EXPRESSION CLONING OF A NOVEL FARNESYLATED PROTEIN, RDJ2, ENCODING A DNAJ PROTEIN HOMOLOG

The CAAX farnesyltransferase is a heterodimeric enzyme that attaches a farnesyl group to a single cysteine in cellular proteins which termin ate in the sequence CAAX, where C is cysteine, A is an aliphatic amino acid, and X is most often methionine or serine, Substrates include th e p21(ras) proteins, nuclear lamins, and a series of retinal proteins. To date, a limited number of substrates for the farnesyltransferase h ave 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, tog ether with enzymatic in vitro [H-3]farnesyl radiolabeling, as a means of identifying novel farnesylated proteins. One candidate cDNA was ful ly cloned and found to be a homologue of the Escherichia coil heat sho ck gene dnaJ. The predicted amino acid sequence of this protein was fo und 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 protei n farnesylation. (C) 1997 Academic Press.