2 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. Xlca ax-1 is a maternally expressed protein that during development is asso ciated with the basal lateral membrane of polarized epithelial cells. It is enriched in the tubule cells of the adult kidney and several oth er organs that are involved in osmoregulation. Xlcaax-1 has a C-termin al CAAX sequence (CVVM) identical to that of N-ras, followed by two cy steines that are potential palmitoylation sites and a polybasic domain . Mutants were constructed that either deleted specific domains or cha nged specific amino acids of the consensus sequences in or near the CA AX motif. Synthetic mRNAs were injected into Xenopus oocytes and their protein products analyzed for their ability to associate with the ooc yte plasma membrane. A mutation changing cysteine-588 of the CAAX box to serine or the inhibition of prenylation by lovastatin eliminated th e membrane association of the protein. Mutation of either of the upstr eam cysteines (either 585 or 587) also inhibited the association of xl caax-1 with the membrane. Unlike Ras, however, deletion of the polybas ic domain had no effect on membrane binding. In addition, we show that xlcaax-1 binds ATP but not GTP.