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 precursor protein to its interaction with membrane lipids is unclear. To address this issue, we examined the interaction of the precursor to mitochondrial aspartate aminotransferase (pmAAT) and of a synthetic peptide corresponding to the 29-residue presequence peptide (mAAT-pp) with anionic phospholipid vesicles. The affinity of mAAT-pp and pmAAT for anionic vesicles is nearly identical. Results obtained by analyzing the effect of mAAT-pp or full-length pmAAT on either the permeability or microviscosity of the phospholipid vesicles are consistent with only a shallow insertion of the presequence peptide in the bilayer. Analysis of the quenching of Trp-17 fluorescence by brominated phospholipids reveals that this presequence residue inserts to a depth of approximately 9 A from the center of the bilayer. Furthermore, in 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 presequence to model membranes. In the presence of vesicles, mAAT-pp adopts considerable alpha-helical structure. Hydrolysis by trypsin after Arg-8 results in the dissociation of the remaining 21-residue C-terminal peptide fragment from the membrane bilayer, suggesting that the N-terminal portion of the presequence is essential for membrane binding. Based on these results, we propose that the presequence peptide may contain dual recognition elements for both the lipid and import receptor components of the mitochondrial membrane.
Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri, Kansas City, Missouri 64110, USA.
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D001219:Aspartate Aminotransferases / Q000737:chemistry / Q000378:metabolism*; D002942:Circular Dichroism; D004792:Enzyme Precursors / Q000378:metabolism*; D005454:Fluorescence Polarization; D007700:Kinetics; D008567:Membranes, Artificial; D008928:Mitochondria / Q000201:enzymology*; D017433:Protein Structure, Secondary; D014357:Trypsin / Q000378:metabolism
D004792:Enzyme Precursors; D008567:Membranes, Artificial; D001219:Aspartate Aminotransferases; D014357:Trypsin