Interaction of the precursor to mitochondrial aspartate aminotransferase and its presequence peptide with model membranes

The possible contribution of the mature portion of a mitochondrial precurso r protein to its interaction with membrane lipids is unclear. To address th is issue, we examined the interaction of the precursor to mitochondrial asp artate aminotransferase (pmAAT) and of a synthetic peptide corresponding to the 29-residue presequence peptide (mAAT-pp) with anionic phospholipid ves icles. The affinity of mAAT-pp and pmAAT for anionic vesicles is nearly ide ntical. Results obtained by analyzing the effect of mAAT-pp or full-length pmAAT on either the permeability or microviscosity of the phospholipid vesi cles are consistent with only a shallow insertion of the presequence peptid e in the bilayer. Analysis of the quenching of Trp-17 fluorescence by bromi nated phospholipids reveals that this presequence residue inserts to a dept h of approximately 9 Angstrom hom the center of the bilayer, Furthermore, i n membrane-bound pmAAT or mAAT-pp, both Arg-8 and Arg-28 are accessible to the solvent. These results suggest that the presequence segment lies close to the surface of the membrane and that the mature portion of the precursor protein has little effect on the affinity or mode of binding of the preseq uence to model membranes. Tn the presence of vesicles, mAAT-pp adopts consi derable a-helical structure. Hydrolysis by trypsin after Arg-g results in t he dissociation of the remaining 21-residue C-terminal peptide fragment fro m the membrane bilayer, suggesting that the N-terminal portion of the prese quence is essential for membrane binding. Based on these results, we propos e that the presequence peptide may contain dual recognition elements for bo th the lipid and import receptor components of the mitochondrial membrane.