Expression cloning of a novel farnesylated protein, RDJ2, encoding a DnaJ protein homologue.
The CAAX farnesyltransferase is a heterodimeric enzyme that attaches a farnesyl group to a single cysteine in cellular proteins which terminate in the sequence CAAX, where C is cysteine, A is an aliphatic amino acid, and X is most often methionine or serine. Substrates include the p21ras proteins, nuclear lamins, and a series of retinal proteins. To date, a limited number of substrates for the farnesyltransferase have been identified, predominantly by demonstration of the attachment of a farnesyl group to previously identified cDNA clones which encode proteins containing an appropriate carboxyl-terminal tetrapeptide. We describe here the use of a cDNA fusion protein expression library, together with enzymatic in vitro [3H]farnesyl radiolabeling, as a means of identifying novel farnesylated proteins. One candidate cDNA was fully cloned and found to be a homologue of the Escherichia coli heat shock gene dnaJ. The predicted amino acid sequence of this protein was found to terminate with the tetrapeptide Cys-Ala-His-Gln, which conforms to the consensus sequence for recognition by farnesyltransferase, and was shown to undergo in vivo farnesylation. This farnesylated protein, designated RDJ2 (rat DnaJ homologue 2), is a novel and ubiquitously expressed DnaJ homologue and is the newest member of the subfamily of DnaJ-related proteins which are posttranslationally modified by protein farnesylation.
Andres
D A
DA
Department of Biochemistry, University of Kentucky, College of Medicine, Lexington 40536-0084, USA. dandres@pop.uky.edu
Shao
H
H
Crick
D C
DC
Finlin
B S
BS
D019883:Alkyl and Aryl Transferases / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000235:genetics; D029968:Escherichia coli Proteins; D005204:Farnesol / Q000031:analogs & derivatives*; D015870:Gene Expression; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000235:genetics*; D008798:Mevalonic Acid / Q000378:metabolism; D008969:Molecular Sequence Data; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D017368:Protein Prenylation; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012717:Sesquiterpenes
D018076:DNA, Complementary; C494635:DnaJ protein, E coli; C494643:Dnaja2 protein, rat; D029968:Escherichia coli Proteins; D050956:HSP40 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D011106:Polyisoprenyl Phosphates; D011993:Recombinant Fusion Proteins; D012717:Sesquiterpenes; D005204:Farnesol; C004808:farnesyl pyrophosphate; D019883:Alkyl and Aryl Transferases; C064911:p21(ras) farnesyl-protein transferase; D008798:Mevalonic Acid