Search for Novel Stress-responsive Protein Components Using a Yeast Mutant Lacking Two Cytosolic Hsp70 Genes, SSA1 and SSA2

  • Matsumoto, Rena (Graduate School of Science and Technology, Chiba University) ;
  • Rakwal, Randeep (Human Stress Signal Research Center (HSS), AIST) ;
  • Agrawal, Ganesh Kumar (Research Laboratory for Agricultural Biotechnology and Biochemistry (RLABB)) ;
  • Jung, Young-Ho (Department of Molecular Biology, College of Natural Science, Sejong University) ;
  • Jwa, Nam-Soo (Department of Molecular Biology, College of Natural Science, Sejong University) ;
  • Yonekura, Masami (Food Function Laboratory, School of Agriculture, Ibaraki University) ;
  • Iwahashi, Hitoshi (Human Stress Signal Research Center (HSS), AIST) ;
  • Akama, Kuniko (Graduate School of Science and Technology, Chiba University)
  • Received : 2006.02.03
  • Accepted : 2006.03.28
  • Published : 2006.06.30

Abstract

Heat shock proteins (Hsp) 70 are a ubiquitous family of molecular chaperones involved in many cellular processes. A yeast strain, ssa1/2, with two functionally redundant cytosolic Hsp70s (SSA1 and SSA2) deleted shows thermotolerance comparable to mildly heatshocked wild type yeast, as well as increased protein synthesis and ubiquitin-proteasome protein degradation. Since mRNA abundance does not always correlate well with protein expression levels it is essential to study proteins directly. We used a gel-based approach to identify stress-responsive proteins in the ssa1/2 mutant and identified 43 differentially expressed spots. These were trypsin-digested and analyzed by nano electrospray ionization liquid chromatography tandem mass spectrometry (nESI-LC-MS/MS). A total of 22 non-redundant proteins were identified, 11 of which were confirmed by N-terminal sequencing. Nine proteins, most of which were up-regulated (2-fold or more) in the ssa1/2 mutant, proved to be stress-inducible proteins such as molecular chaperones and anti-oxidant proteins, or proteins related to carbohydrate metabolism. Interestingly, a translational factor Hyp2p up-regulated in the mutant was also found to be highly phosphorylated. These results indicate that the cytosolic Hsp70s, Ssa1p and Ssa2p, regulate an abundance of proteins mainly involved in stress responses and protein synthesis.

Keywords

Cytosolic Hsp70;Edman Sequence;Gel-based Proteome;Hyp2p;Tandem Mass Spectrometry;Yeast

Acknowledgement

Supported by : Korea Science and Engineering Foundation

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