BIOCHEMICAL AND FUNCTIONAL-CHARACTERIZATION OF A RECOMBINANT GTPASE, RAB5, AND 2 OF ITS MUTANTS

Biochemical, structural, and functional properties of Rab5 wild-type ( WT) protein were compared with those of Q79L and N133I mutants. The de tergent holamidopropyl)dimethylammonio]-1-propanesulfonate increased g uanine nucleotide binding to RabB WT approximate to 10-fold. The singl e-step catalytic rate of RabB WT exceeded that of Q79L 12.2-fold, but the steady-state GTPase rate was only 2.8-fold greater because GDP dis sociation was rate limiting and GDP dissociation was 3.6-fold slower t han for Q79L. In contrast, dissociation rates of GTP were indistinguis hable. Binding to RabB N133I was not detectable. GTP protected RabB WT and Q79L from any apparent proteolysis by trypsin. A 20-kDa fragment was the major product of digestion in the presence of GDP, and 12- and 8-kDa fragments were the major products in the absence of added guani ne nucleotides. RabB N133I underwent no apparent proteolysis with 10 m M GTP or GDP, suggesting a ''triphosphate'' conformation may be induce d in Rab5 N133I by either GTP or GDP. Partially geranylgeranylated Rab 5 WT stimulated endosome fusion in vitro, whereas unmodified RabB WT d id not. Processed RabB Q79L failed to inhibit endosome fusion, and Rab B N133I could not be geranylgeranylated. These findings identify bioch emical and structural features of RabB proteins, providing data for th e interpretation of functional assays.