Biochemical and functional characterization of a recombinant GTPase, Rab5, and two of its mutants.
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.
Hoffenberg
S
S
Department of Medicine, Baylor College of Medicine, Houston, Texas 77030.
Sanford
J C
JC
Liu
S
S
Daniel
D S
DS
Tuvin
M
M
Knoll
B J
BJ
Wessling-Resnick
M
M
Dickey
B F
BF
D000595:Amino Acid Sequence; D001483:Base Sequence; D002793:Cholic Acids / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004926:Escherichia coli; D020558:GTP Phosphohydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins
D002793:Cholic Acids; D003902:Detergents; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate