Biochemical and Functional Characterization of a Recombinant GTPase, Rab5, and Two of Its Mutants

Abstract

Biochemical, structural, and functional properties of Rab5 wild-type (WT) protein were compared with those of Q79L and N133I mutants. The detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate increased guanine nucleotide binding to Rab5 WT approximately 10-fold. The single-step catalytic rate of Rab5 WT exceeded that of Q79L 12.2-fold, but the steady-state GTPase rate was only 2.8-fold greater because GDP dissociation was rate-limiting and GDP dissociation was 3.6-fold slower than for Q79L. In contrast, dissociation rates of GTP were indistinguishable. Binding to Rab5 N133I was not detectable. GTP protected Rab5 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 guanine nucleotides. Rab5 N133I underwent no apparent proteolysis with 10 mM GTP or GDP, suggesting a "triphosphate" conformation may be induced in Rab5 N133I by either GTP or GDP. Partially geranylgeranylated Rab5 WT stimulated endosome fusion in vitro, whereas unmodified Rab5 WT did not. Processed Rab5 Q79L failed to inhibit endosome fusion, and Rab5 N133I could not be geranylgeranylated. These findings identify biochemical and structural features of Rab5 proteins, providing data for the interpretation of functional assays.

GTPase mechanism and function: new insights from systematic mutational analysis of the phosphate-binding loop residue Ala30 of Rab5

“…mutants, were the same as the wt protein in terms of GTP binding and dissociation rates (Figures 3C and 3D). The ratio of GTP hydrolysis rate to GTP dissociation rate for Rab5 is high (0.0034 s −" as against 0.0042 min −" ) [31]. The 3-fold increase in the GTP dissociation rate might contribute to, but cannot account for, the 12-fold decrease in the GTPase activity of the Pro$!…”

“…Because purified Rab proteins often contain tightly bound nucleotides [31,32], the difference in binding rate might reflect a difference in nucleotide dissociation rate. This contention was confirmed by direct measurement of the GTP dissociation rate ( Figure 3D).…”

“…mutants. The biological function of Rab5 is to regulate endocytosis by promoting early endosome fusion [31,35,36], and possibly vesicle budding at the plasma membrane [37]. As a result, overexpression of Rab5 in cultured cells increased the endocytic activity by 2-fold, as determined by HRP uptake [23,24].…”

“…The biological function of Rab5 is to promote early endosome fusion [31,35,36]. A recent study also suggests the involvement of Rab5 in vesicle budding from the plasma membrane [37].…”

Rabaptin-5-independent Membrane Targeting and Rab5 Activation by Rabex-5 in the Cell

“…Rabex-5 is a guanine nucleotide exchange factor (GEF) for Rab5 (Horiuchi et al, 1997), a small GTPase regulating early endosome fusion and endocytosis (Gorvel et al, 1991;Bucci et al, 1992;Li et al, 1994;Hoffenberg et al, 1995;Li and Liang, 2001). Interestingly, Rabex-5 was originally purified as a soluble complex with Rabaptin-5, and immunodepletion of this complex can reduce early endosome fusion in vitro (Horiuchi et al, 1997).…”