Abstract
Ssq1, a specialized yeast mitochondrial Hsp70, plays a critical role in the biogenesis of proteins containing Fe-S clusters through its interaction with Isu, the scaffold on which clusters are built. Two substitutions within the Ssq1 substrate binding cleft, both of which severely reduced affinity for Isu, had very different effects in vivo. Cells expressing Ssq1(F462S), which had no detectable affinity for Isu, are indistinguishable from Deltassq1 cells, underscoring the importance of the Ssq1-Isu1 interaction in vivo. In contrast, cells expressing Ssq1(V472F), whose affinity for Isu is at least 10-fold lower than that of wild-type Ssq1, had only moderately reduced Fe-S enzyme activities and increased iron levels and grew similarly to wild-type cells. Consistent with the reduced affinity for Isu, the ATPase activity of Ssq1(V472F) was stimulated less well than that of Ssq1 upon addition of Isu and Jac1, the J-protein partner of Ssq1. However, higher concentrations of Jac1 or Isu1, which form a stable complex, could compensate for this defect in stimulation of Ssq1(V472F). Expression of Isu1 was up-regulated 10-fold in ssq1(V472F) compared with wild-type cells, suggesting that formation of a Jac1-Isu1 complex can overcome a lowered affinity of Ssq1 for Isu in vivo as well as in vitro.
Highlights
Chaperones of the Hsp70 family function in a number of essential cellular roles, including de novo protein folding, refolding of unfolded proteins, and protein translocation across intracellular membranes [1,2,3]
Phenotypic Effects of Alterations in the Substrate Binding Cleft of Ssq1—To investigate the functional importance of the interaction of Ssq1 with its protein substrate, Isu1, we introduced alterations in Ssq1 at residues analogous to those previously shown to be important for the ability of other Hsp70s to bind substrate proteins (29 –31)
The null phenotype of a ssq1 mutant encoding a single amino acid alteration in the predicted substrate binding cleft of Ssq1 underscores the importance of substrate binding in the physiological function of this Hsp70. This result is not surprising, as interaction with substrate proteins is fundamental to molecular chaperone function [1, 2]
Summary
Chaperones of the Hsp70 family function in a number of essential cellular roles, including de novo protein folding, refolding of unfolded proteins, and protein translocation across intracellular membranes [1,2,3]. As expected of a J-protein partner, Jac1 stimulates the ATPase activity of Ssq1 [13] and promotes formation of stable Ssq1-Isu complex in the presence of ATP. To test whether binding of Ssq1 wild-type and mutant proteins to the immobilized P-PVK substrate was sequence-specific, increasing concentrations of peptide were passed over the chip together with Ssq1.
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call