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The protein import system of the yeast mitochondrial inner membrane includes at least three membrane proteins that presumably form a transmembrane channel as well as several chaperone proteins that mediate the import and refolding of precursor proteins. We show that one of the membrane proteins, Isp45, spans the mitochondrial inner membrane yet is extracted from this membrane at high pH. Solubilization of mitochondria with a nonionic detergent releases Isp45 as a complex with the chaperones mitochondrial hsp7O (mhsp7O) and GrpEp. Both chaperones reversibly dissociate from Isp45 upon addition of ATP or adenosine 5'-[ythioltriphosphate, suggesting that dissociation requires the binding of ATP. Control experiments indicate that the interaction between mhsp7O and Isp45 occurs in the intact mitochondria. We propose that Isp45 lines the inside of a proteinaceous channel across the inner membrane and that it is the membrane anchor for an ATP-driven "import motor" composed of mhsp7O and GrpEp. This arrangement is reminiscent of the protein transport systems of the yeast endoplasmic reticulum and the bacterial plasma membrane.The mitochondrial inner and outer membranes both have their own protein import system (1, 2). Although these two systems can act independently, they cooperate during the import of precursor proteins into the matrix. Insertion of a matrix-targeting presequence into the inner membrane is driven by the electrochemical potential across that membrane; the remainder of the polypeptide chain is then apparently pulled into the matrix by the ATP-dependent action of mitochondrial hsp70 (mhsp70) and its cochaperone, GrpEp (3, 4). (8).In this paper, we show that Isp45 is an unusual membrane protein; it spans the inner membrane, even though it can be extracted at alkaline pH. Upon solubilization of mitochondria with a nonionic detergent, Isp45 is released as a complex with mhsp70 and GrpEp. This complex reversibly dissociates upon addition of ATP. We suggest that the dynamic complex composed of Isp45, mhsp70, and GrpEp represents a forcegenerating motor that mediates the energy-dependent transport of precursors across the mitochondrial inner membrane.MATERIALS AND METHODS General Methods. Mitochondria were prepared from wildtype Saccharomyces cerevisiae strain D273-10B or YZIM6 and purified on a Nycodenz gradient (B.S.G. and L. Pon, unpublished data). Radiolabeled mitochondria (specific activity, 4 x 108 cpm/mg) were isolated from cells grown overnight to an A600 of 0.7 in low-sulfate medium supplemented with 2% lactate and 50 ,Ci of Na235SO4 per ml (1 Ci = 37 GBq) and were mixed with a 10-fold excess of unlabeled mitochondria that had been purified on a Nycodenz gradient. Published methods were used for generating mitoplasts by osmotic shock (9) and for treating mitochondria or mitoplasts for 20 min on ice with 50-100 ,ug of proteinase K per ml (10). mhsp70 was prepared as described (11). Hexahistidinetagged mhsp70 was isolated from mitochondria purified on a Nycodenz gradient (B.S.G. and L. Pon, unpub...

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