Two upstream cysteines and the CAAX motif but not the polybasic domain are required for membrane association of Xlcaax in Xenopus oocytes.
We have analyzed the role of several protein motifs in controlling the membrane association of the xlcaax-1 protein in Xenopus oocytes. Xlcaax-1 is a maternally expressed protein that during development is associated with the basal lateral membrane of polarized epithelial cells. It is enriched in the tubule cells of the adult kidney and several other organs that are involved in osmoregulation. Xlcaax-1 has a C-terminal CAAX sequence (CVVM) identical to that of N-ras, followed by two cysteines that are potential palmitoylation sites and a polybasic domain. Mutants were constructed that either deleted specific domains or changed specific amino acids of the consensus sequences in or near the CAAX motif. Synthetic mRNAs were injected into Xenopus oocytes and their protein products analyzed for their ability to associate with the oocyte plasma membrane. A mutation changing cysteine-588 of the CAAX box to serine or the inhibition of prenylation by lovastatin eliminated the membrane association of the protein. Mutation of either of the upstream cysteines (either 585 or 587) also inhibited the association of xlcaax-1 with the membrane. Unlike Ras, however, deletion of the polybasic domain had no effect on membrane binding. In addition, we show that xlcaax-1 binds ATP but not GTP.
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Department of Molecular Genetics, University of Texas M.D. Anderson Cancer Center, Houston 77030.
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D005260:Female; D006160:Guanosine Triphosphate / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009865:Oocytes / Q000378:metabolism*; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014162:Transfection; D014981:Xenopus / Q000235:genetics; D029867:Xenopus Proteins
D008565:Membrane Proteins; C068700:PALM3 protein, Xenopus; D011106:Polyisoprenyl Phosphates; D012333:RNA, Messenger; D011994:Recombinant Proteins; D029867:Xenopus Proteins; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D004247:DNA