The full length of a mitochondrial presequence is required for efficient monolayer insertion and interbilayer contact formation.

The peptide specificity of both presequence-monolayer interactions and the ability of presequences to induce interbilayer contacts between large unilamellar vesicles was investigated. A range of different synthetic peptides that are documented for their mitochondrial protein import abilities were used for this purpose. Both monolayer insertion and vesicle aggregation were found to be strongly dependent on the primary structure of the studied presequence peptides. The combination of monolayer data and results of vesicle aggregation experiments leads to the overall suggestion that monolayer insertion and interbilayer contact formation are mechanistically related. For maximal effects the full length of a presequence peptide is required. The cardiolipin specificity of presequence-induced interbilayer contact formation previously reported was found to be a more general property among presequence peptides. The peptide's ability to induce vesicle-vesicle contacts seems to parallel the efficiency of its import ability into mitochondria. These results lead to an extended hypothesis on the role of presequence-induced contact site formation during the mitochondrial protein import process.