©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Homologous Proteins with Different Affinities for groEL
THE REFOLDING OF THE ASPARTATE AMINOTRANSFERASE ISOZYMES AT VARYING TEMPERATURES (*)

(Received for publication, October 12, 1994)

Joseph R. Mattingly Jr. Ana Iriarte Marino Martinez-Carrion (§)

From the Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri, Kansas City, Missouri 64110-2499

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 °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 °C but arresting it completely at 30 °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 °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.


INTRODUCTION

Naturally occurring isozymes which partition into separate intracellular compartments provide unique systems to study the structural features which determine the cellular destination of proteins. We have recently reported the distinct folding behavior of the mitochondrial and cytosolic isozymes of aspartate aminotransferase (AspAT) (^1)after synthesis in cell-free extracts(1, 2) . The cytosolic isozyme (cAspAT) appears to fold quite rapidly when synthesized at 30 °C in vitro in rabbit reticulocyte lysate (RRL)(1) . By contrast, the mitochondrial isozyme, which is synthesized in the cytosol as a precursor (pmAspAT) with an amino-terminal targeting signal, folds at a much slower rate in RRL and then only at reduced temperatures. However, pmAspAT folds rapidly after import into mitochondria and processing to its mature form at 30 °C(2). The divergent behavior of pmAspAT and cAspAT in RRL might be simply explained by differences in the intrinsic propensities of the two proteins to fold, were it not for the fact that the rates at which the chemically denatured proteins refold in vitro at 10 °C are not so dissimilar(1) . One likely explanation for the divergent behavior of the two isozymes in RRL is that the nascent proteins interact differently with molecular chaperones in the cytosol. Proving this hypothesis still awaits the isolation of the AspAT folding intermediates and the identification of any chaperone components with which they may be associated. However, assessing the effect of a well characterized chaperone system on the in vitro refolding of the two isozymes can provide a test for the feasibility of our earlier hypothesis and may reveal some functional properties that might prove useful in identifying these yet unknown chaperones.

The chaperones we have chosen, groEL and groES from Escherichia coli, are prototypical molecular chaperones, having been the most intensively studied members of this expanding class of proteins (for reviews see (3, 4, 5, 6) ). Our choice is due in large part to the ease and quantity in which they can be obtained. However, since groEL and groES are structurally and functionally (7, 8) homologous to the intramitochondrial hsp60 and hsp10 chaperones which are thought to participate in the folding of translocated proteins, this choice provides a physiologically relevant basis for understanding the intramitochondrial folding of mAspAT. (For a recent review of intramitochondrial protein folding, see (9) .) In addition, groEL shares a considerable degree of quarternary structural homology with the TCP-1 ring complex of the eukaryotic cytosol (10, 11, 12) as well as limited sequence homology with TCP-1(13) . This multicomponent protein, while demonstrated to act as a chaperone for a limited number of proteins in vitro, may function as a general chaperone and could conceivably assist the folding of AspAT. Consequently, while recognizing that a protein may react with molecular chaperones differently depending on whether it is refolding in vitro, emerging from the ribosome, or crossing the inner mitochondrial membrane into the matrix, we propose that this system is adequate for judging the general tendency of the two AspAT isozymes to associate with chaperones as they are synthesized in an intracellular environment.

Of more general interest, however, is the possibility that examining the interaction of groEL and groES with m- and cAspAT may provide new insights into the specificity and mechanism of this chaperone family. The groE proteins have been shown to modulate the in vitro refolding of a wide range of purified proteins, proteins from diverse sources and with significantly different physical characteristics. The initial step in all these processes is the formation of a complex between groEL and the refolding protein (reviewed in (14) ). However, conditions for release of the bound protein from groEL are almost as varied as the proteins themselves and range from simply changing the temperature to requiring the addition of groES and ATP with concomitant ATP hydrolysis. The degree of folding of the discharged protein can also vary. Because the isozymes of AspAT have a nearly identical spatial arrangement of their peptide backbones once folded (15) and have a high degree of amino acid homology (approx50%), they seem especially appropriate as the beginning for a more systematic study of the structural factors which determine the affinity of groEL and similar chaperones for unfolded proteins.


EXPERIMENTAL PROCEDURES

Materials

In the context of this report, mAspAT refers to the product from the trypsin digestion of the precursor form of rat mitochondrial aspartate aminotransferase (pmAspAT) expressed in E. coli. This protein differs from the naturally occurring mAspAT by having an additional alanine residue at the amino terminus. mAspAT was purified essentially as described for pmAspAT(2) . Similarly, cAspAT refers to the product of trypsin digestion of a previously described chimeric protein (pcAspAT) containing the targeting presequence of pmAspAT fused to the amino terminus of rat cAspAT(1) . This cAspAT protein has an additional Ala-Met at the amino terminus of what would be the true cAspAT isolated from its natural source. pcAspAT was subcloned into pET3A and expressed in E. coli BL21(DE3) pLysS (16) in the same fashion as pmAspAT. pcAspAT was expressed at approximately the same levels as pmAspAT and could be completely resolved from endogenous E. coli transaminase activity by a combination of DEAE-Sepharose and hydroxylapatite chromatography. The protein exhibited a single band upon silver staining a sample subjected to SDS-polyacrylamide gel electrophoresis. Radiolabeled mAspAT was prepared by expression in bacteria growing in minimal media to which [S]methionine (TranS-label, ICN Biomedicals Inc.) was added after induction with isopropyl-beta-D-thiogalactopyranoside. More details regarding the purification and expression of these proteins are available upon request from the authors. Protein concentrations were estimated using the absorbance of the pyridoxal-5`-phosphate cofactor bound to either m- or cAspAT, a molar absorption coefficient of 8500 M cm, and M = 44,597 for mAspAT or M = 46,399 for cAspAT. groEL and groES were overexpressed using the pGroESL (17) plasmid and purified as described by the originators of the construct. The concentrations of groEL or groES were determined by using the extinction coefficients provided by the Lorimer laboratory (^2)and confirmed for our particular preparation by amino acid analysis.

Denaturation and Refolding of AspAT Isozymes

mAspAT was denatured by incubating a solution containing 1 mg/ml mAspAT, 4 M GdnHCl (Heico Chemical's extreme purity), 100 mM HEPES (adjusted to pH 7.5 with KOH), 10 mM dithiothreitol, and 0.1 mM EDTA for 30 min at room temperature(18) . The unfolded protein was then kept at 0 °C until used. (This was never more than 7 h.) cAspAT was similarly denatured except its concentration was 1.5 mg/ml, and the GdnHCl concentration was increased to 6 M. For refolding mAspAT, a 2-µl aliquot of the denatured protein was diluted 40-fold by adding it to 78 µl of refolding buffer (100 mM KHEPES, 1 mM dithiothreitol, 0.1 mM EDTA, pH 7.5) in a siliconized polypropylene microtube while vortexing. For refolding cAspAT, reactions were prepared in the same fashion except that the denatured protein was diluted 60-fold by adding 2 µl to 118 µl of refolding buffer. The nominal, final AspAT protein concentrations in these experiments was 25 µg/ml, and the final concentration of GdnHCl was 0.1 M. Unless otherwise indicated, reactions containing other components such as groEL or groEL, groES, and MgATP had those substances present in the refolding buffer before adding the denatured AspATs. MgATP was added to a final concentration of 10 mM from a stock solution containing 0.2 M ATP and 0.2 M MgCl(2).

In constant temperature refolding experiments, the refolding buffer was equilibrated to the temperature desired for the reaction. For temperature-shifted refolding experiments, the buffer was initially at 0 °C (the tube was equilibrated in an aluminum block on ice), and the addition and mixing was done in a cold room at approx5 °C. The reaction vessel was then transferred to a water bath at the final desired temperature. The time which elapsed between dilution and transfer to the water bath was never more than 1 min. The time required for temperature equilibration was estimated by using a thermocouple to follow the temperature of a 100-µl aliquot of water in a microtube that was transferred from a 0 °C water bath to one at either 20, 40, or 60 °C. We found that it took approx0.4, approx0.7, and approx 1.2 min, respectively, to reach the final temperatures. However, since the rate of temperature change decreases as the temperature difference decreases, most of the temperature change occurs much faster. For instance, when the initial temperature difference is 60 °C, the time required to reach approx55 °C is approximately the same as the time required to go from 55 to 60 °C.

The refolding of the transaminases was assessed by monitoring the recovery of catalytic activity in a microassay. 5-µl aliquots of the refolding reaction were added to 200-µl aliquots of the previously described assay solution (2) in a microassay plate equilibrated to 32 °C. The assay reagent also contained 0.15% (w/v) bovine serum albumin which increased the observed activity, presumably by lessening nonspecific adsorption of the enzymes to the microassay plate. The rate of change in absorbance at 340 nm was measured for 30-60 s using a Molecular Dynamics plate reader and associated software. This value was corrected for background activity in the absence of added transaminase and converted to milliunits. As illustrated in Fig. 3C, a certain amount of the refolding protein appears to be adsorbed to the reaction vessel; experiments with the native protein have revealed a similar tendency, and this effect is not readily mitigated by any agent other than nonionic detergents. We did not attempt to measure the exact amount of non-adsorbed protein in each refolding reaction. Consequently, we decided not to calculate a specific activity because we only knew the amount of protein added to the reaction vessel, not the amount available for reaction in solution.


Figure 3: The inhibitory effect of groEL on the refolding of mAspAT and pmAspAT. Panel A shows the time course of reactivation of mAspAT at 0 °C in refolding reactions containing increasing amounts of groEL. The ratios of groEL to mAspAT protomers are , 0; bullet, 3; up triangle, 6; , 9; box, 14; and circle, 21. The data are fitted to a kinetic model with two sequential first-order reactions. The rate constants are identical within experimental uncertainty for all reactions that yield an appreciable amount of refolding (ratios leq14, data not shown). Panel B plots the maximum activity recovered as a function of the groEL/mAspAT ratio. bullet represents data from panel A for mAspAT, and box indicates data from a similar experiment for pmAspAT. Panel C shows the effect of groEL on the amount of refolding mAspAT which remains in solution. Standard refolding reactions at 0 °C containing the indicated amounts of groEL were spiked with a measured amount of radiolabeled mAspAT, and then an aliquot was immediately removed and counted. The amount of radiolabel recovered is presented as a percentage of the amount added to the reaction. Panel D shows a continuation of the experiment of panel C. Similar reactions were shifted to 37 °C for 10 min and then centrifuged. The radiolabel present in the supernatant is compared with the amount present before centrifugation.



The maximum activity recovered in a particular reaction, as well as the rate constant, were determined by assaying the refolding reaction at various times and fitting the data to a first-order reaction (A = A(o){1 - e}) using the Levenberg-Marquardt algorithm as implemented in the commercial program DeltaGraph (DeltaPoint, Inc.).

Determining the Thermal Stability of the Chaperone Activity of groEL + groES

74-µl aliquots of groEL and groES in refolding buffer (each at a protomer concentration of 8.4 µM) were heated for 10 min at the temperatures indicated in Fig. 2and then cooled for approx2.5 min (the minimum necessary to ensure proper temperature equilibration) or 30 min at 0 °C. Then 2 µl of denatured mAspAT (with a protomer concentration of 22.4 µM) was added followed by 4 µl of 0.2 M MgATP. The refolding reactions were then shifted to a water bath at 37 °C where they were allowed to proceed. After 33 min (5 times the t measured in another experiment with untreated groEL and groES), the reactions were shifted to 0 °C, and replicate 5-µl aliquots were assayed for transaminase activity. After assaying for regain of transaminase activity, a 15-µl aliquot of each reaction was removed and denatured with an equal volume of SDS sample buffer. The remaining sample was centrifuged for 30 min at approx16,000 times g, and a 15-µl aliquot of the supernatant was denatured with SDS sample buffer. The samples were electrophoresed, the gels were stained with Coomassie Blue, and the quantity of groEL and mAspAT in each sample was determined by densitometry using a Molecular Dynamic Personal Densitometer and ImageQuant software.


Figure 2: The effect of prior heating on the chaperone activity of groEL and groES. The ability of groEL and groES to support the reactivation of mAspAT at 37 °C was measured using a mixture of groEL and groES that had been heated to the indicated temperatures and then immediately cooled to leq approx10 °C. The amount of groEL that remained in solution after centrifugation of the heated chaperones was also determined as was the amount of mAspAT that remained soluble in the various refolding reactions. The error bars indicate the average deviation for duplicate reactions performed using the same stock solution of denatured mAspAT.



Determining the Effect of groEL on the Solubility of Refolding mAspAT at 37 °C

Denatured radiolabeled mAspAT (2.5 µl) was added to refolding buffer (97.5 µl) containing various amounts of groEL initially at 0 °C. 20-µl aliquots of the reactions were removed and added, rinsing the tip, to 100 µl of 1% SDS in a scintillation vial. The reactions were heated for 10 min at 37 °C, centrifuged (30 min at approx16,000 times g), then aliquots of the supernatant were similarly removed. The amount of radiolabel in the first aliquots was determined and compared to the average amount in aliquots of the undiluted denatured mAspAT stock solution in order to assess the amount of refolding mAspAT which was adsorbed to the pipette tips or reaction vessel. The amount of radiolabel in the second aliquots was determined and compared to that in the first in order to determine the amount of refolding mAspAT which was protected from thermally induced aggregation by groEL.


RESULTS

Temperature Effects on the in Vitro Refolding of mAspAT

Rat mAspAT completely unfolds in 4 M GdnHCl but refolds when the chaotrope is diluted under appropriate conditions(18) . Fig. 1A shows that the temperature of the refolding reaction is one of the more important factors influencing the yield of properly refolded mAspAT. In this figure, the yields at the various temperatures are expressed relative to the yield of a reaction at 0 °C. In general, the relative yield decreases as the temperature of the refolding reaction increases. However, the magnitude of this effect depends on the experimental protocol. In constant temperature experiments, the denatured protein is diluted directly into refolding buffer at the indicated temperatures. In temperature shift experiments, the denatured protein is first diluted into refolding buffer at 0 °C, and then this reaction is quickly (leq1 min) transferred to a water bath at the indicated final temperatures. The experimental protocol has no significant effect on the yield of completely refolded mAspAT from 0 to 15 °C. But, between 20 and 30 °C, the yield is significantly greater when the reaction is shifted to the final temperature rather than directly added to buffer at the indicated temperature. Regardless of how the experiment is done, though, GdnHCl-denatured mAspAT fails to refold at physiological temperatures.


Figure 1: The effect of temperature on the rate and yield of m- and cAspAT refolding. Panels A and B show the maximum recovery of catalytic activity in reactions containing mAspAT and cAspAT, respectively, in the absence of chaperones. Activity is expressed as a percentage of the activity recovered in the reaction at 0 °C. The maximum yield relative to an equivalent amount of native protein under the same conditions has been previously reported to be approx70% for mAspAT at 10 °C in a constant temperature reaction(18) ; a similar yield is observed for cAspAT (data not shown). Panels C and D show the maximum recovery of catalytic activity in reactions containing mAspAT (C) or cAspAT (D) in the presence of groEL and groES at a ratio of 14 groEL protomers/AspAT protomer. groES was added in 2-fold excess with respect to groEL and the reactions contained 10 mM MgCl(2) and 10 mM ATP. box indicates reactions in which denatured protein was diluted into refolding buffer pre-equilibrated to the indicated temperature. circle indicates reactions in which denatured protein was diluted into refolding buffer initially at 0 °C then quickly shifted to the indicated temperature. Error bars indicate the average deviation of measurements from duplicate reactions.



In contrast to the dependence of refolding yield on experimental protocol, the rate constants for the recovery of catalytic activity are essentially independent of protocol (data not shown). Careful examination of the time course of activity recovery in reactions at 0 °C reveals a distinct initial lag (see Fig. 3A), consistent with a kinetic mechanism involving two sequential first-order reactions. Similar results were reported for reactions at 10 °C(18) . At 0 °C the rate for the faster of the two reactions (k = 0.042 min) is approximately eight times greater than that for the slower reaction (k = 0.00532 min). As the temperature increases, the lag becomes less discernible. This is likely the result of our inability to acquire activity measurements with sufficient rapidity at the beginning of the reaction rather than the result of a change in kinetic mechanism, although the latter is certainly also possible.

Temperature Effects on the in Vitro Refolding of cAspAT

The cytosolic isozyme of AspAT requires a higher concentration of GdnHCl to unfold than does mAspAT (6 Mversus 4 M, data not shown). But, like the mitochondrial isozyme, denatured cAspAT can also regain catalytic activity when the denaturant is diluted. The refolding of cAspAT is also impaired as the reaction temperature increases, but somewhat less so than that of mAspAT (Fig. 1B). When denatured cAspAT is directly diluted into buffer at various temperatures, the yield of refolded protein gradually declines more or less linearly over the temperature range of 0-40 °C. This is in contrast to the yield of mAspAT which remains relatively constant from 0 to 15 °C and then diminishes sharply from 15 to 35 °C (Fig. 1A). The net result of this behavior is that a very small amount of cAspAT (approx20%) manages to fold properly at approx35 °C, while no mAspAT is able to do so.

The refolding behavior of cAspAT is quite different if the alternative protocol is used and cAspAT is allowed to refold for a short time at 0 °C prior to shifting the reaction to a higher temperature (Fig. 1B). As the final reaction temperature is increased from 0 to approx30 °C, the yield of reactivated protein increases by about 1.5-fold relative to the yield at 0 °C. This suggests that temperature may accelerate the rate of productive folding reactions to a greater extent than the rate of nonproductive folding processes, at least within this temperature range. As the temperature is increased beyond 30 °C, all of this increased yield is lost and the yield continues to decrease further until the relative extent of reactivation is only 50% at 45 °C. Between 45 and 60 °C, the slope of the curve decreases, but the yield continues to decline until only 20% is reactivated at 60 °C.

As noted for mAspAT, the rates at which cAspAT recovers catalytic activity are also unaffected by the experimental protocol (data not shown). However, unlike the biphasic recovery of catalytic activity noted for mAspAT at low temperatures, the recovery of activity by cAspAT is adequately described by a single first-order reaction at all the temperatures examined (see Fig. 4A). The activation energies calculated for the rate-limiting steps in the reactivation of m- and cAspAT are fairly similar (37 kcal/mol and 45 kcal/mol, respectively) with the end result that the rate constant for cAspAT refolding remains approx 2.4-fold greater than that for mAspAT throughout the 0-20 °C temperature range.


Figure 4: The inhibitory effect of groEL on the refolding of cAspAT. Panel A, the rate of regain of transaminase activity at 0 °C is compared for reactions containing no groEL (box) or a 14:1 ratio of groEL protomers to cAspAT protomers (circle). The data are fitted to a single first-order reaction in both cases. Panel B, cAspAT refolding reactions with (circle) and without (box) a 14:1 ratio of groEL were examined as in panel A, but at varying temperatures, and the maximum transaminase activity was determined. In this experiment, denatured cAspAT was added to refolding reactions previously equilibrated to the indicated temperature.



Temperature Effects on the Yield of Refolded mAspAT in Reactions Containing groE

If the inability of mAspAT to refold in vitro at physiological temperatures extends to in vivo folding, some sort of chaperones are probably required for the proper folding of mAspAT in vivo. Indeed, current models for the import and folding of certain mitochondrial precursor proteins posit the involvement of intramitochondrial analogs of hsp70 and groEL/groES (9) . The combined action of groEL, groES, and MgATP allows more mAspAT to refold at higher temperatures than otherwise would in the absence of chaperones (Fig. 1C). Furthermore, unlike the yield in unassisted refolding reactions, the yield in chaperone facilitated refolding reactions is barely influenced by the experimental protocol. Within experimental uncertainty, the yield in reactions containing the chaperones remains more or less constant from 0 to 30 °C for either type of experiment (Fig. 1C), whereas, in a reaction lacking chaperones, the yield at 30 °C is reduced by at least half in temperature-shifted reactions and is nearly abolished in constant temperature reactions (Fig. 1A). At 35 °C there may be some small effects from experimental protocol on the reactivation yields in chaperone-assisted reactions. Above 35 °C, the yield begins to decrease precipitously, and by 45 °C, the yield has decreased to zero regardless of the experimental protocol or the presence of chaperones. Thus, chaperoneassisted mAspAT refolding at physiological temperatures is quite efficient relative to unassisted refolding.

Although no folding of mAspAT is noted in temperature-shifted reactions containing groEL, groES, and MgATP at 45 °C, subsequently lowering the temperature to 35 °C allows refolding to proceed. However, the amount of mAspAT which does refold when the temperature of the reaction is lowered depends upon the length of time which it spent in the 45 °C bath, declining exponentially with a half-life of about 4.6 min. (This time is short relative to the approx 0.8 min that a mock reaction containing a thermocouple required to go from the initial 0 to 45 °C.) The short lived protection against denaturation at 45 °C afforded to refolding mAspAT by groEL and groES is nonetheless substantial when compared to refolding reactions lacking chaperones. In that case, no mAspAT whatsoever refolds when a reaction is first incubated for as little as 2 min in the 45 °C bath and then shifted to 0 °C.

Temperature Effects on the Yield of Refolded cAspAT in Reactions Containing groE

In a temperature-shifted refolding reaction, the yield of reactivation of cAspAT at 35 °C is nearly the same as at 0 °C, which can be interpreted as indicating that the in vivo folding of cAspAT might not require chaperone assistance. However, the low 20% yield of reactivated cAspAT produced in a constant temperature refolding reaction at 35 °C suggests that, indeed, chaperones might be required for its in vivo folding. In an attempt to clarify this question, we tested whether a particular chaperone, groE, is able to facilitate the refolding of cAspAT in an in vitro model system.

As illustrated above, the effect of temperature on the yield of reactivated enzyme is largely independent of the experimental protocol in groE-assisted mAspAT refolding reactions. However, in groE-assisted cAspAT refolding reactions, there are measurable differences between the yields in constant temperature and temperature-shifted reactions (Fig. 1D). Above 15 °C, the yield in a temperature-shifted reaction is always greater than that in the corresponding constant temperature reaction. Despite the presence of groE, the yield in constant temperature cAspAT reactions decreases gradually throughout the temperature range of 0 to 40 °C although this declining yield is less than observed in the absence of chaperones. For instance, at nearly physiological temperatures (35 °C), the yield in the chaperone-assisted reaction is almost three times greater than the yield in the reaction without chaperones. This groE mediated improvement is even greater at 40 °C since the yield with groE present is 60% but is nearly zero without chaperones. Above 40 °C, the yield decreases more rapidly as the temperature increases, but a significant amount of reactivation occurs even at 45 °C. No refolding is observed at 50 °C. In conclusion, the effect of groE on the reactivation of cAspAT is most noticeable in constant temperature reactions where it effects a considerable improvement in yield.

In temperature-shifted, chaperone-assisted reactions, groE does not always have a beneficial effect on the reactivation yield. Although the yield of reactivated cAspAT in refolding reactions lacking chaperones appears to increase between 0 and 20-30 °C (Fig. 1B), the relative yield in chaperone-assisted reactions remains essentially constant over this temperature range (Fig. 1D). Thus the groE chaperones appear to deleteriously affect reactivation at approx30 °C. At 40 °C, groE effects a marginal improvement in the yield of reactivated cAspAT and above 40 °C, the chaperone-assisted refolding yield decreases sharply and either is equal to or less than the refolding yield in the absence of groE.

It is surprising perhaps to note that no cAspAT refolds in temperature-shifted reactions at 60 °C containing groE (Fig. 1D) but that 20% refolds in the absence of chaperones (Fig. 1B). This seems to indicate that the chaperone combination inhibits the small amount of refolding which occurs as the reaction lacking chaperones is heated from 0 to 60 °C. This apparent inhibition of refolding by chaperones at high temperature is somewhat reversible. If such a reaction is only incubated a short time in the 60 °C bath and then cooled to 0 °C, a significant amount of refolded protein is obtained, with the amount recovered depending on the time the reaction spent in the 60 °C bath. The potential to refold at a lower temperature is lost with a half-life of approx1.9 min (data not shown). (Approximately 1.2 min is required for the sample to reach 60 °C.)

Temperature Effects on the Rates of groE-mediated in Vitro Refolding Reactions

The activation energy for groEl/groES-assisted reactivation of the AspAT isozymes was calculated from the temperature dependence of the refolding rates. The activation energy for chaperone-assisted mAspAT refolding is somewhat higher than observed for unassisted mAspAT refolding (53.3 versus 36 kcal/mol). The calculated activation energy for cAspAT in the presence of chaperones is approximately the same as observed for unassisted cAspAT refolding (approx46 kcal/mol in both cases). Refolding rates are uniformly slower in the presence of chaperones (Table 1).



Thermal Stability of the Chaperone Activity of groE

While the groEL/groES chaperones allow chemically denatured m- and cAspAT to refold at higher temperatures than they otherwise would, cAspAT can still refold at slightly higher temperatures than mAspAT. This difference in the maximum temperature at which groEL can still successfully promote refolding of the two isozymes suggests that the ability of groEL to prevent thermally induced detours from the correct refolding path is determined at least in part by the refolding protein itself. However, the thermal stability of groEL may be limiting the temperature range over which it can function. To test this hypothesis, we attempted to determine the effect of temperature on the ability of groEL and groES to function as chaperones.

Refolding reactions containing groEL and groES but no AspAT were heated at the temperatures indicated in Fig. 2then cooled to 0 °C. Denatured mAspAT was then added immediately after cooling, and the refolding reaction was shifted to 37 °C, a temperature at which mAspAT requires chaperone assistance to refold. The amount of reactivated mAspAT was then used as an index of the chaperone activity of groEL and groES. The chaperone activity gradually decreases as groEL and groES are heated, but substantial activity is retained even after heating at 65 °C (Fig. 2). Heating at 75 °C causes a precipitous and complete loss in chaperone activity. groEL and groES not only lose the ability to foster the release of a folded protein after heating to 75 °C, but groEL also loses its capacity to prevent thermally induced aggregation of the refolding mAspAT (Fig. 2). These results indicate that neither groEL nor groES undergo any irreversible thermal transitions at temperatures up to 65 °C. Supporting this hypothesis is the observation that groEL irreversibly loses its ATPase activity at approximately 70 °C (data not shown) and also shows an apparently irreversible thermal transition at this temperature when examined by differential scanning calorimetry. (^3)Despite the loss of functional properties after heating at 75 °C, most of the groEL protein remains soluble (Fig. 2), suggesting that denaturation does not lead to extensive exposure of hydrophobic surfaces which might cause aggregation. Any slowly reversible thermal perturbations in groEL/groES chaperone activity can also apparently be ruled out since similar results are obtained if the heated groEL/groES mixture is maintained at 0 °C for 30 min prior to use in the refolding reactions. However, rapidly reversible changes may still occur.

groEL Alone Affects the Refolding of mAspAT and cAspAT Differently

In refolding reactions with the full assemblage of groEL, groES, and MgATP, more cAspAT than mAspAT refolds at higher temperatures. Otherwise, the effects of groE on the refolding of the two isozymes are remarkably similar. However, mAspAT and cAspAT interact quite differently with groEL alone. Fig. 3A shows the inhibitory effect of groEL on the reactivation of GdnHCl-denatured mAspAT at 0 °C. The yield of catalytically active protein decreases as the concentration of groEL increases until inhibition is nearly complete at a groEL to pmAspAT protomer to protomer ratio of about 21 (Fig. 3B). Interpretation of this apparent stoichiometry is complicated by a variety of observations. First, this ratio is calculated on the basis of the amount of denatured mAspAT added to the refolding reaction. However, approximately 25% of the denatured protein added to a refolding reaction in the absence of groEL seems to be adsorbed to the reaction vessel (Fig. 3C). The amount of denatured mAspAT not adsorbed coincides with the previously reported approx70% recovery of catalytic activity observed in the absence of chaperones(18) . But as the amount of groEL in the reaction increases, the amount of adsorbed mAspAT diminishes. Second, as pointed out by others(20) , the stoichiometry may be determined at least in part by a kinetic partitioning effect, that is, the amount of groEL needed to stop refolding is not so much determined by the ratio of refolding protein to groEL in the final complex as it is by the influence of the groEL concentration on the rate of the complex formation relative to the rates of folding or nonproductive aggregation. However, the stoichiometry is not greatly affected by allowing refolding to briefly proceed before adding groEL (data not shown). An alternative method for determining the stoichiometry is presented in Fig. 3D. If a refolding reaction lacking groEL is centrifuged shortly after shifting to 37 °C, approx80% of the non-adsorbed mAspAT is sedimented. However, as increasing amounts of groEL are added to the reaction, less mAspAT aggregates until none at all sediments in reactions with a groEL to mAspAT ratio of approx14, again basing the concentration of mAspAT on the amount of protein added to the reaction. The affinity of groEL for refolding mAspAT appears to be high, since the mAspATbulletgroEL complex remains intact during size exclusion chromatography in refolding buffer, rate zonal centrifugation in a 10-30% glycerol gradient, and anion-exchange chromatography on a MonoQ column (data not shown).

Unlike the complete inhibition noted for mAspAT, groEL affects only the rate of cAspAT refolding in reactions at 0 °C (Fig. 4A). As the temperature of the refolding reaction is increased from 0 to 16 °C, groEL still causes little or no inhibition in cAspAT refolding (Fig. 4B). But as the temperature is increased from 16 to 30 °C, the refolding of cAspAT is nearly completely inhibited. Lowering the temperature of a reaction initially at 35 to 0 °C allows refolding to proceed, however, the yield is adversely affected as the time spent at 35 °C increases, with only 50% recovered after as little as 15 min at 35 °C (data not shown). In order to check the stoichiometry required for maximal inhibition of cAspAT refolding by groEL, refolding reactions containing various ratios of groEL to cAspAT were done at 30 °C. The result of this experiment is similar to that seen with mAspAT, in that the maximal effect exerted by groEL is observed at a stoichiometry of somewhat less than 14 to 1, again basing that ratio on the amount of protein added to the reaction (Fig. 4D).

groES Releases AspAT from Complexes with groEL

mAspAT or cAspAT species whose refolding has been arrested by groEL can apparently be released by subsequently adding groES and MgATP, since refolding then proceeds at a rate which is the same as the rate observed when all the chaperone components were present initially. (The refolding buffer already contains K and dithiothreitol, reactants already shown to be necessary for release of other proteins from groEL/groES mixtures.) Fig. 5shows the effects of adding groES and MgATP to a cAspAT refolding reaction at 37 °C and initially containing only groEL. As the interval between formation of the groEL adduct and addition of the missing components increases, the yield of refolded protein diminishes. This process is not rapid, however. The groELbulletcAspAT complex loses its potential to refold with a half-life of approximately 70 min at 37 °C. This is significantly slower than the rate at which the refolding cAspATbulletgroEL complex loses its ability to refold by simply reducing the temperature to 0 °C without addition of groES. The groELbulletmAspAT complex behaves similarly and is only slightly less stable than the cAspAT complex, losing its potential to refold upon addition of groES and MgATP with a half-life of 60 min (data not shown).


Figure 5: The stability of the cAspAT-groEL complex at 37 °C. Denatured cAspAT was diluted into refolding buffer containing groEL at 0 °C, the reaction was transferred to a 37 °C bath, and aliquots were removed at the indicated times and added to another microtube containing groES and MgATP. These reactions were incubated further at 37 °C, and the rate and extent of reactivation was determined (panel A). Panel B shows a plot of the maximum activity recovered as a function of the time which the mAspATbulletgroEL complex was incubated at 37 °C before adding the missing chaperonin components. The data are fit to a first-order reaction.



The Effect of Temperature on Factors Required for the Release of m- and cAspAT from groEL

Just as the AspAT isozymes differ in their interaction with groEL, they also differ in the factors required for their release from binary complexes with groEL. groES and MgATP are not always both required. While groEL inhibits the refolding of mAspAT throughout the temperature range of 0-37 °C, including MgATP along with groEL allows some refolding to occur at low temperatures (Fig. 6A). The yield is small at 0 °C relative to reactions without additions or with the full complement of chaperones and cofactors and decreases by almost half as the temperature increases to 15 °C. No refolding is observed at 30 or 37 °C with only groEL and MgATP present. As with reactions containing only mAspAT and groEL, a timely subsequent addition of groES allows refolding to continue (data not shown). The rate at which refolded mAspAT is released from groEL in the presence of MgATP at 0 and 15 °C is similar to the rate of release when groES is also present, although as noted above, the rates of groE-assisted refolding are somewhat less than the rates of unassisted refolding.


Figure 6: Factors effecting release of m- or cAspAT from groEL. Four sets of refolding reactions were set up for each isozyme, containing either no additions, only groEL, groEL, and MgATP, or the complete complement of chaperonins groEL, groES, and MgATP as indicated. Denatured AspATs were added to the refolding reactions at 0 °C, and then the reactions were transferred to a bath at the indicated temperatures. The rate (fitting the data to a single first-order reaction for both isozymes) and yield were measured for duplicate reactions; error bars indicate the average deviation in these parameters. Panel A shows the results for mAspAT while panel B shows the results for cAspAT. Note that the graph showing the rates of refolding at 0 °C has a different scale on the y axis.



Fig. 6B recalls the previously noted observation that the inclusion of groEL in the refolding reaction progressively diminishes the yield of reactivated cAspAT as the reaction temperature increases. In addition, it shows that at temperatures where cAspAT reactivation is observed in the presence of groEL, the rate of refolding is significantly lower than the rate of groEL/groES/MgATP-assisted refolding. This rate, in turn, is itself always less than the rate of unassisted refolding. In contrast to the limited amount of mAspAT apparently released from groEL by MgATP, the yield of reactivated cAspAT at any temperature between 0 and 37 °C is almost the same in reactions containing only two components (groEL and MgATP) or containing the full complement (groEL, groES, and MgATP). But as observed for mAspAT, the rate of refolding in the presence of groEL and MgATP is similar to the rate of groEL/groES/MgATP-assisted refolding at 0 and 15 °C. However, at 30 and 37 °C, cAspAT refolding in reactions containing only groEL and MgATP becomes substantially slower than fully assisted refolding.


DISCUSSION

Because of the uncertainties involved, extrapolating from conditions required for in vitro refolding to predictions of in vivo protein folding mechanisms is a precarious endeavor. First among these uncertainties is a fundamental difference between the initial state of a protein when it is being synthesized and when its completely synthesized version is refolding from a disordered conformation. Complete folding of a protein probably requires the arrangement of local elements of secondary structure appearing early in the process into a finished tertiary or quarternary structure. Sequential folding of proteins concomitant to their vectorial synthesis in vivo(21) would substantially reduce the range of conformations available to the polypeptide. As a result of this, the possibility for intra- or intermolecular misfolding may be greater for a refolding protein in vitro than for a protein which folds as it is synthesized. It is, therefore, possible that assistance might be required for in vitro refolding but not for in vivo folding. However, the order of synthesis does not seem to be critical for the proper in vitro refolding or in vivo folding of at least several mutated proteins having circularly permuted sequences(22, 23, 24, 25) .

An even greater uncertainty in predicting the requirements for the in vivo folding of a particular protein arises from the relative complexity of the intracellular milieu in which folding occurs. The environment in which proteins are synthesized and fold is replete with different chaperones, each with the potential to uniquely influence the folding pathway by selectively interacting with some or all of the reactive surfaces present in incompletely folded proteins. Furthermore, proteins such as mAspAT must undergo a translocation process to arrive at the cellular compartments where they ultimately fold. This may involve additional levels of folding regulation. Despite these pitfalls, we believe that the results presented in this work on the in vitro refolding of AspAT isozymes, both in the presence and absence of chaperones, provide further insight into the distinct features of the in vivo folding of these two homologous proteins.

It is possible that our inability to observe the refolding of mAspAT at physiological temperature (37 °C) is the result of our choices in experimental conditions such as protein concentration, pH, or ionic strength. However, the groEL/groES chaperone pair facilitate refolding regardless of these reaction conditions. Taken alone, these data strongly argue for a similar chaperone requirement in the in vivo folding of mAspAT. In fact, the folding of mAspAT which has been synthesized in RRL shows a similar temperature dependence(2) . However, the protein synthesized in RRL retains a potential to fold when conditions become suitable, for example, after shifting to a lower temperature or after import into mitochondria, while the denatured protein apparently irreversibly loses this ability after brief exposure to physiological temperatures. This is similar to the ability of groEL to protect refolding mAspAT from irreversible aggregation at 37 °C and yet allow refolding to continue when groES and MgATP are added.

Analysis of the in vitro refolding of denatured cAspAT to assess whether cAspAT might require chaperones for proper folding in vivo does not lead to a conclusion as easily as it does for mAspAT. This is primarily because the efficiency at which cAspAT refolds at various temperatures depends so strongly on the experimental protocol. In a constant temperature experiment at physiological temperature, little or no cAspAT refolds. In a temperature shift experiment at the same temperature, a substantial amount of cAspAT refolds. Part, but not all, of this difference in yield can be ascribed to activity being recovered while the sample equilibrates to the higher final temperature. For instance, the reactivation rate of cAspAT at 30 °C (k = 0.32 min or t approx2.2 min) is significant relative to the rate of thermal equilibration, thus some refolding will occur while the sample is warming from 0 to 30 °C. Since no refolding is observed in constant temperature experiments above 30 °C, at temperatures of approx45 °C or greater most of the activity is probably recovered while the samples are equilibrating to the final temperature. As the differential between the initial and final temperature shrinks, the time required for equilibration decreases and so very little reactivation is expected to occur while the sample equilibrates from 0 to 20-25 °C. Nevertheless, the yield of refolded cAspAT in a temperature-shifted experiment is at least double that in a constant temperature reaction at 25 °C. This can not be due solely to refolding which occurs during equilibration of the sample to a new temperature.

What is the basis for the increased yield in temperature-shifted experiments? In vitro refolding reactions seem to be characterized by a competition between two rapid processes one of which leads to the formation of an intermediate with regions of compact local structure and which has the potential to continue folding properly and another which leads to irreversible aggregation. When a denatured protein is diluted into a refolding reaction at increasing temperatures, the proportion of protein diverted into nonproductive species would most likely increase because of increased nonspecific hydrophobic interactions. In contrast, diluting the denatured protein into a reaction initially at 0 °C might maximize the yield of the early on-path intermediates. Thus, the thermal stability of these intermediates may determine the yield of refolded protein when the reaction is shifted to higher temperatures. If this interpretation is true, the cAspAT early folding intermediate or intermediates apparently have a greater degree of thermal stability than the corresponding mAspAT intermediate(s), since folding occurs at significantly higher temperatures than for mAspAT. This apparently greater thermal stability of the cAspAT versus mAspAT folding intermediates parallels the greater thermal stability of completely folded cAspAT relative to mAspAT(19) . However, it appears that refolding cAspAT is more susceptible to thermally induced off-path events than mAspAT since the yield of folding differs so drastically in the two types of refolding reactions. In the absence of chaperones, experimental protocol has a more pronounced effect on the reactivation yield of cAspAT than mAspAT, but both are affected. In the presence of chaperones, the yield of reactivated mAspAT is largely independent of experimental protocol. Nevertheless, temperature-shifted cAspAT reactions containing chaperones still produce more refolded protein than reactions not shifted to a higher final temperature. This suggests that groEL cannot greatly affect the thermally induced off-path reactions of refolding cAspAT. These reactions probably occur faster than groEL can intercept and stabilize a refolding cAspAT in order to effectively prevent temperature-induced misfolding.

Can observations regarding the in vitro refolding of cAspAT lead to a prediction as to whether its in vivo folding might require chaperone assistance? This depends upon which conditions provide a better model for in vivo folding, the constant temperature or the temperature shift protocol. We have advanced the argument that proteins in the early stages of synthesis may be less prone to aggregation than a complete version of the protein which is beginning to refold in vitro. If this is indeed true, conditions which minimize hydrophobic interactions at the very beginning of the in vitro refolding reaction might provide the same advantage as progressive synthesis in vivo. A low initial temperature in the refolding reaction might allow local regions of secondary and even tertiary structure to form rapidly and perhaps sequester interactive hydrophobic surfaces that would otherwise lead to irreversible aggregation. Consequently, results from the temperature shift protocol may provide a more useful model upon which to base our predictions. Since significant amounts of cAspAT can refold at physiological temperatures in temperature-shifted reactions without chaperones, it is possible that cAspAT may not absolutely require chaperones to assist its folding in vivo. However, our results also indicate that if chaperones functionally similar to groEL and groES were present in the cytosol, they might somewhat enhance the yield of cAspAT folding at certain temperatures.

Although refolding c- and mAspAT both interact with groEL in vitro, it is clear that there are significant differences in the affinity of those interactions. One way of judging the affinity of groEL for a refolding protein is by the rigor of the conditions required for release of the protein in a more folded state. Proteins seem to fall into one of three general groups. Those with the highest affinity, such as Rubisco (17) and the mitochondrial enzymes rhodanese (20, 26) , citrate synthase(27, 28) , ornithine transcarbamylase(29) , and malate dehydrogenase(30, 31, 32) , are discharged from their complexes with groEL only in the presence of both groES and ATP. There is a growing number of proteins for which groES and ATP are not absolutely required perhaps because they bind to groEL with lower affinity. ATP or certain analogs can effect release of the E. coli proteins pre-beta-lactamase (33) and tryptophanase (39) or cytosolic dihydrofolate reductase(34) , lactate dehydrogenase(35, 36) , glutamine synthetase(37, 38) , and enolase (40) from complexes with groEL; groES is not necessary. However, in most instances, the combination of groES and ATP still provides a higher yield of refolded protein or a faster rate of release from groEL. Still other unfolded proteins seem to transiently interact with groEL and appear to require no additional factors for release in a folded conformation(41, 42, 43, 44) . In this group, which includes glucose-6-phosphate dehydrogenase, groEL only slows their rate of refolding while not affecting the yield and MgATP reduces this inhibitory effect. Although mAspAT does not fit neatly into any of these categories, it most closely resembles those in the first group which includes other mitochondrial proteins with the highest affinity for groEL. The peculiarity which distinguishes mAspAT from other members of this group is that at 15 °C and more so at 0 °C, a small proportion of the groEL-bound mAspAT can refold in the presence of only MgATP. This suggests that there may be a range of affinities within these groupings, that mAspAT is perhaps at the low end, and that lowering the temperature can reduce the affinity to such an extent that groES is not required. By contrast, cAspAT is similar to other nonmitochondrial proteins (41, 42, 43, 44) in that groEL slows the rate of refolding, increasing temperature increases the affinity of these proteins for groEL, and that ATP alone releases the bound protein.

The various patterns suggest that the mechanism for proteins binding to and being released from groEL is determined more by the nature of the refolding protein than by groEL itself and that each protein may have a unique set of requirements for its release. Proteins with the greatest risk of nonproductive aggregation, presumably caused by having the most extensive exposure of reactive surfaces while refolding, might be expected to have the highest affinity for groEL. Thus refolding mAspAT, which is more susceptible to thermally induced misfolding than cAspAT, apparently has a higher affinity for groEL than does cAspAT, which is better able to refold at higher temperatures. Conversely, well folded proteins would be expected to have little affinity for groEL. Indeed, a close relationship has been found between extent of folding and binding to groEL(35, 41) .

The basis for temperature modulating the binding of a protein to groEL is somewhat unclear. Temperature may affect the conformation of groEL itself. Temperature-induced changes in the emission maximum of fluorescently labeled groEL seem to favor this possibility(45) . However, the introduction of the fluorescent group by sulfhydryl modification may alter the functional properties of groEL(46) . Alternatively, raising the temperature may alter the conformation of the protein bound to groEL. This may involve either changes in the conformation of a single species, the average conformation of a family of conformations, or even a shift in the distribution of a several heterogenous species. Such a conformational change might result in the exposure of additional hydrophobic surfaces which in turn could increase the affinity toward groEL. The small amount of mAspAT which can refold in the presence of groEL and MgATP at low temperature may support this explanation. However, the relatively constant yield seen in the unassisted, temperature-shifted refolding experiments for cAspAT at 0 to 30 °C and for mAspAT from 0 to 15 °C argues against such conformational changes occurring in this temperature range as they would cause aggregation and lower yields. A simpler interpretation for the increase in binding affinity at higher temperatures, particularly for cAspAT, is that hydrophobic interactions may be primarily responsible for the binding of proteins to groEL. Since hydrophobic interactions are strengthened with increasing temperature, it is not surprising that proteins should show increased affinity for groEL at increasing temperatures. We believe that an increased strength of hydrophobic interactions between groEL and regions of either a single cAspAT folding intermediate or of a fixed population of several intermediates is an adequate description of the effect of temperature on the inhibition of refolding caused by groEL. More data are required to evaluate the applicability of this hypothesis to mAspAT.

The temperature-dependent increase in the affinity of groEL for refolding cAspAT could possibly limit the temperature range over which groEL and groES can facilitate refolding. Indeed, the amount of refolding is actually less at temperatures above 45 °C in temperature-shifted reactions containing chaperones than in those without. In addition, mAspAT or cAspAT rapidly lose their ability to refold when shifted to a lower temperature after incubation with groEL and groES at high temperature. These results do not seem to be caused by thermal instability of the chaperones themselves but rather may result from dissociation of bound AspAT and ensuing aggregation or a gradual increase in affinity for groEL such that groES and MgATP can no longer effect release, and the protein essentially becomes irreversibly bound. If the latter explanation should prove true, it would suggest that groEL might also have the physiological role of sequestering irreversibly damaged proteins and preventing them from interfering with cellular activities.

A number of models have been proposed for the mechanism by which groEL facilitates protein folding, and they all postulate that nucleotides and/or groES cooperatively modulate the affinity of groEL for the bound, incompletely folded protein and thereby allow the bound protein to undergo cycles of dissociation and rebinding. This process continues until the refolding protein no longer has any affinity for groEL. While there is more or less general agreement on this much of the mechanism, the extent to which the bound protein dissociates remains a point of contention. The bound protein may dissociate completely, continue refolding while free of the chaperones, and, if still not completely refolded, rebind to groEL. However, complete dissociation does not appear to be an absolute requirement for such a process. If groEL and the refolding protein interact at multiple sites, as is likely the case, a change in affinity for the refolding protein may result in the loss of some but not all of these contacts. This partial release may be sufficient to allow refolding to continue by degrees as well. Our data for the groEL/ES-assisted refolding of mAspAT are difficult to reconcile with the complete release hypothesis unless rebinding of mAspAT to groEL is more rapid than any nonproductive misfolding or that, as proposed, an incompletely folded protein might be released from any direct contact from groEL but be physically constrained within the groEL double toroid(47) .

Notwithstanding the intimate details regarding protein release, one would expect from this mechanism that chaperone-assisted folding would be slower than unassisted refolding if refolding proceeds through an identical series of structural rearrangements in both cases and if on-path folding intermediates are bound before they can be diverted toward a nonproductive folding pathway. Our data fulfill this expectation in that the rate for unassisted refolding of cAspAT is always greater than the rate of groEL/groES/MgATP-assisted refolding (Table 1). This is also the case for mAspAT at 0 and 15 °C. However, at 30 °C, the rate of assisted mAspAT refolding is greater than that of unassisted refolding. This reversal in relative rates correlates with the observation that the rates for unassisted refolding seem to reach a plateau as the reaction temperature increases, becoming increasingly less than the rates predicted from the Arrhenius plot of the rates at 15 °C and below (data not shown). All these facts together suggest that groEL, in addition to preventing on-path intermediates from misfolding, at high temperatures may preferentially interact with folding intermediates which have begun to stray from the productive folding path but have not yet advanced to a state of irreversible misfolding. In so doing, groEL redirects them onto the correct path and increases the overall rate of refolding(36) .

Recent experiments have suggested that under non-permissive conditions which do not permit unassisted refolding, groES may have an integral role in the release of proteins from groEL in a state committed to proper folding(48) . Our results suggest that groES may have an integral role under permissive conditions as well: the bulk of groEL-bound mAspAT requires groES and MgATP to refold, even at temperatures which are permissive for unassisted folding. However, our results also seem to indicate that the affinity of the protein for groEL, rather than the conduciveness of the refolding environment, dictates the need for groES. For instance, the positive effect of groES on the rate of cAspAT reactivation in the presence of groEL and MgATP is more pronounced under conditions which enhance the affinity of cAspAT for groEL such as an increase in the temperature of the reaction (Fig. 6B). groES has no effect on the yield of cAspAT refolding.

All the criteria we have discussed indicate that the complex of groEL with refolding cAspAT is not as stable as that with mAspAT. This lends further credence to our previous hypothesis, based on studies with the proteins translated in RRL, that newly synthesized cAspAT has less tendency to associate with cytosolic chaperones than nascent mAspAT (1) . In addition, the modulating effect of temperature on the binding of refolding cAspAT and, to a lesser extent, mAspAT provides a paradigm for understanding the fact that mAspAT is able to fold in RRL only after the temperature is lowered after synthesis. Decreasing the temperature could well diminish the affinity of some presumed cytosolic chaperone for mAspAT and cause the gradual release of a folded protein. Under normal physiological conditions, the protein would be kept in an incompletely folded conformation such as thought to be necessary for translocation.

Although the chaperones ultimately responsible for the slow folding of mAspAT or its precursor form after synthesis in reticulocyte lysate (1, 2) have yet to be identified, our current results suggest that they may have functional properties similar to those of groEL. While hsp70 has been shown to interact with nascent mAspAT in reticulocyte lysate (49) and to be necessary for maintaining mitochondrial precursor proteins in an import competent state, other factors are also required for efficient import (50, 51, 52) or folding(49) . groEL differs in at least one important respect from small chaperones such as hsp70 in that the much larger groEL molecule is potentially capable of sequestering the folding protein within its central cavity (53) and interacting with many more regions of the folding protein molecule than a single hsp70 molecule could. Perhaps a toroidal chaperone such as the yeast hsp104 (54) or a member of the TCP-1/TF55 chaperone family is responsible for the arrest of mAspAT folding at physiological temperatures in reticulocyte lysate.

What is the physical basis for the different affinity of groEL for the two AspAT isozymes refolding in vitro or, by extension, the differing affinity of functionally similar chaperones potentially present in the eukaryotic cytosol as the proteins are being synthesized? Binding to groEL seems to impose an alpha-helical structure on certain small peptides, and amino acid side chains seem to be more immobilized than the backbone(55, 56) . However, an all beta-protein also has been shown to bind to groEL when refolding(42) . At least in their folded states, c- and mAspAT are equivalent in the proportion of helical and beta-sheet structure which they contain, so this is not a likely factor in determining the strength of the interaction with groEL. Hydrophobic interactions have been proposed to be the primary basis for complex formation. Yet the number of amino acid residues which could be considered hydrophobic are nearly the same for c- and mAspAT (Table 2), with cAspAT actually containing somewhat more of the most hydrophobic residues than mAspAT. Furthermore, the distribution of these hydrophobic residues in the primary sequence of the two isozymes is similar(1) . These parameters alone are insufficient to explain the differences in affinity for groEL which we observe. If hydrophobic interactions are indeed the primary mode by which peptides bind to groEL, the hydrophobic side chains available for interacting with groEL are evidently arrayed in a more favorable geometry in the mAspAT folding intermediate than in the cAspAT intermediate. This implies that the structures of the c- and mAspAT folding intermediates which are captured by groEL differ by substantially more than the structures of the finished proteins, which are practically identical. The 2-fold greater refolding rate of GdnHCl-denatured cAspAT is consistent with different refolding pathways for the two proteins. Perhaps the slightly greater number of phenylalanine and tryptophan residues in cAspAT facilitates the collapse of the unfolded protein to a compact structure in which fewer hydrophobic residues are exposed. In vivo, these inherent differences in the folding mechanism of the two isozymes could be exaggerated by the interaction with other chaperones early in the synthesis process.



Recent data indicate that polar amino acid residues can also bind to groEL(57) . This observation suggests that the marked difference in pI of the final folded state for the two isozymes might contribute to their differential interaction with groEL. In the native state, mAspAT is a basic protein while cAspAT has a slightly acidic isoelectric point (58) . This difference in net charge may exist in the refolding proteins as well. groEL is negatively charged at the pH of our refolding experiments. Consequently, it is not inconceivable that electrostatic interactions may also contribute to a faster initial binding of mAspAT to groEL or an ultimately greater stability of the mAspATbullet groEL complex relative to that of cAspAT-groEL. The observation that the inhibitory effect of lipid vesicles on the refolding of mAspAT increases with their anionic lipid content (^4)supports this hypothesis. Proteins originating from mitochondria and chloroplasts seem to fall in the category of refolding proteins which have the highest affinity for groEL(37) . Furthermore, mitochondrial isozymes tend to be more positively charged than their cytosolic counterparts(59) . These observations suggest that the potential contribution of charge to the interaction of proteins with groEL might also have a role in directing translocated proteins toward a chaperone system which slows or prevents their premature folding in the cytosol. On the other hand, mitochondria contain a member of the hsp60/hsp10 family which is highly homologous to the E. coli groEL/groES system. These mitochondrial chaperonins appear to participate in the folding of translocated proteins in the matrix compartment(9) . Thus, the greater need of mAspAT for folding helpers such as groEL/groES for in vitro refolding may very well reflect a certain specificity of these types of chaperones toward proteins which undergo their final folding steps in the same cell compartment where they reside. Consequently, they are unable to function as efficiently with proteins residing in a different cell location. However, due to the limited specificity toward potential substrates which is characteristic of all chaperones, they are capable of binding cytosolic proteins when presented to them under certain experimental conditions. Thus, whether the modulation of c- and mAspAT folding by groEL is more relevant to the role of mitochondrial hsp60 in the folding of mAspAT in the matrix or to that of the putative cytosolic chaperonin TCP-1 in the dissimilar fate of the two isozymes in the cytoplasm remains to be established.

A definitive answer to the question of why refolding mAspAT binds more tightly to groEL than its homologous isozymic counterpart awaits an analysis of the structure of the complex between groEL and its passenger protein. Such a study can provide additional insights into the molecular mechanism of groEL in particular and chaperone action in general.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL-38412 and GM-38341. This work was presented in part at the 1992 ASBMB Fall Symposium, October 2-5, 1992, Keystone, CO and at the Seventh Symposium of The Protein Society, July 24-28, 1993, San Diego, CA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 816-235-5246; Fax: 816-235-5158.

(^1)
The abbreviations used are: AspAT, rat aspartate aminotransferase; cAspAT refers to the cytosolic isozyme; mAspAT refers to the mitochondrial isozyme; pmAspAT refers to the precursor form of mAspAT; GdnHCl, guanidine hydrochloride; groE refers to the combination of groEL and groES; RRL, rabbit reticulocyte lysate.

(^2)
G. H. Lorimer, personal communication.

(^3)
Mark T. Fisher, personal communication.

(^4)
F. Doñate, A. Iriarte, and M. Martinez-Carrion, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank George H. Lorimer and Anthony A. Gatenby of the Dupont Company for providing the pGroESL plasmid and the details of their purification protocol for groEL and groES. We also thank Mark T. Fisher for his helpful comments and the sharing of information prior to publication and Lydia Mandel for assisting in the amino acid analysis of groES and groEL.


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