Isoprenylation of Rab1B is impaired by mutations in its effector domain.
Low molecular mass GTP-binding proteins encoded by the Rab gene family are posttranslationally modified by a specific geranylgeranyltransferase (GGTase II), which catalyzes the thioether linkage of geranylgeranyl isoprenoids to cysteines within one of the following carboxyl-terminal sequence motifs: GGCC, CXC, CCSN. Short peptides containing these sequences are poor substrates for isoprenylation in vitro, suggesting that structural domains remote from the carboxyl terminus are required for interactions between Rab proteins and GGTase II. To begin to define these domains, deletions and point mutations were created within the Rab1B gene, and the ability of the mutant translation products to undergo isoprenylation was evaluated in reticulocyte lysates. Deletion of amino acids 2-9 diminished but did not eliminate isoprenylation of Rab1B, suggesting that the extreme amino-terminal region is not absolutely required for interaction with GGTase II. Longer deletions in the amino-terminal region, which probably disrupt the overall conformation of Rab1B, completely prevented isoprenylation. Site-directed mutations predicted to lie in the amino-terminal variable region (Y5N), the beta 3 strand (Q60E), and Loop 7 (A110D) of the Rab1B structure did not reduce isoprenylation. However, two mutations (I41N, D44N) in the effector domain, which appears to mediate interactions with proteins that stimulate GTP hydrolysis or GDP dissociation, essentially abolished the ability of Rab1B to undergo isoprenylation. These findings imply that the effector domain plays a key role in the isoprenylation of Rab proteins, either by serving as a prenyltransferase binding site or by facilitating interactions with accessory proteins that allow Rab1B to assume a specific guanine nucleotide-dependent conformation that is recognized by GGTase II.
Wilson
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Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822.
Maltese
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D000596:Amino Acids / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004277:DNA, Single-Stranded; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D017354:Point Mutation; D017368:Protein Prenylation; D017384:Sequence Deletion; D020761:rab1 GTP-Binding Proteins
D000596:Amino Acids; D004277:DNA, Single-Stranded; D019204:GTP-Binding Proteins; C061569:RAB1B protein, rat; D020761:rab1 GTP-Binding Proteins