Mechanism of digeranylgeranylation of Rab proteins. Formation of a complex between monogeranylgeranyl-Rab and Rab escort protein.
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.
Shen
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Department of Molecular Genetics, University of Texas Southwestern Medical Center, Dallas, Texas 75235, USA.
Seabra
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MC
D019883:Alkyl and Aryl Transferases; D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003545:Cysteine; D003902:Detergents / Q000494:pharmacology; D004285:Dogs; D004926:Escherichia coli; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000494:pharmacology; D011485:Protein Binding; D017368:Protein Prenylation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014166:Transferases / Q000378:metabolism*; D014316:Tritium; D020691:rab GTP-Binding Proteins; D020761:rab1 GTP-Binding Proteins
D002352:Carrier Proteins; D003902:Detergents; D010743:Phospholipids; D011994:Recombinant Proteins; D014316:Tritium; D014166:Transferases; D019883:Alkyl and Aryl Transferases; C083291:geranylgeranyltransferase type-I; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D020691:rab GTP-Binding Proteins; D020761:rab1 GTP-Binding Proteins; D003545:Cysteine