(Received for publication, October 12, 1994)
From the
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.
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) ()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 (50%), 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.
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 5 °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
0.4,
0.7, and
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
55 °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;
, 3;
, 6;
, 9;
, 14; and
, 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
14,
data not shown). Panel B plots the maximum activity recovered
as a function of the groEL/mAspAT ratio.
represents data from panel A for mAspAT, and
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{1 - e
}) using the
Levenberg-Marquardt algorithm as implemented in the commercial program
DeltaGraph (DeltaPoint, Inc.).
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
10 °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.
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 70% 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
and 10 mM ATP.
indicates reactions in which denatured protein was
diluted into refolding buffer pre-equilibrated to the indicated
temperature.
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.
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 30 °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 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 () or a 14:1 ratio of groEL protomers to cAspAT
protomers (
). The data are fitted to a single first-order reaction
in both cases. Panel B, cAspAT refolding reactions with
(
) and without (
) 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.
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
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.
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 30 °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 1.9 min (data not shown). (Approximately 1.2
min is required for the sample to reach 60 °C.)
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. ()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.
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).
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
mAspATgroEL complex was incubated at 37 °C before adding the
missing chaperonin components. The data are fit to a first-order
reaction.
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.
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
2.2 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
45 °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--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 -helical
structure on certain small peptides, and amino acid side chains seem to
be more immobilized than the backbone(55, 56) .
However, an all
-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
-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 mAspAT 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 (
)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.