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

doi:10.1074/jbc.270.10.5048

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

From the (1)Departments of Medicine and
(2)Cell Biology, Baylor College of Medicine, Houston, Texas 77030, the
(3)Department of Nutrition, Harvard School of Public Health, Boston, Massachusetts 02115, and the
(4)Division of Pulmonary and Critical Care, VA Medical Center, Houston, Texas 77030

^§To whom correspondence and reprint requests should be addressed:
Div. of Pulmonary and Critical Care Medicine, VA Medical Center, Bldg. 109, Rm. 106, 2002 Holcombe Blvd., Houston, TX 77030.
Tel.: 713-794-7794; Fax: 713-794-7853.

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 ≈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.

Footnotes

↵^¶ A fellow of the American Heart Association (Massachusetts Affiliate).
↵** Recipient of a Junior Faculty Award from the American Cancer Society.
↵* This work was supported by United States Public Health Service Award HL43161 and a Veterans Administration Merit Review Award (to B. F. D.), a grant-in-aid from the American Heart Association, Texas Affiliate (to B. J. K), and Grants 3381 from the Council for Tobacco Research U. S. A., and CB-15 from the American Cancer Society (to M. W. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

WT

wild-type

CHAPS

3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate

PAGE

polyacrylamide gel electrophoresis

GTPS

guanosine 5′-O-(3-thiotriphosphate)

GAP

GTPase activating protein.
- Received May 23, 1994.
- Revision received December 13, 1994.