ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 346, No. 1, October 1, pp. 113–124, 1997 Article No. BB970296 Expression Cloning of a Novel Farnesylated Protein, RDJ2, Encoding a DnaJ Protein Homologue 1 Douglas A. Andres, 2 Haipeng Shao, Dean C. Crick, and Brian S. Finlin Department of Biochemistry, University of Kentucky, College of Medicine, Lexington, Kentucky 40536-0084 Received June 2, 1997, and in revised form July 10, 1997 isoprenoids, either 15-carbon farnesyl or 20-carbon ger- The CAAX farnesyltransferase is a heterodimeric en- anylgeranyl, can be found attached to proteins in thio- zyme that attaches a farnesyl group to a single cysteine ether linkage to conserved cysteine residues at or near in cellular proteins which terminate in the sequence their carboxyl terminus. Most prenylated proteins are CAAX, where C is cysteine, A is an aliphatic amino acid, thought to serve as regulators of signal transduction and X is most often methionine or serine. Substrates and membrane trafficking, and prenylation has been include the p21 ras proteins, nuclear lamins, and a series shown to promote both the protein–protein and pro- of retinal proteins. To date, a limited number of sub- tein – membrane interactions of these molecules (2, 3). strates for the farnesyltransferase have been identified, Prenylation provides a mechanism for the membrane predominantly by demonstration of the attachment of localization of proteins which lack a transmembrane a farnesyl group to previously identified cDNA clones domain and appears to be a prerequisite for their in which encode proteins containing an appropriate car- vivo activity. boxyl-terminal tetrapeptide. We describe here the use Three distinct protein prenyltransferases catalyzing of a cDNA fusion protein expression library, together these modifications have been identified (4, 5). Two ger- with enzymatic in vitro [ 3 H]farnesyl radiolabeling, as a anylgeranyltransferases (GGTases) 3 have been charac- means of identifying novel farnesylated proteins. One terized and are known to modify distinct protein sub- candidate cDNA was fully cloned and found to be a ho- strates. The CAAX GGTase (also known as GGTase- mologue of the Escherichia coli heat shock gene dnaJ. 1) geranylgeranylates proteins which end in a CAAL The predicted amino acid sequence of this protein was sequence, where C is cysteine, A is usually an aliphatic found to terminate with the tetrapeptide Cys-Ala-His- amino acid, and L is leucine (1, 5). CAAX GGTase sub- Gln, which conforms to the consensus sequence for rec- strates include the g subunits of many heterotrimeric ognition by farnesyltransferase, and was shown to un- GTP-binding regulatory proteins (G proteins) and Ras- dergo in vivo farnesylation. This farnesylated protein, related small G proteins such as Rac1, Rac2, and designated RDJ2 (rat DnaJ homologue 2), is a novel and ubiquitously expressed DnaJ homologue and is the Rap1B (4). Rab GGTase (also known as GGTase-2) cat- newest member of the subfamily of DnaJ-related pro- alyzes the attachment of two geranylgeranyl groups to teins which are posttranslationally modified by protein paired carboxyl-terminal cysteines in most members farnesylation.  1997 Academic Press of the Rab family of GTP-binding proteins (6). These Key Words: protein isoprenylation; farnesyltransfer- proteins terminate in a CC or CXC motif, where X is a ase; DnaJ. small hydrophobic amino acid. Less common in cells is the modification of substrates by CAAX farnesyltrans- ferase (FTase) (7). All known farnesylated proteins ter- minate in a tetrapeptide CAAX box, wherein C is cys- Covalent modification of proteins by isoprenoid lipids (prenylation) is now known to be a common posttrans- teine, A is an aliphatic amino acid, and X has been shown to be a C-terminal methionine, serine, gluta- lational event in mammalian cells (1, 2). Two distinct mine, cysteine, or alanine (8). Known farnesylated pro- teins include p21 ras , nuclear lamins A and B, the a and 1 This work was supported in part by NIH Grant EY11231 and aided by Grant IRG-77653 from the American Cancer Society. The GenBank accession number for rat RDJ2 is U95727. 3 Abbreviations used: FTase, protein farnesyltransferase; GGTase, 2 To whom correspondence should be addressed at Department of Biochemistry, College of Medicine, University of Kentucky, 800 Rose protein geranylgeranyltransferase; HMG – CoA, 3-hydroxy-3-methyl- glutaryl – coenzyme A; IPTG, isopropyl-b-D-thiogalactopyranoside; Street, Lexington, KY 40536-0084. Fax: (606) 323-1037. E-mail: dandres@pop.uky.edu. kb, kilobase(s). 113 0003-9861/97 $25.00 Copyright  1997 by Academic Press All rights of reproduction in any form reserved. AID ABB 0296 / 6b3f$$$121 08-29-97 22:15:43 arcal