Li G, Barbieri M A, Colombo M I, Stahl P D
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110.
J Biol Chem. 1994 May 20;269(20):14631-5.
Rab5 is a Ras-like small GTPase that regulates early events of endocytosis. Previous work indicates that two GTP-binding defective Rab5 mutants (Rab5:S34N and Rab5:N133I) are dominant inhibitors of endocytosis. In this report, we have initiated experiments to address the structural features necessary for the inhibitory activity of these two Rab5 mutants. Second-site mutations were introduced into Rab5:S34N and Rab5:N133I, respectively, and the resulting double mutants were expressed in cultured BHK-21 cells via a Sindbis virus expression vector. Endocytic activity of the cells was monitored by following the uptake of a fluid-phase endocytic marker (horseradish peroxidase). The effects of the Rab5 mutants on endosome fusion in vitro were also examined. Truncation of the C-terminal isoprenylation motif CCSN abolished the inhibitory activity of both Rab5:S34N and Rab5:N133I. The same held true when the secondary mutation was a substitution mutation (F57S) in the effector domain. Another substitution mutation in this region (I53A) had no effect on the inhibitory activity of either Rab5:S34N or Rab5:N133I. The final mutation (R81A) was created immediately downstream of the second GTP binding motif (WDTAGQER), i.e. in the loop 4 region based on the structural model of Ras. This mutation greatly decreased the isoprenylation of Rab5:N133I and its inhibitory activity on endocytosis. It is believed that Rab5 function requires protein-protein interactions with Rab5-specific regulators and effectors. Some of these interactions are disrupted by Rab5:S34N and Rab5:N133I. By analogy to Ras, both Rab5:S34N and Rab5:N133I are likely to sequester a Rab5-specific guanine nucleotide exchange factor. This interaction requires the effector domain Phe57 residue and C-terminal isoprenylation of Rab5.
Rab5是一种Ras样小GTP酶，可调节内吞作用的早期事件。先前的研究表明，两种GTP结合缺陷型Rab5突变体（Rab5:S34N和Rab5:N133I）是内吞作用的显性抑制剂。在本报告中，我们已启动实验，以研究这两种Rab5突变体抑制活性所需的结构特征。分别将第二位点突变引入Rab5:S34N和Rab5:N133I，并通过辛德毕斯病毒表达载体在培养的BHK-21细胞中表达所得的双突变体。通过跟踪液相内吞标记物（辣根过氧化物酶）的摄取来监测细胞的内吞活性。还检测了Rab5突变体对体外内体融合的影响。C末端异戊二烯化基序CCSN的截短消除了Rab5:S34N和Rab5:N133I的抑制活性。当第二位点突变是效应器结构域中的取代突变（F57S）时，情况也是如此。该区域中的另一个取代突变（I53A）对Rab5:S34N或Rab5:N133I的抑制活性均无影响。最后一个突变（R81A）位于第二个GTP结合基序（WDTAGQER）的紧邻下游，即基于Ras的结构模型位于环4区域。该突变大大降低了Rab5:N133I的异戊二烯化及其对内吞作用的抑制活性。据信Rab5功能需要与Rab5特异性调节剂和效应器进行蛋白质-蛋白质相互作用。其中一些相互作用被Rab5:S34N和Rab5:N133I破坏。与Ras类似，Rab5:S34N和Rab5:N133I都可能隔离一种Rab5特异性鸟嘌呤核苷酸交换因子。这种相互作用需要Rab5的效应器结构域苯丙氨酸57残基和C末端异戊二烯化。
