Novel interaction of the Hsp90 chaperone machine with Ssl2, an essential DNA helicase in Saccharomyces cerevisiae

Abstract

Hsp90 is an essential molecular chaperone that is critical for the activity of diverse cellular proteins. Hsp90 functions with a number of co-chaperone proteins, including Sti1/Hop. We conducted a genetic screen in Saccharomyces cerevisiae to isolate mutations that exhibit enhanced growth defects in the absence of STI1. We obtained mutations in genes encoding components of the Hsp90 chaperone machine, HSC82, CPR7 and YDJ1, and two essential genes, SSL2 and UTP21, not previously linked to Hsp90. Ssl2, the yeast homologue of XPB, is an ATP-dependent DNA helicase that is a component of the TFIIH multiprotein complex and has dual functions in transcription and DNA repair. In order to determine whether Ssl2 function is dependent on Hsp90, we further examined the interaction between Ssl2 and Hsp90. Multiple mutant alleles of SSL2 exhibited a pronounced growth defect when co-expressed with a mutant allele of Hsp90. In addition, isolation of Ssl2 protein resulted in the co-purification of Hsp90 and Sti1, suggesting that Ssl2 and Hsp90 are in the same protein complexes in vivo. These results suggest a novel role for Hsp90 in the essential cellular functions of transcription and DNA repair.

Fig. 1

SL182 contains a mutation in SSL2. a Domain structure of Ssl2. Ssl2 contains six conserved helicase motifs (shown in black). The location of the single amino acid mutation (ssl2-L691I) in strain SL182 is indicated. b Growth of sti1 screening strain JJ47 and original SL182 candidate strain with (+STI1) or without the pRS316 ADE3STI1 plasmid. c Growth of ssl2-L691I mutant allele in ssl2 strain (JJ546) or ssl2sti1 strain (JJ830). A plasmid expressing wild-type (WT) or mutant ssl2 alleles was transformed into the indicated strains and transformants were grown in the presence of 5-FOA to counterselect for YCp50-SSL2. Indicated yeast strains were serially diluted ten-fold, spotted onto YPD and grown for 2 days at the indicated temperature

Fig. 2

Additional ssl2 alleles exhibit STI1-dependent growth. a The location of additional ssl2 alleles described in the text are indicated. b Growth of indicated ssl2 mutant alleles in ssl2 strain (JJ546) or ssl2 sti1 strain (JJ830). Mutant ssl2 alleles were transformed into the indicated strains and transformants were grown in the presence of 5-FOA to counterselect for YCp50-SSL2. Indicated yeast strains were serially diluted ten-fold, spotted onto YPD and grown for 2 days at the indicated temperature

Fig. 3

The ssl2 alleles exhibit Hsp90-dependent growth. Left panel hsc82 hsp82 ssl2/Ycp50-SSL2 strains expressing HSC82 (JJ835;+) or hsc82-G309S (JJ839; −) were transformed with ssl2 alleles and plated onto medium containing 5-FOA to counterselect for YCp50-SSL2. Right panel hsc82 hsp82 ssl2/Ycp50-SSL2 strains expressing HSP82 (JJ869; +) or hsp82-G313S (JJ870; −) were transformed with ssl2 alleles and plated onto medium containing 5-FOA to counterselect for YCp50-SSL2. Transformants were grown overnight, serially diluted ten-fold, spotted onto YPD and grown for 2 days at 30°C

Fig. 4

Deletion of STI1 enhances the senstitivity of some ssl2 strains to UVC. Mutant ssl2 alleles were transformed into strain JJ546 (STI1+) or JJ830(STI1−) and transformants were grown in the presence of 5-FOA to counterselect for YCp50-SSL2. The indicated yeast strains were serially diluted ten-fold, spotted onto YPD, exposed to the indicated doses of UVC and grown for 2 days in the dark at 30°C

Fig. 5

Strains with mutations in the Hsp90 chaperone machine exhibit enhanced sensitivity to UVC. The indicated yeast strains were serially diluted ten-fold, spotted onto YPD, exposed to the indicated doses of UVC and grown for 2 days in the dark at 30°C. Top panel Strain JJ546 expressing WT SSL2 or ssl2-XP, middle panel strain JJ816 (hsc82 hsp82) expressing WT HSC82, hsc82-G309S, WT HSP82 or hsp82-G313S, bottom panel WT (PJ43-1B), sti1 (JJ97), sba1 (YF223) and sba1sti1 (JJ808)

Fig. 6

Interaction of Flag-Ssl2 with Hsp90. Left panel Affinity isolation of Flag-Ssl2 out of yeast lysates. Lanes 1, 2 Yeast lysates from ssl2 strains expressing wild-type Ssl2 (−) or Flag-Ssl2 (+) were analyzed by SDS-PAGE and immunoblot analysis. Lanes 3, 4 Proteins retained on the anti-Flag affinity resin were resuspended in sample buffer and analyzed by SDS-PAGE and immunoblot analysis: lane 3 Flag-Ssl2 (+) cells expressing Cal-N-Flag Ssl2, lane 4 cells expressing wild-type Ssl2 (−). Immnoblot analysis was performed using antibodies that recognized Flag-Ssl2 (α-Flag), Hsc82/Hsp82 (α-Hsp90) or Sti1. Right panel Interaction of Flag-Ssl2 with Hsp90 in rabbit reticulocyte lysate. ³⁵S-labeled Flag-Ssl2 was synthesized in rabbit reticulocyte lysates (RL). Lane 1 RL from control in vitro synthesis reaction with the pCAL-N-Flag vector. Lane 2 RL showing the migration of synthesized radiolabeled Flag-Ssl2. Equal amounts of Flag-Ssl2 were added to fresh RL prior to immunoprecipitation of protein complexes with antibodies specific for: lane 3 no antibody, lane 4 α-Flag, lane 5 α-Hsp90, lane 6 α-Hop. Samples were separated by SDS-PAGE and autoradiographed to detect radiolabeled Flag-Ssl2