A multisubunit complex of outer and inner mitochondrial membrane protein translocases stabilized in vivo by translocation intermediates

Translocation of nuclear encoded preproteins into the mitochondrial matrix requires the coordinated action of two translocases: one (Tom) located in t he outer mitochondrial membrane and the other (Tim) located in the inner me mbrane. These translocases reversibly cooperate during protein import. We h ave previously constructed a chimeric precursor (pPGPrA) consisting of an a uthentic mitochondrial precursor at the N terminus (Delta(1)-pyrroline-5-ca rboxylate dehydrogenase, pPut) linked, through glutathione S-transferase, t o protein A. When pPGPrA is expressed in yeast, it becomes irreversibly arr ested during translocation across the outer and inner mitochondrial membran es. Consequently, the two membranes of mitochondria become progressively "z ippered" together, forming long stretches in which they are in close contac t (Schulke, N., Sepuri, N. B. V., and Pain, D. (1997) Proc. Natl. Acad. Sci . U. S. A. 94, 7314-7319), We now demonstrate that trapped PGPrA intermedia tes hold the import channels stably together and inhibit mitochondrial prot ein import and cell growth. Using IgG-Sepharose affinity chromatography of solubilized zippered membranes, we have isolated a multisub unit complex th at contains all Tom and Tim components known to be essential for import of matrix-targeted proteins, namely Tom40, Tom22, Tim17, Tim23, Tim44, and mat rix-localized Hsp70. Further characterization of this complex may shed ligh t on structural features of the complete mitochondrial import machinery.