Signal anchor sequence insertion into the outer mitochondrial membrane. Comparison with porin and the matrix protein targeting pathway

We have addressed the question of overlap between the pathways for protein insertion into the outer mitochondrial membrane and import to the matrix compartment, using competition studies in vitro. A synthetic peptide corresponding to the matrix-targeting signal of pre-ornithine carbamyl transferase competed for outer membrane insertion of porin but did not compete for membrane insertion of outer membrane signal anchor-containing proteins. Conversely, however, a synthetic peptide corresponding to the signal anchor sequence of Tom70 competed for import of all proteins examined. Both peptides competed for a step beyond receptor binding. Import of all precursors examined was inhibited by antibodies raised against the import receptor Tom20. Following binding to the surface of the organelle, outer membrane integration of porin was sensitive to depletion of nucleoside triphosphates by apyrase, whereas signal anchor protein insertion was not. The results demonstrate that outer membrane signal anchor insertion overlaps with a general insertion pathway. However, it exhibits both properties and steps that differ from the pathway followed by porin and matrix-targeted protein.