(Received for publication, July 21, 1995; and in revised form, August 14, 1995)
From the
Chaperonins are known to facilitate protein folding, but their
mechanism of action is not well understood. The fact that target
proteins are released from and rebind to different chaperonin molecules
(``cycling'') during a folding reaction suggests that
chaperonins function by unfolding aberrantly folded molecules, allowing
them multiple opportunities to reach the native state in bulk solution.
Here we show that the cycling of -tubulin by cytosolic chaperonin
(c-cpn) can be uncoupled from the action of cofactors required to
complete the folding reaction. This results in the accumulation of
folding intermediates which are chaperonin-bound, stable, and
quasi-native in that they bind GTP nonexchangeably. We present evidence
that these intermediates can be generated without the target protein
leaving c-cpn. These data show that, in contrast to prevailing models,
target proteins can maintain, and possibly acquire, significant
native-like structure while chaperonin-bound.
The final stage in the flow of genetic information from DNA to expressed proteins is the folding of each protein into the three-dimensional structure that specifies its biological activity. In principle, such folding reactions can occur spontaneously, since all the necessary information required to determine the final folded structure is contained within the primary sequence of amino acids. However, under physiological conditions, constraints of temperature and the tendency of unfolded proteins to aggregate are such that many proteins must undergo facilitated folding via interaction with protein complexes termed chaperonins(1, 2, 3, 4, 5, 6, 7, 8, 9, 10) . These protein complexes take the form of toroidal structures that facilitate protein folding in an ATP-dependent manner. For example, the prokaryotic chaperonin GroEL facilitates the folding of a range of proteins in E. coli(6, 27) , often in conjunction with the cochaperonin GroES(1, 4) . There is evidence that GroES, which is itself a heptameric ring(12) , functions at least in part by interacting with the ends of the GroEL cylinder, such that it modulates and coordinates the hydrolysis of ATP by GroEL(11, 13, 14, 15) .
Many models depict facilitated folding occurring within the central cavity that is present in all chaperonins, thereby protecting the target protein from interaction with other proteins in the general milieu; release then occurs following acquisition of the native structure(3, 8, 9, 10, 16) . Recently, however, this concept has been challenged by evidence that target proteins can jump between different chaperonin molecules during a folding reaction (``cycling'')(11, 17) . Thus, the function of chaperonins might be to unfold and release proteins that have misfolded. In this view, protein folding occurs spontaneously in solution, while the function of the chaperonin is to ``recycle'' aberrantly folded molecules so that they can return to a potentially productive pathway. Accordingly, a given target molecule might require multiple rounds of interaction with different chaperonin molecules before partitioning to the native state.
To
understand the mechanism of chaperonin-mediated folding, it is
essential to know the states of folding intermediates during the
cycling process. However, such intermediates are usually difficult to
study because of their heterogeneity, transient existence, and
pronounced tendency to aggregate. To examine the states of folding
intermediates produced during chaperonin-mediated folding, we took
advantage of the observation that the facilitated folding of
-tubulin by cytosolic chaperonin (c-cpn) (
)cannot
proceed to the native state in the absence of specific protein
cofactors(18, 19) . This system allowed us to
establish the existence of a novel class of chaperonin-bound
quasi-folded intermediates that are generated during ATP-dependent
facilitated folding.
The facilitated folding of -actin requires ATP-dependent
interaction with c-cpn, the eukaryotic cytosolic homolog of
GroEL(20) . In contrast, the facilitated folding of
- and
-tubulin requires interaction with both c-cpn and additional
protein cofactors(18, 19) . To see whether it might be
possible to uncouple the ATP-driven c-cpn-mediated reaction from the
action of these cofactors, an
-tubulin folding reaction was done
in which
-tubulin
c-cpn binary complexes were incubated with
ATP and GTP alone. After 45 min, the ATP-dependent reaction was
quenched by the addition of hexokinase and glucose. Cofactors and
carrier native tubulin were then added, and the reaction was allowed to
continue at 30 °C. We found that the ATP-driven reaction is almost
as efficient when uncoupled from the action of cofactors, since the
amount of native tubulin product was very similar in a parallel
reaction that contained cofactors at the outset of the incubation with
ATP and GTP (Fig. 1a, lanes 1 and 2).
A control folding reaction in which glucose and hexokinase were
included at the outset resulted in no detectable product, demonstrating
the effectiveness of the ATP quench in arresting c-cpn-mediated folding (Fig. 1a, lane 3). The ATP-dependent reaction
that generates the species upon which cofactors act is slow, requiring
about 45 min to reach equilibrium (Fig. 1, b and c), while the action of cofactors on this intermediate seems
to be very fast, requiring at most only a few seconds at 30 °C (Fig. 1d).
Figure 1:
Accumulation of
intermediates in the c-cpn-mediated folding of -tubulin. a, the nucleotide-driven c-cpn-mediated
-tubulin folding
reaction can be uncoupled from the action of cofactors required to
generate native product. Analysis of the products of
-tubulin
folding reactions containing c-cpn, ATP, and GTP. Lane 1,
control folding reaction supplemented with cofactors and native carrier
tubulin (18, 19) and incubated for 45 min at 30
°C; lane 2, folding reaction incubated for 45 min at 30
°C, quenched with hexokinase, and incubated for an additional 2 min
in the presence of cofactors and native carrier tubulin; lane
3, reaction identical with that shown in lane 1, but
quenched with hexokinase immediately following presentation of the
target protein. b, the intermediates that are the substrates
upon which cofactors act accumulate slowly during ATP exchange and
hydrolysis.
-Tubulin
c-cpn binary complexes were incubated at
30 °C for the times shown in the presence of ATP and GTP, quenched
with hexokinase, and incubated for an additional 2 min in the presence
of cofactors and carrier native tubulin. c, quantitative
analysis of the data shown in b (averaged from four
experiments and with the plateau level of native tubulin rationalized
to 100%). d, cofactors act rapidly on intermediates
accumulated as a result of incubation with ATP and GTP.
-Tubulin/c-cpn binary complexes were incubated in the presence of
ATP and GTP for 45 min at 30 °C. The reaction was quenched with
hexokinase, supplemented with cofactors and carrier native tubulin, and
aliquots were withdrawn from the reaction at the times shown. e, half-life of accumulated
-tubulin folding
intermediates.
-Tubulin folding reactions were incubated for 45
min at 30 °C in the presence of ATP and GTP. The reactions were
quenched with hexokinase, and the incubation continued at 30 °C. At
the times shown, aliquots were withdrawn, supplemented with cofactors
and carrier native tubulin, and incubated for an additional 2 min. f, semi-log plot of the data shown in e (averaged
from four experiments and with the initial yield of native tubulin
rationalized to 100%); arrow shows t
. Upper and lower arrows in a, b, d, and e show the location of the
-tubulin
c-cpn binary complex and native tubulin,
respectively.
To determine the stability of the subset
of ATP-generated intermediates that can be converted to native
-tubulin molecules by the action of cofactors, we incubated
-tubulin
c-cpn binary complexes with ATP and GTP for 45 min
to generate these intermediates, quenched the ATP-dependent reaction
with hexokinase and glucose, and continued the incubation at 30 °C
for increasing times before completing the reaction by adding cofactors
and native carrier tubulin. We found that the subset of intermediates
that can be converted to native molecules by the action of cofactors is
quite stable, with a half-life of about 50 min at 30 °C (Fig. 1, e and f). We define this
subpopulation of end states as I
(for intermediates,
quasi-native (see below)). The existence of these stable intermediates
provides a unique opportunity to study the mechanism of
chaperonin-mediated folding.
To see whether I intermediates exist bound to c-cpn or free in solution, an
-tubulin folding reaction containing c-cpn, ATP, and GTP was
incubated for 45 min at 30 °C, quenched with hexokinase, and
applied to a sucrose gradient (Fig. 2a). Following
centrifugation, fractions from the gradient were analyzed in two ways:
without further incubation and following incubation with cofactors and
native carrier tubulin. We found that correctly folded tubulin was
generated only by addition of cofactors to those fractions that contain
-tubulin-chaperonin binary complex (Fig. 2b).
Similarly, when the products of a quenched
-tubulin folding
reaction done without cofactors were applied to a Superose 6 gel
filtration column, only fractions that co-eluted with c-cpn were active
in the generation of native tubulin upon incubation with cofactors
(data not shown). These results demonstrate that there is a tight
association of c-cpn and I
-tubulin end states upon
which cofactors act.
Figure 2:
Characterization of quasi-folded
-tubulin folding intermediates. a, sucrose gradients used
for the size fractionation of I
intermediates. Numbers denote the percentage of sucrose contained in successive layers. b, analysis on 4.5% nondenaturing polyacrylamide gels (18, 20) of the products of reactions done using
fractions recovered from sucrose gradients.
-Tubulin folding
reactions done with c-cpn in the presence of Mg-ATP and Mg-GTP were
quenched by the addition of glucose and hexokinase, and the products
were applied to sucrose gradients (as shown in a). Following
centrifugation, fractions were incubated in two different reactions:
alone(-) or supplemented with cofactors and carrier native
tubulin (18, 20) (+). Upper and lower arrows denote the locations of
-tubulin
c-cpn
binary complexes and native tubulin, respectively. c, I
-tubulin folding intermediates do not cycle in the presence
of ATP. Quantitative analysis of the yield of
-tubulin
c-cpn
binary complexes (solid bars) and native tubulin (hatched
bars) produced in folding reactions in which I
was
formed at 30 °C for 45 min and diluted in the presence of ATP and
GTP to varying extents so as to preclude efficient
cycling(22) . Following incubation at 30 °C for an
additional 30 min, cofactors and native carrier tubulin (18) were added and the incubation continued for an additional
2 min.
Two kinds of experiments showed that
-tubulin I
intermediates are not cycled by c-cpn in
the presence of ATP. First, they survive incubation under dilute
conditions where there is a concomitant loss of counts from the binary
complex (Fig. 2c). Under these conditions, there is
insufficient chaperonin in the reaction to capture released molecules
before they aggregate or adhere to the reaction vessel
walls(22) . The loss of label from the binary complex is a
result of the release of cycling intermediates under these dilute
conditions, and not the disintegration of chaperonin, since we found no
loss of radioactivity when the
-tubulin
c-cpn binary complex
was diluted and incubated with ADP. Secondly, we found no effect on the
yield of native
-tubulin produced when stable
-tubulin end
states were incubated with ATP in the presence of a large molar excess
of a mitochondrial chaperonin trap for the capture of non-native forms (22) before completing the reaction by addition of cofactors
(data not shown).
To investigate the extent of native-like structure
in -tubulin target molecules acquired as a consequence of their
interaction with c-cpn, we compared the extent of target protein
protease resistance in
-tubulin
c-cpn binary complexes that
had been incubated with or without ATP and GTP. As controls, we first
established the resistance to proteolysis of urea-unfolded
-tubulin diluted into buffer alone, as well as the resistance of
native tubulin synthesized by in vitro translation. Under the
conditions used in our experiments, no intact tubulin survived beyond
the initial addition of protease when the target protein was diluted
into buffer (Fig. 3a). In contrast, there was no
significant loss of intact
-tubulin in a parallel experiment done
with native tubulin (Fig. 3b). In experiments to
measure the proteolytic sensitivity of target protein
c-cpn binary
complexes, we found that resistance to proteolysis of c-cpn-bound
-tubulin increased significantly upon incubation with ATP and more
so upon incubation with both ATP and GTP (Fig. 3, c-e). These data imply that
-tubulin end states
generated as a result of ATP-dependent cycling with c-cpn are more
extensively folded than molecules that have not been cycled.
Figure 3:
Resistance to proteolysis of -tubulin
folding intermediates. a and b, resistance to
proteolysis of unfolded
-tubulin diluted directly into folding
buffer (a) or
-tubulin synthesized by translation in
vitro and incorporated into tubulin heterodimers (b). c-e, resistance to proteolysis of
-tubulin
c-cpn binary complexes incubated for 45 min at 30
°C with ADP and GTP (c), ATP alone (d), or ATP
and GTP (e). Reactions were quenched with hexokinase, and
aliquots were incubated with proteinase K for the times shown (in
minutes). Reaction products were analyzed on 10%
Tricine-SDS-polyacrylamide gels(26) . Arrows mark the
location of intact
-tubulin. f and g,
quantitation of data shown in a and b (f)
and c-e (g), in each case averaged from three
experiments; radioactivity contained in target protein at t = 0 is normalized to 100%.
, unfolded target protein
diluted into folding buffer;
,
-tubulin synthesized by
translation in vitro;
,
,
,
-tubulin
target protein bound to c-cpn and incubated with ADP alone, ATP alone,
or ATP and GTP, respectively.
The
/
-tubulin heterodimer binds two molecules of GTP, one of
which is exchangeable and is located on the
-subunit (the E-site),
and a second that is nonexchangeable, located on the
-subunit (the
N-site)(25) . Our observation that
-tubulin
c-cpn
binary complexes become more resistant to proteolysis upon incubation
with both ATP and GTP (compared to incubation with ATP alone) (Fig. 3, d and e) implies that at least one
function of GTP binding is to stabilize tubulin molecules during their
facilitated folding. To probe the state of
-tubulin I
intermediates in terms of their nonexchangeable (N-site) GTP
binding properties(25) , we performed c-cpn-mediated folding
reactions in the presence of ATP and
-[
P]GTP, using unlabeled unfolded
-tubulin as target protein. The incorporation of bound, labeled
GTP into c-cpn
-tubulin binary complex is ATP- and target
protein-dependent (Fig. 4a). When these binary
complexes were incubated with cofactors and native carrier tubulin in
the presence of excess unlabeled GTP, we found nonexchangeable label in
association with both c-cpn and native tubulin (Fig. 4b, lanes 1 and 2). This
GTP-labeled tubulin was native as shown by its ability to copolymerize
with authentic brain tubulin. These experiments demonstrate that
labeled GTP is bound to the N-site of I
(which gives rise
to labeled native tubulin), as well as to the N-site of other
intermediates which remain c-cpn-associated in the presence of
cofactors. We conclude that I
and other
-tubulin
folding intermediates are so extensively native-like that they contain
the GTP binding pocket.
Figure 4:
-Tubulin folding intermediates
contain nonexchangeably bound GTP. Incorporation of nonexchangeable GTP
into
-tubulin folding intermediates and native
-tubulin. a, incorporation of GTP into
-tubulin
c-cpn binary
complex. Lane 1, control reaction in which purified c-cpn
without added target protein was incubated with ATP and
-[
P]GTP for 45 min at 30 °C. Lanes
2 and 3, c-cpn-mediated folding reactions in which
unlabeled
-tubulin
c-cpn binary complexes were incubated for
45 min at 30 °C with either
-[
P]GTP
alone (lane 2) or
-[
P]GTP and ATP (lane 3). b, incorporation of nonexchangeable GTP
into native
-tubulin. c-cpn-mediated folding reactions in which
c-cpn alone (lanes 1, 3, and 5) or unlabeled
-tubulin
c-cpn binary complexes (lanes 2, 4, and 6) were diluted 10-fold into folding buffer
containing ATP and
-[
P]GTP and supplemented
with either 0.2 µM c-cpn (lanes 1 and 2)
or a 10-fold molar excess of trap for the capture of non-native cycling
intermediates (22) (lanes 3 and 4). Following
incubation at 30 °C, native tubulin was discharged by the addition
of cofactors(18) . Upper and lower arrows show the location of
-tubulin
c-cpn binary complexes and
native tubulin, respectively.
When c-cpn is diluted sufficiently to
preclude efficient cycling and in the presence of a mitochondrial
chaperonin (mt-cpn) trap for the capture of released non-native target
protein(22) , labeled GTP can still become incorporated into
-tubulin intermediates (Fig. 4b, lanes
4-6). These data suggest that the nonexchangeable GTP
binding site can form while the target protein is chaperonin-bound. GTP
is not acquired before binary complex formation, since the complex is
formed in the absence of GTP. Nor is GTP (labeled or unlabeled)
acquired after the addition of cofactors: when folding reactions
containing
-[
P]GTP and unfolded
[
S]methionine-labeled
-tubulin were
quenched with unlabeled GTP before the addition of cofactors, each mole
of labeled native tubulin produced contained 1 mol of nonexchangeably
bound labeled GTP.
Like -tubulin, the GroEL-mediated folding of
rhodanese (3) and the c-cpn-mediated folding of
-tubulin (
)also proceed via the formation of chaperonin-bound
protease-resistant intermediates. These data suggest that
chaperonin-bound quasi-native intermediates are an important feature of
the mechanism of facilitated folding (although there is normally no
accumulation of such intermediates). The existence of such highly
structured c-cpn-bound intermediates is surprising in view of current
models of GroEL-mediated protein folding(11, 17) , but
is consistent with our observation that different chaperonins produce
distinct spectra of folding intermediates (22) . Furthermore,
our evidence that
-tubulin can acquire its GTP binding pocket via
a single cycle of interaction with c-cpn suggests that, in contrast to
the prevailing view, some target protein folding occurs either on the
chaperonin surface or in its central cavity, rather than in bulk
solution.