Document Detail


Prenylation of a Rab1B mutant with altered GTPase activity is impaired in cell-free systems but not in intact mammalian cells.
MedLine Citation:
PMID:  8836150     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Previous studies have reached differing conclusions as to whether or not guanine-nucleotide-dependent conformational changes affect the ability of Rab proteins to undergo post-translational modification by Rab:geranylgeranyltransferase (Rab-GGTase). We now show that the ability of a Rab1B mutant [Q67L (Gln-67-->Leu)] with reduced intrinsic GTPase activity to undergo geranylgeranylation in cell-free assays depends on the guanine nucleotide composition of the system. When GTP is the predominant nucleotide in the assay, Rab1BQ67L is a poor substrate. However, when GDP is present and GTP is omitted, prenylation of the Q67L mutant is comparable with that of the wild-type (WT) protein. These studies, coupled with the poor prenylation of Rab1BWT in the presence of the non-hydrolysable GTP analogue guanosine 5'-[gamma-thio]triphosphate, support the notion that Rab-GGTase prefers substrates in the GDP conformation. When the abilities of Rab1BQ67L and Rab1BWT to undergo prenylation were compared by metabolic labelling of transiently expressed proteins in cultured human 293 cells, we did not observe a decline in prenylation of the mutant protein as predicted on the basis of the cell-free assays. Moreover, the Q67L mutant was comparable with the wild-type Rab1B in its ability to associate with co-expressed Rab GDP dissociation inhibitors in 293 cells. These findings raise the possibility that unidentified proteins present in intact cells may compensate for the reduced intrinsic GTPase activity of the Q67L mutant, allowing a significant proportion of the nascent Rab1BQ67L to assume a GDP conformation. The differential prenylation of Rab1BQ67L in cell-free systems versus intact cells underscores the importance of evaluating the post-translational modification of specific Rab mutants in vivo, where poorly characterized regulatory proteins may have a significant effect on GTPase activity or nucleotide exchange rates.
Authors:
A L Wilson; K M Sheridan; R A Erdman; W A Maltese
Publication Detail:
Type:  In Vitro; Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  The Biochemical journal     Volume:  318 ( Pt 3)     ISSN:  0264-6021     ISO Abbreviation:  Biochem. J.     Publication Date:  1996 Sep 
Date Detail:
Created Date:  1996-11-20     Completed Date:  1996-11-20     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  2984726R     Medline TA:  Biochem J     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  1007-14     Citation Subset:  IM    
Affiliation:
Weis Center for Research, Geisinger Clinic, Danville, PA 17822-2616, USA.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Animals
Base Sequence
Cattle
Cell Line
Cell-Free System
DNA Primers / genetics
GTP Phosphohydrolases / genetics*,  metabolism*
GTP-Binding Proteins / genetics*,  metabolism*
Guanine Nucleotide Dissociation Inhibitors*
Guanosine Diphosphate / metabolism
Guanosine Triphosphate / metabolism
Humans
Mutagenesis, Site-Directed
Point Mutation
Protein Prenylation
Rats
Recombinant Proteins / genetics,  metabolism
Reticulocytes / metabolism
rab1 GTP-Binding Proteins*
Grant Support
ID/Acronym/Agency:
CA34569/CA/NCI NIH HHS
Chemical
Reg. No./Substance:
0/DNA Primers; 0/GDP dissociation inhibitor 1; 0/Guanine Nucleotide Dissociation Inhibitors; 0/Recombinant Proteins; 146-91-8/Guanosine Diphosphate; 86-01-1/Guanosine Triphosphate; EC 3.6.1.-/GTP Phosphohydrolases; EC 3.6.1.-/GTP-Binding Proteins; EC 3.6.1.-/RAB1B protein, rat; EC 3.6.5.2/rab1 GTP-Binding Proteins
Comments/Corrections

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