Adamson P, Paterson H F, Hall A
Section of Cell and Molecular Biology, Chester Beatty Laboratories, Institute of Cancer Research, London, United Kingdom.
J Cell Biol. 1992 Nov;119(3):617-27. doi: 10.1083/jcb.119.3.617.
The three mammalian ras proteins associated 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-rasB) or a palmitoylation site (p21Ha-ras and p21N-ras). 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-rasB or P21N-ras/Ha-ras. 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, rap1, has been localized to the Golgi (Beranger et al., 1991). None of the mammalian rho-like subfamily has yet been localized. Three forms (A, B, and C) of p21rho, the prototype of this family are known; the COOH termini of p21rhoA and p21rhoC resemble p21K-rasB with a polybasic domain, whereas p21rhoB resembles p21N-ras/Ha-ras with two cysteine residues as potential palmitoylation sites. Despite this similarity to the p21ras 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 p21rhoA and p21rhoC is cytosolic whereas p21rhoB is associated with early endosomes and a pre-lysosomal compartment. Along with the results obtained with chimeric molecules using heterologous proteins attached to rho COOH termini, this suggests that the p21rho proteins cycle on and off the plasma membrane and this may have important implications for their biological function.
三种哺乳动物Ras蛋白与质膜特异性相关，这对它们的生物学活性至关重要。蛋白质极端COOH末端编码的两个信号决定了这种细胞定位；一个CAAX框与一个多碱性结构域（p21K-rasB）或一个棕榈酰化位点（p21Ha-ras和p21N-ras）相结合。小GTP结合蛋白Ras超家族的Ras样和Rho样亚家族的所有成员也都有CAAX框，其潜在的第二位点序列类似于p21K-rasB或P21N-ras/Ha-ras。然而，它们是否都位于质膜上还完全不清楚，事实上，一种Ras样蛋白rap1已被定位到高尔基体（Beranger等人，1991年）。哺乳动物的Rho样亚家族中还没有一种被定位。已知该家族的原型p21rho有三种形式（A、B和C）；p21rhoA和p21rhoC的COOH末端类似于带有多碱性结构域的p21K-rasB，而p21rhoB类似于带有两个半胱氨酸残基作为潜在棕榈酰化位点的p21N-ras/Ha-ras。尽管与p21ras蛋白有这种相似性，但Rho蛋白已从多种组织的颗粒和胞质部分中纯化出来。为了明确地定位这三种Rho蛋白，我们使用了一种表位标记方法，并结合对活细胞的显微注射。我们发现，所有这三种蛋白的一小部分定位于质膜，但大多数p21rhoA和p21rhoC位于胞质中，而p21rhoB与早期内体和一个前溶酶体区室相关。连同使用连接到rho COOH末端的异源蛋白的嵌合分子所获得的结果，这表明p21rho蛋白在质膜上循环进出，这可能对它们的生物学功能有重要影响。
