We have investigated the unusual import pathway of cytochrome c oxidase subunit Va (COXVa) into the yeast mitochondrial inner membrane by use of mutants that lack import receptors or are defective in matrix hsp70. (i) Mitochondria lacking the receptor MOM72 are not impaired in import of COXVa. Mitochondria lacking the main receptor MOM19 are moderately reduced in import of COXVa; this, however, is caused by a reduction of the inner membrane potential and not by a lack of specific receptor functions. (ii) Mitochondria defective in the unfoldase function of matrix hsp70 efficiently import COXVa, whereas mitochondria defective in the translocase function of the hsp70 are blocked in import of COXVa. A COXVa construct where the internal hydrophobic sorting signal is placed close to the presequence does not require either hsp70 function.

These results demonstrate that import of COXVa does not require MOM19 or MOM72, but they unexpectedly reveal a strong dependence on the translocase function of matrix hsp70. Two important implications about the characterization of mitochondrial protein import in general are obtained. First, the interpretation of import results with mutants lacking MOM19 have to consider effects on the membrane potential. Second, the distance between a matrix targeting sequence and a hydrophobic sorting sequence within a precursor appears to determine if the inner membrane sorting machinery can substitute for the translocase function of hsp70 or not.

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