Abstract
The cytosolic (cAAT) and mitochondrial (mAAT) isozymes of eukaryotic aspartate aminotransferase share a high degree of sequence identity and almost identical three-dimensional structure. The rat liver proteins can be refolded and reassembled into active dimers after unfolding at low pH. However, refolding of the mitochondrial form after unfolding at pH 2.0 is arrested in the presence of hsp70, whereas this chaperone does not affect the refolding of the cytosolic isozyme unfolded under similar conditions. To elucidate the nature of the differential interaction between hsp70 and the two transaminase forms, we have characterized their refolding from their acid-unfolded states. The recovery of activity of the cytosolic enzyme is monophasic and can be adequately described by a single first-order reaction. By contrast, two sequential first-order rate-limiting steps can be detected for the refolding and reactivation of the mitochondrial protein. The overall refolding pathway of mAAT includes a very fast collapse to an intermediate with 80% of the secondary structure of the active dimer. This is followed by a slow isomerization to form assembly-competent monomers that rapidly associate to form an inactive dimer and a final structural rearrangement of the dimer to the native conformation. Analysis of the interaction of hsp70 with intermediates along the folding pathway of mAAT shows that the polypeptide loses its ability to bind to the chaperone after it has proceeded through the first isomerization/fast dimerization steps. Thus it appears that only the first collapsed intermediate states in the folding of mAAT bind hsp70. By contrast a faster refolding of cAAT from this collapsed state could explain, at least in part, the inability of hsp70 to bind this isozyme.
Highlights
Proteins that are synthesized in the cytoplasm but reside in other cellular locations must traverse membranes to reach their final destination
1 The abbreviations used are: hsp70, 70-kDa heat shock protein; AAT, aspartate aminotransferase; cAAT, cytosolic aspartate aminotransferase; eAAT, aspartate aminotransferase form E. coli; mAAT, mitochondrial aspartate aminotransferase; pmAAT, precursor to mitochondrial aspartate aminotransferase; GdnHCl, guanidine hydrochloride; protein family, have been implicated in the binding of these incompletely folded precursors to presumably prevent their aggregation while maintaining them in a loose, import-competent conformation [5,6,7,8], that is folding to the native conformation is delayed until the protein reaches its final destination
CAAT remains in the cytoplasm while mAAT is post-translationally transported into the mitochondrial matrix
Summary
Materials and Protein Purification—Aspartic acid, cysteine sulfinic acid, and a-ketoglutarate were purchased from Sigma. To study the effect of hsp on the refolding of the enzymes, different concentrations of hsp were added to the refolding mixture either previous to the addition of the unfolded proteins or at different times after the initiation of refolding. Analysis of the Complex hsp70zpmAAT—The complex between hsp and refolding pmAAT was isolated by centrifugation on a top-bench centrifuge (Marathon 14K/M, Fisher Instruments) at 10,000 rpm for 20 min, and the pellet and supernatant were analyzed on 12% SDS-PAGE gels. A time course of the kinetic mechanism shown in Reaction 2 (see “Discussion”) was simulated using the KINSIM program [38], and the rate constants for reactivation of the PLP form of pmAAT are shown, assuming diffusion controlled rates for the bimolecular steps and that activity is recovered only with the final phase of folding. The quality of the fit was judged by visual analysis of scatter diagrams of the residuals (difference between the experimental and fitted values) and by calculating the x2 statistic [39] using the Minitab program (Minitab Inc.) to verify whether there are significant differences between the experimental and calculated data
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
