Search for novel stress-responsive protein components using a yeast mutant lacking two cytosolic Hsp70 genes, SSA1 and SSA2.
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 heat-shocked 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.
Matsumoto
Rena
R
Graduate School of Science and Technology, Chiba University, Chiba 263-8522, Japan. matsumoto-rn@aist.go.jp
Rakwal
Randeep
R
Agrawal
Ganesh Kumar
GK
Jung
Young-Ho
YH
Jwa
Nam-Soo
NS
Yonekura
Masami
M
Iwahashi
Hitoshi
H
Akama
Kuniko
K
D000251:Adenosine Triphosphatases / Q000235:genetics* / Q000502:physiology; D003600:Cytosol / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics* / Q000502:physiology; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000502:physiology; D009154:Mutation / Q000502:physiology*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D014176:Protein Biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism; D015854:Up-Regulation
D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; C542288:SSA2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex; D000251:Adenosine Triphosphatases; C095973:SSA1 protein, S cerevisiae