PubMed · 4558599
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R R Grams, E A Johnson, E S Benson. 1972. Laboratory data analysis system. IV. Multivariate diagnosis.. https://doi.org/10.1093/ajcp%2F58.2.201
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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.
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Cytosolic aspartate aminotransferase (cAspAT) is regulated by glucocorticoids in rat liver and kidney. Part of this regulation is mediated by an unusual glucocorticoid-responsive element (GRE)-like sequence called GRE A. GRE A is composed of two overlapping imperfect GREs, each comprising a conserved half-site (half-sites 1 and 4 respectively) and a poorly conserved half-site (half-sites 2 and 3 respectively). The sequence binds co-operatively two dimers of the glucocorticoid receptor (GR) and mediates efficient glucocorticoid regulation of gene expression. Analysis of deletions of the cAspAT gene promoter and subcloning of GRE A upstream of the thymidine kinase promoter indicate that this sequence is responsive to glucocorticoids, but not to androgens. Electrophoretic mobility shift assays indicate that the GRE A unit does not bind the androgen receptor (AR). The modification of three nucleotides in the poorly conserved half-sites 2 and 3, converting GRE A into two overlapping high-affinity GREs (ov-cGRE), resulted in co-operative binding of the AR. Furthermore, ov-cGRE efficiently mediated androgen regulation of the thymidine kinase promoter. A single base modification in half-site 2 or 3 in GRE A allowed the binding of the AR as one or two dimers respectively, and restored transcriptional activation by androgens only in the latter case. Thus the poor affinity of the AR for half-sites 2 and 3 prevented its binding to GRE A, indicating that the overlapping GRE A sequence of the cAspAT gene promoter discriminates a glucocorticoid-mediated from an androgen-mediated response.