STRUCTURAL FEATURES OF THE GTP-BINDING DEFECTIVE RAB5 MUTANTS REQUIRED FOR THEIR INHIBITORY ACTIVITY ON ENDOCYTOSIS

Rab5 is a Ras-like small GTPase that regulates early events of endocyt osis. Previous work indicates that two GTP-binding defective Rab5 muta nts (Rab5:S34N and Rab5:N133I) are dominant inhibitors of endocytosis. In this report, we have initiated experiments to address the structur al features necessary for the inhibitory activity of these two Rab5 mu tants. Second-site mutations were introduced into Rab5:S34N and Rab5:N 133I, respectively, and the resulting double mutants were expressed in cultured BHK-21 cells via a Sindbis virus expression vector. Endocyti c activity of the cells was monitored by following the uptake of a flu id-phase endocytic marker (horseradish peroxidase). The effects of the Rab5 mutants on endosome fusion in vitro were also examined. Truncati on of the C-terminal isoprenylation motif CCSN abolished the inhibitor y activity of both Rab5:S34N and Rab5:N133I. The same held true when t he secondary mutation was a substitution mutation (F57S) in the effect or domain. Another substitution mutation in this region (I53A) had no effect on the inhibitory activity of either Rab5:S34N or Rab5:N133I. T he final mutation (R81A) was created immediately downstream of the sec ond GTP binding motif (WDTAGQER), i.e. in the loop 4 region based on t he structural model of Pas. This mutation greatly decreased the isopre nylation of Rab5:N133I and its inhibitory activity on endocytosis. It is believed that Rab5 function requires protein-protein interactions w ith Rab5-specific regulators and effecters. Some of these interactions are disrupted by Rab5:S34N and Rab5:N133I. By analogy to Ras, both Ra b5:S34N and Rab5:N133I are likely to sequester a Rab5-specific guanine nucleotide exchange factor. This interaction requires the effector do main Phe(57) residue and C-terminal isoprenylation of Rab5.