Localization of ral, a small Mr GTP-binding protein, to collecting duct cells in bovine and rat kidney.
Plasma membranes from bovine kidney cortex were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Blotting with [alpha-32P]GTP and [35S]GTP gamma S demonstrated specific binding to three and six distinct protein bands, respectively, in the 20,000- to 29,000-Mr range. This indicated the presence of small Mr GTP binding proteins (smg) in bovine kidney cortex. Only one smg with 28,000 Mr was labeled with hydrolysis-resistant GTP photoaffinity probe p3-(4-azidoanilido)-p1-5GTP (AAGTP). The major smg in platelet membranes that binds GTP on nitrocellulose blots has been identified as ral-Mr 29,000. With the use of an antiserum against the ral A gene product, one of the smg with Mr of 29,000 present in bovine renal cortical plasma membranes was identified as ral. Ral was absent from glomerular homogenate, suggesting that it is localized to the tubular segments of the nephron. Ral was detected only in the particulate fraction and not the cytosol. Further subcellular localization of ral was investigated by immunohistochemical staining. Anti-ral antibody immunostained the apical and basolateral membranes of cells in the cortical and medullary collecting ducts in a speckled pattern in the bovine kidney. In the rat kidney, however, uniform linear staining of cortical and medullary collecting ducts predominantly localized to the apical membrane was observed. To date, no function has been assigned to ral. Localization of the ral gene product to the collecting duct suggests a specific functional role for this GTP-binding protein.
Gupta
A
A
Renal Division, Jewish Hospital of St. Louis, Missouri.
Bastani
B
B
Chardin
P
P
Hruska
K A
KA
D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry; D005455:Fluorescent Antibody Technique; D019204:GTP-Binding Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D015151:Immunoblotting; D007672:Kidney Cortex / Q000737:chemistry*; D007685:Kidney Tubules, Collecting / Q000737:chemistry*; D008970:Molecular Weight; D010777:Photochemistry; D051381:Rats
D000345:Affinity Labels; D001386:Azides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C031907:GTP gamma-4-azidoanilide; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins