Sequence-specific interaction between mitochondrial Fe-S scaffold protein Isu and Hsp70 Ssq1 is essential for their in vivo function.
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.
Dutkiewicz
Rafal
R
Department of Molecular and Cellular Biology, University of Gdansk, 24 Kladki, 80-822 Gdansk, Poland.
Schilke
Brenda
B
Cheng
Sara
S
Knieszner
Helena
H
Craig
Elizabeth A
EA
Marszalek
Jaroslaw
J
D000251:Adenosine Triphosphatases / Q000378:metabolism; D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D018840:HSP70 Heat-Shock Proteins; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism*; D046911:Macromolecular Substances; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D018840:HSP70 Heat-Shock Proteins; C121520:ISU1 protein, S cerevisiae; D007506:Iron-Sulfur Proteins; C115914:JAC1 protein, S cerevisiae; D046911:Macromolecular Substances; D024101:Mitochondrial Proteins; D018832:Molecular Chaperones; D010455:Peptides; C115913:SSQ1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D000251:Adenosine Triphosphatases