Tim9p, an essential partner subunit of Tim10p for the import of mitochondrial carrier proteins

Tim10p, a protein of the yeast mitochondrial intermembrane space, was shown previously to be essential for the import of multispanning carrier proteins from the cytoplasm into the inner membrane. We now identify Tim9p, another essential component of this import pathway. Most of Tim9p is associated with Tim10p in a soluble 70 kDa complex. Tim9p and Tim10p co‐purify in successive chromatographic fractionations and co‐immunoprecipitated with each other. Tim9p can be cross‐linked to a partly translocated carrier protein. A small fraction of Tim9p is bound to the outer face of the inner membrane in a 300 kDa complex whose other subunits include Tim54p, Tim22p, Tim12p and Tim10p. The sequence of Tim9p is 25% identical to that of Tim10p and Tim12p. A Ser 67 →Cys 67 mutation in Tim9p suppresses the temperature‐sensitive growth defect of tim10‐1 and tim12‐1 mutants. Tim9p is a new subunit of the TIM machinery that guides hydrophobic inner membrane proteins across the aqueous intermembrane space.