Mutagenesis of a functional chimeric gene in yeast identifies mutations in the simian virus 40 large T antigen J domain Sheara W. Fewell, James M. Pipas, and Jeffrey L. Brodsky† Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260 Communicated by Mary Edmonds, University of Pittsburgh, Pittsburgh, PA, December 14, 2001 (received for review July 2, 2001) Simian virus 40 large T antigen contains an amino terminal J Escherichia coli (27) and the J domains from two human DnaJ domain that catalyzes T antigen-mediated viral DNA replication homologs can substitute for the amino terminus of T antigen in and cellular transformation. To dissect the role of the J domain in promoting viral DNA replication (21) or enhancing Tst-1 tran- these processes, we exploited the genetic tools available only in scription factor activity (28). Because T antigen inactivates the yeast Saccharomyces cerevisiae to isolate 14 loss-of-function normal cellular growth controls by altering the retinoblastoma point mutations in the T antigen J domain. This screen also (Rb) family of tumor suppressors (pRb, p107, and p130) and p53 identified mutations that, when engineered into simian virus 40, (26, 29–31), we proposed that T antigen performs its diverse resulted in T antigen mutants that were defective for the ability to functions by recruiting cellular hsc70 to modulate the activities support viral growth, to transform mammalian cells in culture, to of these multiprotein complexes (32). However, validation of this dissociate the p130–E2F4 transcription factor complex, and to ‘‘chaperone model’’ requires the correlation of in vivo T antigen- stimulate ATP hydrolysis by hsc70, a hallmark of J domain-contain- mediated phenotypes with biochemical measurements of T ing molecular chaperones. These data correlate the chaperone antigen chaperone activity. To accomplish this goal, we estab- activity of the T antigen J domain with its roles in viral infection and lished a genetic screen in yeast from which T antigen mutants cellular transformation and support a model by which the viral J compromised for SV40 growth and the ability to promote domain recruits the cytoplasmic hsc70 molecular chaperone in the cellular transformation were identified. Biochemical measure- host to rearrange multiprotein complexes implicated in replication ments of the purified wild-type and mutant T antigens indicated and transformation. More generally, this study presents the use of that these mutants were defective for the stimulation of hsc70 a yeast screen to identify loss-of-function mutations in a mamma- ATPase activity. This study also describes the random mutagen- lian virus and can serve as a widely applicable method to uncover esis of a J domain toward the goal of identifying noncomple- domain functions of mammalian proteins for which there are yeast menting mutants. homologues with selectable mutant phenotypes. Materials and Methods sp40–hsc70 molecular cochaperones modulate the confor- Plasmid Construction and Genetic Screen. The Saccharomyces cer- Hmations of polypeptide substrates to perform diverse func- evisiae strain ACY17b (MAT␣ ade2–1 leu2–3 112, his3–11 15 tions including the folding of nascent polypeptides, the transport trp1–1 ura3–1 can1–100 ydj1–2::HIS3 LEU2::ydj1–151; ref. 33) of proteins across organellar membranes, and the rearrangement was manipulated by standard procedures (34). The chimeric of multiprotein complexes (1). Members of the hsp40 family are T-Ydj1 fusion was created by sequential PCRs. In the first round, Ϸ defined molecularly by the presence of an 70-aa motif called oligonucleotides TJ1A (see Table 2, which is published as the J domain and biochemically by their ability to stimulate ATP supporting information on the PNAS web site, www.pnas.org, for hydrolysis by hsc70 partner chaperones. Hsc70s bind and release oligonucleotide sequences) and TJ3B were used to amplify DNA polypeptide substrates through the coordinated actions of their encoding the T antigen J domain with an EcoRI site at the 5Ј end amino-terminal ATPase and carboxyl-terminal substrate- and a short sequence complementary to the region downstream binding domains; transient interactions between ATP-bound of the YDJ1 J domain at the 3Ј end. In the second PCR step, hsc70 and polypeptide substrates are stabilized upon ATP DNA encoding residues 82–409 of YDJ1 from pAV6 (obtained hydrolysis (2–4). The J domain of hsp40 chaperones directly from A. Caplan, Mount Sinai School of Medicine, New York) interacts with the ATPase domain of hsc70s and regulates the and a NotI site at the 3Ј end was amplified with oligonucleotides affinity of hsc70 for its substrates by stimulating ATP hydrolysis TJ3A and TJ2B. In the final PCR, the products from the first two (5–7). Other domains in hsp40s may bind specific substrates and amplifications were fused, and the resulting fragment was di- target them to hsc70 (8–11). gested with EcoRI and NotI and cloned into the EcoRI and NotI Structural studies on four hsp40 homologs define a common sites of pYes2 (Invitrogen) to create pT-Ydj1. Mutant T-Ydj1 element in the J domain (12–15). J domains consist of four constructs were generated similarly with dl1135 (35), P43L͞ ␣-helices arranged such that the antiparallel second and third K45N, or D44E͞G47R (provided by Kathy Rundell, Northwest- helices form a finger-like projection. An invariant HPD se- ern University School of Medicine, Chicago) T antigen con- quence located in the loop connecting the second and third helices is critical for the interaction with and stimulation of hsc70 structs as template DNA in the initial PCR. The mutations in the (16, 17), but additional residues on the surface of helix II may J domains in each of these constructs were confirmed by DNA also contact hsc70 (18). sequence analysis. The large tumor antigen of simian virus 40 (SV40) T antigen To screen for T antigen J domain mutants, the J domain in contains a J domain that is essential for most aspects of SV40 infection and contributes to the transformation of cultured Abbreviations: SV40, simian virus 40; Rb, retinoblastoma. rodent cells and the promotion of tumorigenesis in transgenic †To whom reprint requests should be addressed at: 257 Crawford Hall, University of mice (19–23). Wild-type T antigen associates directly with hsc70 Pittsburgh, Pittsburgh, PA 15260. E-mail: [email protected]. in vivo and in vitro (24, 25) and is capable of stimulating hsc70 The publication costs of this article were defrayed in part by page charge payment. This ATPase activity in vitro (22, 26). In addition, the T antigen J article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. domain can functionally replace the J domain of DnaJ in §1734 solely to indicate this fact. 2002–2007 ͉ PNAS ͉ February 19, 2002 ͉ vol. 99 ͉ no. 4 www.pnas.org͞cgi͞doi͞10.1073͞pnas.042670999 Downloaded by guest on September 28, 2021 pT-Ydj1 was mutagenized by modified error-prone PCR (36). confirmed by Immunoblot analysis using mAb419, and stored at Two sets of PCRs containing A or G at a final concentration of Ϫ20°C. Wild type and each mutant T antigen were purified at 20 ␮M and the other deoxynucleotides at 200 ␮M were per- least twice for biochemical studies. At initial time points, yeast- formed with oligonucleotides SWF3 and SWF4 and TaqDNA purified T antigen stimulated Hsc70 ATPase activity comparably polymerase (Roche Molecular Biochemicals). DNA from mul- to T antigen from insect cells (40), and more than 90% of the tiple reactions was purified from agarose gels and cotransformed Hsc70-bound ATP was hydrolyzed after a 10-min incubation with a gapped version of pT-Ydj1 into ACY17b. To generate the with either yeast or insect-purified T antigen (compare figure 4 gapped pT-Ydj plasmid, a Bgl11 site was introduced just down- to figure 7 in ref. 26). stream of the T antigen J domain with the Quickchange Mu- tagenesis Kit (Stratagene) and oligonucleotides SWF1 and Hsc70 ATPase and Gel-Shift Assays. Single turnover ATPase ex- SWF2. The resulting plasmid was digested with EcoR1 and BglII, periments and E2F gel-shift assays were performed as de- purified, and transformed into yeast. Cells containing plasmids scribed (26). repaired by homologous recombination between the mu- tagenized J domain fragment and the gapped vector (37) were Results selected by plating transformation mixtures onto synthetic com- The T Antigen J Domain Functionally Replaces the J Domain of YDJ1. plete medium lacking uracil. Transformants were subsequently To test our chaperone model for T antigen function and to screened for growth on galactose-containing medium at 25°C, initiate a structure–function analysis of the T antigen J domain, 35°C, and 37°C. Of a total of 1,800 transformants screened, 115 we required a pool of unique J domain mutants. Because candidates were selected. Expression of the T-Ydj1 mutants was traditional methods for isolating mutations are labor intensive or subsequently examined by immunoblot analysis using mAb419 generate compound mutations (19), we developed a genetic that recognizes the T antigen J domain (38). From this analysis, screen that exploited a functional chimeric gene in yeast to Ϸ50% of the mutant proteins were not expressed and were not rapidly identify chaperone-defective point mutations in the T examined further. Plasmids in the remaining temperature- antigen J domain. In principle, our screen should be generally sensitive candidates were rescued from yeast, amplified in bac- applicable to investigators who want to generate a large set of teria, and reintroduced into ACY17b to confirm the loss-of- random mutations in a defined domain in a mammalian gene as function phenotype, resulting in 41 mutants. DNA sequencing long as mutation of a yeast homologue displays a tractable using the Amplicycle sequencing kit (Perkin–Elmer) identified phenotype. 14 unique point mutations in the T antigen J domain.