The homologous cytosolic and mitochondrial isozymes of aspartate aminotransferase (c- and mAspAT, respectively) seem to follow very different folding pathways after synthesis in rabbit reticulocyte lysate, suggesting that the nascent proteins interact differently with molecular chaperones (Mattingly, J. R., Jr., Iriarte, A., and Martinez-Carrion, M. (1993) J. Biol. Chem. 268, 26320-26327). In an attempt to discern the structural basis for this phenomenon, we have begun to study the effect of temperature on the refolding of the guanidine hydrochloride-denatured, purified proteins and their interaction with the groEL/groES molecular chaperone system from Escherichia coli. In the absence of chaperones, temperature has a critical effect on the refolding of the two isozymes, with mAspAT being more susceptible than cAspAT to diminishing refolding yields at increasing temperatures. No refolding is observed for mAspAT at physiological temperatures. The molecular chaperones groEL and groES can extend the temperature range over which the AspAT isozymes successfully refold; however, cAspAT can still refold at higher temperatures than mAspAT. In the absence of groES and MgATP, the two isozymes interact differently with groEL, groEL arrests the refolding of mAspAT throughout the temperature range of 0-45 degrees C. Adding only MgATP releases very little mAspAT from groEL; both groES and MgATP are required for significant refolding of mAspAT in the presence of groEL. On the other hand, the extent to which groEL inhibits the refolding of cAspAT depends upon the temperature of the refolding reaction, only slowing the reaction at 0 degrees C but arresting it completely at 30 degrees C. MgATP alone is sufficient to effect the release of cAspAT from groEL at any temperature examined; inclusion of groES along with MgATP has no effect on the refolding yield but does increase the refolding rate at temperatures greater than 15 degrees C. These results demonstrate that groEL can have significantly different affinities for proteins with highly homologous final tertiary and quarternary structures and suggest that dissimilarities in the primary sequence of the protein substrates may control the structure of the folding intermediates captured by groEL and/or the composition of the surfaces through which the folding proteins interact with groEL.

The precursor (pmAspAT) and mature (mAspAT) forms of mitochondrial aspartate aminotransferase interact with hsp70 very early during translation when synthesized in either rabbit reticulocyte lysate or wheat germ extract (Lain, B., Iriarte, A., and Martinez-Carrion. (1994) J. Biol. Chem. 269, 15588 -15596). The nature of the structural elements responsible for recognition and binding of this protein to hsp70 has been studied by examining the folding and potential association with the chaperone of several engineered forms of this enzyme. Whereas pmAspAT and mAspAT bind hsp70 very early during translation, the cytosolic form of this enzyme (cAspAT) does not interact with hsp70. A fusion protein consisting of the mitochondrial presequence peptide attached to the amino terminus of cAspAT associates with hsp70 only after the protein has acquired its native-like conformation, apparently through binding to the presequence exposed on the surface of the folded protein. Deletion of the amino-terminal segment of mAspAT or its replacement with the corresponding domain from the cytosolic isozyme eliminates the cotranslational binding of hsp70 to the mitochondrial protein. We conclude that both the presequence and NH 2 -terminal region of pmAspAT represent recognition signals for binding of hsp70 to the newly synthesized mitochondrial precursor. Results from competition studies with synthetic peptides support this conclusion. The ability of hsp70 to discriminate between these two highly homologous proteins probably involves the recognition of specific sequence elements in the NH 2 -terminal portion of the mitochondrial protein and may relate to their separate localization in the cell. A slower folding rate and higher affinity for cytosolic chaperones may represent evolutionary adaptations of translocated mitochondrial proteins to ensure their efficient importation into the organelle.

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