spanning segments and shows 48% identity and 64% similarity to the yeast protein. Despite this homology, however, the mouse cDNA does not complement the phenotype of a yeast strain in which the VMA16 gene has been disrupted. Northern blot analysis demonstrated that the 21-kDa subunit is expressed in most tissues examined and showed an expression pattern almost identical to that of the 16-kDa proteolipid subunit (subunit c). The presence of multiple mRNA species suggests the existence of alternatively spliced forms of the 21-kDa subunit which, from Southern blot analysis, are derived from a single gene. Promoter analysis using the luciferase reporter gene revealed that a region 186 bases upstream of the initiation site is sufficient to show a low level of transcriptional activity but that transcription is significantly enhanced by inclusion of the region -186 to -706. The 21-kDa protein was Myc-tagged and the 16-kDa protein was HA-tagged and the tagged proteins were co-expressed in COS-1 cells in order to study their intracellular localization by immunofluorescence microscopy. Both proteins showed significant punctate and perinuclear staining and were predominantly co-localized throughout the cell, consistent with their presence in the same V(0) complexes. Selective permeabilization of cells with digitonin (to permeabilize the plasma membrane) or Triton X-100 (to permeabilize both intracellular and plasma membranes) followed by immunofluorescence microscopy revealed that the carboxyl terminus of the 21-kDa subunit is exposed on the cytoplasmic side of the membrane whereas the carboxyl terminus of the 16-kDa subunit is located on the lumenal side of the membrane.
Nishi
T
T
Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Kawasaki-Nishi
S
S
Forgac
M
M
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D005816:Genetic Complementation Test; D051379:Mice; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D017830:Octoxynol / Q000494:pharmacology; D010641:Phenotype; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D006180:Proton-Translocating ATPases / Q000737:chemistry*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014158:Transcription, Genetic; D014162:Transfection; D025262:Vacuolar Proton-Translocating ATPases
C506980:Atp6v0b protein, mouse; D018076:DNA, Complementary; D003902:Detergents; D012333:RNA, Messenger; D017830:Octoxynol; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases