Mechanism of Digeranylgeranylation of Rab Proteins

doi:10.1074/jbc.271.7.3692

Mechanism of Digeranylgeranylation of Rab Proteins

FORMATION OF A COMPLEX BETWEEN MONOGERANYLGERANYL-Rab AND Rab ESCORT PROTEIN (*)

From the Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235

^¶To whom correspondence should be addressed:
Dept. of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, TX 75235.
Tel.: 214-648-8643; Fax: 214-648-8804; :seabra{at}utsw.swmed.edu.

Abstract

Rab proteins are Ras-related small GTPases that are digeranylgeranylated at carboxyl-terminal cysteines, a modification essential for their action as molecular switches regulating intracellular vesicular transport. Geranylgeranylation of Rabs is a complex reaction that requires a catalytic Rab geranylgeranyl transferase (GGTase) and a Rab escort protein (REP). REP binds unprenylated Rab and presents it to Rab GGTase. After GG transfer, REP remains associated with diGG-Rab, which leads to insertion of the Rab into a specific membrane. We used recombinant Rab1a single cysteine mutants that accept only one GG group to study the mechanism of the digeranylgeranylation reaction. Using the prenylation assay, gel filtration chromatography, and density ultracentrifugation, we show that REP, but not Rab GGTase, forms a stable complex with unprenylated, monoGG- and diGG-Rab1a. The REP•monoGG-Rab1a complex is stable in the presence of detergents or phospholipids, whereas the REP•diGG-Rab1a complex partially dissociates under these conditions. The stoichiometry of the REP•Rab complex appears to be 1:1 before prenylation. Prenylation induces a change in complex stoichiometry, with the formation of a 2:2 or 2:1 REP•Rab complex. A possible mechanism by which Rab proteins are digeranylgeranylated is suggested by the current studies. We propose that each geranylgeranyl addition is an independent reaction that leads to the production of monoGG-Rab and diGG-Rab, respectively. The stability of the REP•monoGG-Rab complex prevents monoGG-Rab from dissociating from REP prior to the second geranylgeranylation reaction, ensuring efficient digeranylgeranylation of Rab substrates.

Footnotes

↵^§ Supported by a Medical Scientist Training Program grant from the Perot Family Foundation.
↵* This work was supported by research grants from The Foundation Fighting Blindness, National Institutes of Health Grant HL20948, and by the Perot Family Foundation. 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:

GG

geranylgeranyl

GGPP

geranylgeranyl pyrophosphate

GGTase

geranylgeranyl transferase

REP

Rab escort protein

PC

phosphatidylcholine

Sf9

Spodoptera frugiperda-9.
↵²F. Shen and M. C. Seabra, unpublished observations.
- Received July 21, 1995.
- Revision received November 7, 1995.