Intracellular Localization of the P 2 lrh ~ Proteins

The three mammalian ras proteins associate specifically with the plasma membrane and this is essential for their biological activity. Two signals encoded within the extreme COOH terminus of the proteins specify this cellular localization; a CAAX box in combination with either a polybasic domain (p21K-~B) or a palmitoylation site (p21 m-~ and p21N-~). All members of the ras-like and rho-like subfamilies of the ras superfamily of small GTP-binding proteins also have CAAX boxes with potential second site sequences resembling either p21K-~B or P21N-~m-~. However it is not at all clear that they are each located at the plasma membrane, and in fact one of the ras-like proteins , rapl, has been localized to the Golgi (Beranger et al., 1991). None of the mammalian rho-like sub-family has yet been localized. Three forms (A, B, and C) of p21 ~h~ the prototype of this family are known; the COOH termini of p21 rh~ and p21 rh~ resemble p21K-'~B with a polybasic domain, whereas p21 rh~ resembles p21N-r~/m-r~ with two cysteine residues as potential palmitoylation sites. Despite this similarity to the p21 ~ proteins, rho proteins have been purified from both particulate and cytosolic fractions of a variety of tissues. In order to localize definitively the three rho proteins we have used an epitope tagging approach coupled to microinjection of living cells. We show that a small fraction of all three proteins is localized to the plasma membrane but the majority of p21 rh~ and p21 rh~ is cytosolic whereas p21 ~h~ is associated with early endosomes and a pre-lysosomal compartment. Along with the results obtained with chi-meric molecules using heterologous proteins attached to rho COOH termini, this suggests that the p2P h~ proteins cycle on and off the plasma membrane and this may have important implications for their biological function. T HE p21 rh~ proteins are members of a large family of small GTP-binding proteins which share sequence homology (~30%) to p21 ~as (Hall, 1990). They have been described in yeast, Aplysia, and in human cells, where three different rho genes (A, B, and C) have been found by screening a peripheral T-lymphocyte cDNA library with fragments from the Aplysia rho cDNA (Madaule and Axel, 1985). The proteins are expressed in all cell types so far examined (Olofsson et al., 1988). These three mammalian rho proteins are '~80% homologous to each other, and as with the three ras proteins (Ha, Ki, and …