Isu, the scaffold for assembly of Fe-S clusters in the yeast mitochondrial matrix, is a substrate protein for the Hsp70 Ssq1 and the J-protein Jac1 in vitro. As expected for an Hsp70-substrate interaction, the formation of a stable complex between Isu and Ssq1 requires Jac1 in the presence of ATP. Here we report that a conserved tripeptide, PVK, of Isu is critical for interaction with Ssq1 because amino acid substitutions in this tripeptide inhibit both the formation of the Isu-Ssq1 complex and the ability of Isu to stimulate the ATPase activity of Ssq1. These biochemical defects correlate well with the growth defects of cells expressing mutant Isu proteins. We conclude that the Ssq1-Isu substrate interaction is critical for Fe-S cluster biogenesis in vivo. The ability of Jac1 and mutant Isu proteins to cooperatively stimulate the ATPase activity of Ssq1 was also measured. Increasing the concentration of Jac1 and mutant Isu together but not individually partially overcame the effect of the reduced affinity of the Isu mutant proteins for Ssq1. These results, along with the observation that overexpression of Jac1 was able to suppress the growth defect of an ISU mutant, support the hypothesis that Isu is "targeted" to Ssq1 by Jac1, with a preformed Jac1-Isu complex interacting with Ssq1.

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 ⌬ssq1 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.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 (1-3). These various activities rely on the ability of Hsp70s to reversibly bind hydrophobic segments of proteins. Affinity of Hsp70s for substrate depends on the conformation of the C-terminal substrate binding domain, which is regulated by the nucleotide bound to the N-terminal ATPase domain. When ATP is bound to Hsp70, an open conformation of the substrate binding domain allows fast binding and release; when ADP is bound, a closed conformation of the substrate binding domain results in slow binding and release. Under physiological conditions, when ATP concentrations are typically high substrate protein interacts with Hsp70 in the ATP conformation. This interaction is stabilized upon hydrolysis of ATP. The cycle of interaction is completed when ADP is replaced by ATP and the substrate is released.Thus, stimulation of the ATPase activity of Hsp70 is essential for stabilization of the Hsp70-substrate interaction. However, the rate of the intrinsic ATPase activity of Hsp70 is low. ATPase activity is stimulated both by interaction of the substrate in the substrate binding cleft and J-protein co-chaperone interaction with the ATPase domain. Some J-proteins not only stimulate the ATPase activity of their partner Hsp70 but also bind substrates directly. This interaction has led to the suggestion that these proteins play an important role in targeting substrates for Hsp70 binding (4 -7).Biogenesis of Fe-S clusters is a critical step in the maturation of the numerous cellular proteins that contain this prosthetic group. In yeast mitochondria,...