rab GTP-binding proteins with three different carboxyl-terminal cysteine motifs are modified in vivo by 20-carbon isoprenoids.
p21ras and several other ras-related GTP-binding proteins are modified post-translationally by addition of 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoids to cysteines within a conserved carboxyl-terminal sequence motif, Caa(M/S/L), where a is an aliphatic amino acid. Proteins ending with M or S are substrates for farnesyltransferase, whereas those ending with L are modified preferentially by geranylgeranyltransferase. We recently reported that GTP-binding proteins encoded by rab1B (GGCC), rab2 (GGCC), and rab5 (CCSN) are modified by 20-carbon isoprenyl derivatives of [3H]mevalonate when translated in vitro, despite having carboxyl-terminal sequences distinct from the Caa(M/S/L) motif. We now show that these proteins function as specific acceptors for geranylgeranyl in vitro and are modified by 20-carbon isoprenyl groups in COS cells metabolically labeled with [3H]mevalonate. Proteins encoded by rab4 and rab6, with yet another distinct carboxyl-terminal motif (xCxC), are similarly modified by 20-carbon isoprenoids in vitro and in vivo. The geranylgeranyl modification of rab5 protein (CCSN) is catalyzed by an enzyme in brain cytosol but not by a purified geranylgeranyltransferase that modifies GTP-binding proteins with the CaaL motif. Unlike the prenylation of proteins with Caa(M/S/L) termini, the prenylation of rab5 protein is not inhibited by a synthetic peptide based on its carboxyl-terminal sequence (TRNQCCSN). When cellular isoprenoid synthesis is blocked by treatment of cells with lovastatin, rab proteins that are normally localized in membranes of the endoplasmic reticulum, Golgi apparatus, and endosomes accumulate in the cytosol. This change in rab protein localization is reversed by providing cells with mevalonate. These findings suggest that geranylgeranyl modification underlies the ability of rab GTP-binding proteins to associate with intracellular membranes, where they are postulated to function as mediators of vesicular traffic.
Kinsella
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BT
Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822.
Maltese
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WA
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002264:Carboxylic Acids / Q000378:metabolism; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D000882:Haplorhini; D008148:Lovastatin / Q000494:pharmacology; D008798:Mevalonic Acid / Q000378:metabolism; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins
D002264:Carboxylic Acids; D011994:Recombinant Proteins; D004247:DNA; D008148:Lovastatin; D019204:GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins; D003545:Cysteine; D008798:Mevalonic Acid