Role of the mitochondrial Hsp70s, Ssc1 and Ssq1, in the maturation of Yfh1

The mitochondrial matrix of the yeast Saccharomyces cerevisiae contains two molecular chaperones of the Hsp70 class, Ssc1 and Ssq1. We report that Ssc 1 and Ssq1 play sequential roles in the import and maturation of the yeast frataxin homologue (Yfh1). In vitro, radiolabeled Yfh1 was not imported int o ssc1-3 mutant mitochondria, remaining in a protease-sensitive precursor f orm. As reported earlier, the Yfh1 intermediate form was only slowly proces sed to the mature form in Delta ssq1 mitochondria (S. A. B. Knight, N. B. V . Sepuri, D, Pain, and A. Dancis, J. Biol. Chem. 273:18389-18393, 1998). Ho wever, the intermediate form in both wild-type and Delta ssq1 mitochondria was entirely within the inner membrane, as it was resistant to digestion wi th protease after disruption of the outer membrane. Therefore, we conclude that Ssc1, which is present in mitochondria in approximately a 1,000-fold e xcess over Ssq1, is required for Yfh1 import into the matrix, while Ssq1 is necessary for the efficient processing of the intermediate to the mature f orm in isolated mitochondria. However, the steady-state level of mature Yfh 1 in Delta ssq1 mitochondria is approximately 75% of that found in,wild-typ e mitochondria, indicating that this retardation in processing does not dra matically affect cellular concentrations. Therefore, Ssq1 Likely has roles in addition to facilitating the processing of Yfh1. Twofold overexpression of Ssc1 partially suppresses the cold-sensitive growth phenotype of Delta s sq1 cells, as well as the accumulation of mitochondrial iron and the defect s in Fe/S enzyme activities normally Pound in Delta ssq1 mitochondria. Delt a ssq1 mitochondria containing twofold-more Ssc1 efficiently converted the intermediate form of Yfh1 to the mature form. This correlation between the observed processing defect and suppression of in vivo phenotypes suggests t hat Ssc1 is able to carry out the functions of Ssq1, but only when present in approximately a 2,000-fold excess over normal levels of Ssq1.