The cleavable presequence is not essential for import and assembly of the phosphate carrier of mammalian mitochondria but enhances the specificity and efficiency of import.

The phosphate carrier (PiC) of mammalian mitochondria is synthesized with a cleavable presequence, in contrast to other members of the mitochondrial family of inner membrane carrier proteins. The precursor of PiC is efficiently imported, proteolytically processed, and correctly assembled in isolated mitochondria. Here we report that a presequence-deficient PiC was imported with an efficiency of about 50% as compared with the authentic precursor of PiC. This mature-sized PiC was correctly assembled, demonstrating that the presequence is not essential for the assembly pathway. We found the following functions for the PiC presequence. (i) The presequence by itself was able to target a passenger protein to mitochondria with a low efficiency, suggesting that the mammalian PiC contains multiple targeting signals, the more efficient one(s) present in the mature protein part. (ii) Deletion of the presequence allowed a more efficient heterologous import of mammalian PiC into mitochondria from Saccharomyces cerevisiae and Neurospora crassa, indicating an important role of the presequence in determining the specificity of PiC import. (iii) Import of the presequence-deficient PiC required a higher membrane potential across the inner membrane than that of the presequence-carrying form. Therefore, the presequence also enhances the translocation of PiC into the inner membrane.