Abstract
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.
Highlights
The majority of mitochondrial proteins are encoded by nuclear DNA and synthesized in the cytoplasm as precursors containing an N-terminal extension peptide called presequence or signal sequence that targets the passenger protein to mitochondria
We examined the interaction of the precursor to mitochondrial aspartate aminotransferase and of a synthetic peptide corresponding to the 29-residue presequence peptide with anionic phospholipid vesicles
We show that the mature portion does not contribute significantly to the affinity of a mitochondrial precursor for binding to anionic phospholipid vesicles, at least when the protein is in its native state
Summary
Materials—pmAAT was expressed and purified from Escherichia coli as described previously [20]. mAAT was obtained by tryptic treatment of the precursor protein (trypsin:pmAAT molar ratio, 1:100) and a subsequent CM-Sepharose chromatography step [20]. DPH Anisotropy—To measure changes in DPH anisotropy at a constant temperature upon interaction with mAAT-pp or pmAAT, DMPC: DMPG (1:1) SUVs, prepared as described before, were diluted to a lipid concentration of 200 M in lipid buffer and 0.5 l of a solution of DPH in acetonitrile was added (DPH:lipid molar ratio, 1:300). The temperature of the cell compartment in the fluorometer was maintained at 38 °C by a Haake F3 water bath and monitored by a Digi-Sense temperature control with a microprobe inside the cuvette This temperature is above the melting temperature of the SUVs used (23 °C) as reflected in the low value of r (Ϸ0.070) obtained for vesicles alone in the absence of peptide. To calculate the depth of insertion using the “parallax” analysis [27], the following equation was used
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