Sti1 is a novel activator of the Ssa proteins.
The molecular chaperones Hsp70 and Hsp90 are involved in the folding and maturation of key regulatory proteins in eukaryotes. Of specific importance in this context is a ternary multichaperone complex in which Hsp70 and Hsp90 are connected by Hop. In Saccharomyces cerevisiae two components of the complex, yeast Hsp90 (yHsp90) and Sti1, the yeast homologue of Hop, had already been identified, but it remained to be shown which of the 14 different yeast Hsp70s are part of the Sti1 complex and what were the functional consequences resulting from this interaction. With a two-hybrid approach and co-immunoprecipitations, we show here that Sti1 specifically interacts with the Ssa group of the cytosolic yeast Hsp70 proteins. Using purified components, we reconstituted the dimeric Ssa1-Sti1 complex and the ternary Ssa1-Sti1-yHsp90 complex in vitro. The dissociation constant between Sti1 and Ssa1 was determined to be 2 orders of magnitude weaker than the affinity of Sti1 for yHsp90. Surprisingly, binding of Sti1 activates the ATPase of Ssa1 by a factor of about 200, which is in contrast to the behavior of Hop in the mammalian Hsp70 system. Analysis of the underlying activation mechanism revealed that ATP hydrolysis is rate-limiting in the Ssa1 ATPase cycle and that this step is accelerated by Sti1. Thus, Sti1 is a potent novel effector for the Hsp70 ATPase.
Wegele
Harald
H
Institut für Organische Chemie & Biochemie, Technische Universität München, Lichtenbergstrasse 4, 85747 Garching, Germany.
Haslbeck
Martin
M
Reinstein
Jochen
J
Buchner
Johannes
J
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D015374:Biosensing Techniques; D002851:Chromatography, High Pressure Liquid; D004305:Dose-Response Relationship, Drug; D005656:Fungal Proteins / Q000378:metabolism* / Q000502:physiology*; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006360:Heat-Shock Proteins; D006868:Hydrolysis; D007700:Kinetics; D009154:Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D020349:Surface Plasmon Resonance; D013997:Time Factors; D020798:Two-Hybrid System Techniques
D005656:Fungal Proteins; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D006360:Heat-Shock Proteins; C542288:SSA2 protein, S cerevisiae; C090869:SSA3 protein, S cerevisiae; C456269:SSA4 protein, S cerevisiae; C542289:STI1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C095973:SSA1 protein, S cerevisiae