Import of rat mitochondrial citrate carrier (CIC) at increasing salt concentrations promotes presequence binding to import receptor Tom20 and inhibits membrane translocation

Mitochondria contain a family of related carrier proteins that mediate transport of metabolites across the mitochondrial inner membrane. All members of this family are synthesized in the cytosol. We characterized the interactions of newly synthesized rat citrate carrier (CIC) precursor protein (pCIC) with the components of the mitochondrial protein import machinery. pCIC contains both a positively charged presequence of 13 amino acids and internal targeting sequences. We found that the pCIC presequence does not interfere with the import pathway and merely acts as an internal chaperone in the cytosol. Under conditions of increased ionic strength, the pCIC presequence binds to the import receptor Tom20 (translocase of the mitochondrial outer membrane, component of 20 kDa) and accumulates at the mitochondrial surface, thereby delaying pCIC translocation across the mitochondrial outer membrane. Similarly, the presequence of the bovine phosphate carrier (PiC) precursor protein (pPiC) is arrested at the mitochondrial surface when salt concentrations are elevated. In summary, we propose three conclusions: (1) Differently from other mitochondrial pre-proteins, pCIC contains a presequence that does not act as a mediator of protein transport. The presequence is imported as a passenger of the mature protein, which contains the targeting information and does not interfere with the CIC import pathway. (2) The pCIC presequence binds to Tom20 when hydrophobic interactions are facilitated. (3) In general, mitochondrial outer membrane proteins can only act as import receptors if they show sufficiently rapid release of their substrate proteins. The stability of the receptor-substrate complex has to be low enough to allow a continuous flow within a sequence of binding sites.