(Received for publication, August 23, 1994; and in revised form, October 20, 1994)
From the
The folding kinetics of two luciferases were studied after synthesis in reticulocyte lysates to investigate whether molecular chaperones and/or folding catalysts are involved in the folding reactions. Two bacterial luciferases were used as model proteins: heterodimeric Vibrio harveyi luciferase (LuxAB), and a monomeric luciferase fusion protein (Fab2). Data indicate that folding of these enzymes to the native state occurs in the translation system, and that the extent of folding can be quantified. It was found that (i) folding of LuxAB and Fab2 can clearly be separated in time from synthesis, (ii) folding of Fab2 and LuxAB is slow because it involves either transient (Fab2) or permanent (LuxAB) interaction of polypeptides, (iii) preservation of the assembly competent state of LuxA and/or LuxB and folding of Fab2 depend on ATP-hydrolysis, (iv) folding of Fab2 and LuxAB is partially sensitive to cyclosporin A (CsA) and FK506, i.e. inhibitors of two distinct peptidylprolyl cis/trans-isomerases. Thus, bacterial luciferases provide a unique system for direct measurement of the effects of ATP-dependent molecular chaperones on protein folding and enzyme assembly in reticulocyte lysates. Furthermore, these two luciferases provide the first direct evidence documenting the involvement of peptidylprolyl cis/trans-isomerases in protein biogenesis in a eukaryotic cytosol.
Although much is known about the rules and mechanisms governing
protein folding after denaturation and subsequent renaturation
(Jaenicke, 1987), in contrast little is known about protein folding and
subunit assembly following de novo synthesis of polypeptides.
The latter is generally assumed to be assisted by molecular chaperones
and folding catalysts that are present in all organisms and in all
cellular compartments (Ellis and van der Vies, 1991; Gething and
Sambrook, 1992; Georgopoulos and Welch, 1993; Hartl et al.,
1994; Schreiber, 1991). Chaperones, such as the two ATP-dependent
classes of chaperones, the heat shock protein (Hsp) ()families Hsp60 and Hsp70, are believed to prevent
aggregation of unfolded polypeptides by stabilizing folding
intermediates. Folding catalysts, such as peptidylprolyl cis/trans-isomerases (PPIases), are supposed to increase the
rate of slow folding steps. The PPIases do also function as
intracellular receptors for immunosuppressants and are therefore also
named immunophilins (Schreiber, 1991). The PPIases include two
structurally distinct protein families with different, but overlapping
substrate specificities: the cyclophilins are high affinity binding
sites for the drug cyclosporin A (CsA), while the FK506-binding
proteins (FKBPs) bind FK506. In both cases, binding of the drug
inhibits isomerase activity of the enzyme. FK506 inhibits the PPIase
activity of FKBPs, but not of cyclophilins; likewise, CsA does not
inhibit the PPIase activity of FKBPs. Here we directly addressed
questions related to protein folding by employing cell-free translation
systems, such as rabbit reticulocyte lysates. Specifically, we asked
(i) what are the kinetics of folding and assembly of light emitting
model proteins in the reticulocyte lysate, and (ii) are ATP-dependent
molecular chaperones and/or folding catalysts involved in the folding
reactions.
In order to be able to follow the folding kinetics of a
certain protein in a reasonable amount of translation reaction (where
about 10-100 fmol/µl of protein is synthesized), enzymes
catalyzing light emitting reactions were chosen as model proteins. In
the experiments described below two bacterial luciferases were used.
The first luciferase (LuxAB) is a heterodimeric enzyme from Vibrio
harveyi MAV (Waddle et al., 1987; Escher et al.,
1989; Flynn et al., 1993; Ziegler et al., 1993). The
second luciferase is a fusion protein (Fab2) which forms a monomeric
enzyme comprising LuxA and LuxB and a 10-amino acid residue containing
linker peptide between the carboxyl terminus of the -subunit and
the amino terminus of the
-subunit (Escher et al., 1989).
Both luciferases catalyze the oxygen- and FMNH
-dependent
conversion of a long chain aldehyde to the corresponding fatty acid
with concomitant emission of light. In the case of the heterodimer the
two subunits were observed to fold cooperatively after expression in Escherichia coli and after denaturation and subsequent
renaturation (Waddle et al., 1987). Furthermore, it was shown
that after separate expression of the
- and
-subunits in E. coli the subunits can be stabilized in an
assembly-competent state by the bacterial molecular chaperone GroEL
(Flynn et al., 1993). In addition it was observed that upon
denaturation/renaturation there are slow steps in the refolding of
luciferase subunits and in the formation of the heterodimeric
structure, preceding and following the dimerization (Ziegler et
al., 1993). The fusion protein was shown to be
temperature-sensitive with respect to folding in E. coli (Escher et al., 1989). Furthermore, the bacterial
molecular chaperones GroEL/ES were shown to be able to facilitate
folding of Fab2 at the nonpermissive temperature (Escher and Szalay,
1993). In the case of Fab2 we expected to detect a role of molecular
chaperones in the folding of the de novo synthesized enzyme in
a eukaryotic translation system.
The genes coding for LuxA, LuxB, and Fab2 were cloned into plasmids suitable for in vitro transcription. The plasmids were transcribed in vitro, and the transcripts were subsequently used to program translation in rabbit reticulocyte lysates. The kinetics of folding and assembly of the respective enzymes were studied after supplementing the translation mixture with inhibitors of ATP-dependent molecular chaperones and immunophilins. Using this system we were able to demonstrate that folding and assembly of the two luciferases occur in the in vitro translation system, and that the extent of folding and assembly can be quantified. Furthermore, we observed that bacterial luciferases provide a unique system for direct observation of the effects of molecular chaperones and folding catalysts on protein folding and enzyme assembly in eukaryotic cell-free translation systems.
Originally, these studies were carried out under conditions of coexpression of LuxA and LuxB (resulting in similar concentrations of LuxA and LuxB proteins) and at defined temperatures between 23 and 30 °C. In a first set of experiments we observed that the rate of LuxAB and Fab2 folding was slow when compared to the rate of protein synthesis (data not shown). Therefore, in subsequent experiments protein synthesis was inhibited by the addition of cycloheximide and RNase A at an appropriate time of translation. The efficiency of this translation arrest has been previously demonstrated (Wiech et al., 1987) and is shown in Fig. 1, A and B. LuxA plus LuxB proteins and Fab2, respectively, were synthesized at 23, 25, 28, and 30 °C. After 90 or 120 min the translation reaction was inhibited and the incubation for folding was continued at the same temperature. As expected, the yields of the newly synthesized polypeptides increased with increasing temperature until the inhibitors of translation were added (Fig. 1, A and B). Accordingly, the yield of luciferase activity increased with increasing levels of expression in the case of the heterodimeric enzyme (Fig. 1C). However, in the case of the fusion protein the highest yield of luciferase activity was detected after translation and a incubation for folding at 25 °C (Fig. 1D). Taking into account the different expression levels of Fab2 at the various temperatures, i.e. calculating specific enzyme activities (Fig. 1, E and F), it appeared that Fab2 showed an identical temperature-sensitive phenotype for folding which had previously been observed after expression in E. coli (Escher et al., 1989). Thus, optimal specific activity of the enzyme was observed at 23 °C and decreasing levels were detected at increasing temperatures (Fig. 1F). Both luciferases showed slower folding reactions when compared to the rate of translation. At 23 and 25 °C the folding of the heterodimeric as well as that of the monomeric luciferase could almost be completely separated in time from the synthesis of the various polypeptides (Fig. 1, C and D). We note that after synthesis at 23 °C expression efficiency and folding yield were similar for LuxAB and Fab2 (Fig. 1, E and F). When the translation reactions were shifted to 0 °C after inhibition of protein synthesis, no increase in the enzyme activities occurred (data not shown).
Figure 1:
Kinetics for expression and folding of
LuxAB and Fab2 at different temperatures. Plasmids pLX203ab, pLX203-b,
and pLX709fab2 were separately transcribed with T7 polymerase.
Translation in reticulocyte lysates in the presence of transcripts,
coding for LuxA and LuxB (A, C, and E) or Fab2 (B, D, and F), plus
[S]methionine (A and B) or
plus unlabeled methionine (C and D) was carried out
at 23, 25, 28, and 30 °C, as indicated. After 90 and 120 min
(indicated by arrow), respectively, cycloheximide (final
concentration: 100 µg/ml) and RNase A (final concentration: 80
µg/ml) were added to stop the translation reaction and the
incubation was allowed to proceed. At the indicated times aliquots were
withdrawn and analyzed for luciferase content (A and B) and activity (C and D), respectively. In panels A-D data for three temperatures are shown. The apparent
specific activity, shown in E and F, represents the
enzyme activity which was determined at a certain time of the folding
reaction (relative light units (rlu)
10
), divided by the amount of completed polypeptide
chains which were present at the time of translation stop (arbitrary
units).
Therefore, the two bacterial luciferases provide a unique system for studying folding and enzyme assembly in a cell-free translation system, thus eliminating the need for the use of amino acid analogs or the use of truncated polypeptide chains.
LuxA plus LuxB and Fab2, respectively, were synthesized at 23 °C. After 90 or 120 min, the translation reaction was terminated and the translation mixtures were divided into three aliquots. The first aliquot was left undiluted, the second aliquot was diluted 2-fold with the same reticulocyte lysate, and the third aliquot was diluted 2-fold with buffer. Then, the incubation was allowed to continue at 23 °C and the luciferase enzyme activities were monitored. Folding of LuxAB (Fig. 2A) and Fab2 (Fig. 2B) was equally sensitive to dilutions under the two conditions. If similar dilutions were made after completion of folding reactions but prior to the activity measurements, dilutions had no effect on enzyme activity (data not shown). Thus, it appears that both folding of LuxAB and Fab2 polypeptides in the reticulocyte lysate are bimolecular reactions which involve either transient (Fab2) or permanent (LuxAB) interaction of two translation products. The finding that coexpression of either luxA or luxB gene products together with Fab2 led to an increase in the yield of enzymatically active Fab2, regardless of its synthesis in E. coli cells (Escher et al., 1989) or in reticulocyte lysate (data not shown) further supports the interpretation that the fusion protein is able to interact with another protein during its folding. It seems that folding of both luciferases is slow because even in the undiluted translation reactions both the concentrations of the various polypeptides and the chances for two polypeptides to interact are low and limiting for folding. Therefore, the folding kinetics were slowest under conditions of low expression levels, such as was observed at 23 °C (see above). Due to the pronounced sensitivities of the folding reactions to dilution in reticulocyte lysate, we were unable to detect that reticulocyte lysate proteins limited the folding of LuxAB or Fab2 under these experimental conditions.
Figure 2:
Folding of LuxAB and Fab2 is sensitive to
dilution. Plasmids pLX203ab, pLX203-b, and pLX709fab2 were separately
transcribed with T7 polymerase. Translation in reticulocyte lysates in
the presence of transcripts, coding for LuxA and LuxB (A) or
Fab2 (B), plus unlabeled methionine was carried out at 23
°C. After 90 and 120 min (indicated by arrow),
respectively, cycloheximide and RNase A were added and the translation
mixture was divided into three aliquots. One aliquot was not treated
(), the second aliquot was diluted 2-fold with an equal volume
of sodium phosphate buffer (50 mM, pH 7.0, 23 °C)
(
), the third aliquot was diluted with an equal volume of
translation mixture which did not contain any translation products (23
°C) (
). After incubation for the indicated times, aliquots (5
or 10 µl) were withdrawn and analyzed for luciferase activity. We
note that the activity refers to 5 µl of the translation reaction
which was present in 5 or 10 µl of the folding
reaction.
In these experiments the two subunits of the
heterodimeric enzyme were synthesized separately, subsequently
subjected to the various treatments, then combined (resulting in
similar concentrations of LuxA and LuxB) and incubated further. LuxA,
LuxB, and Fab2, respectively, were synthesized at 23 °C. The
translation was allowed to proceed for 35 or 60 min, and then the
translation mixtures were divided into several aliquots. In the first
experiment, the first aliquot was left untreated and the second aliquot
was supplemented with apyrase, an enzyme which catalyzes the hydrolysis
of ATP and ADP. The third aliquot was supplemented with increased
amounts of apyrase (Fig. 3, A and B). The
incubation was continued at 23 °C and the enzyme activities were
monitored under these various conditions. Folding of LuxAB (Fig. 3A) and Fab2 (Fig. 3B) was
similarly sensitive to apyrase treatment. The inhibitory effect of
apyrase was dependent on the apyrase concentration (Fig. 3, A and B) and reached a maximum at around 30 (LuxAB)
and 55% (Fab2) inhibition under these conditions (Fig. 4). In a
follow up experiment, the first aliquot was left untreated, the second
aliquot was supplemented with apyrase, the third aliquot was
supplemented with apyrase plus ATP, and the fourth aliquot was
supplemented with apyrase plus ATPS (Fig. 3, C and D). The effect of apyrase was due to depletion of ATP since
(i) denatured apyrase had no detectable inhibitory effect (data not
shown), and (ii) the apyrase effect was prevented by ATP but not by
ATP
S (Fig. 3, C and D). In a third
experiment, the first aliquot was left untreated, the second aliquot
was supplemented with ATP, and the third aliquot was supplemented with
ATP
S (Fig. 3, E and F). It was found that
ATP
S competed with ATP and had an inhibitory effect on folding of
LuxAB and Fab2, similar to that of apyrase (Fig. 3, E and F). When apyrase was added after completion of LuxAB
or Fab2 folding it had no effect on the enzyme activity (data not
shown). Therefore, these experiments strongly suggest that ATP
depletion does not interfere with the catalytic activity of the two
enzymes, but affects the folding reaction.
Figure 3:
Folding of LuxAB and Fab2 is
ATP-dependent. We note that panels A and B were
derived from different experiments as compared to panels C and E and D and F. In the latter pairs of panels
the same control reactions are shown twice for comparison. Plasmids
pLX203ab and pLX203-b, respectively, were transcribed with T7
polymerase. Translation in reticulocyte lysates in the presence of
transcripts, coding for LuxA or LuxB, plus unlabeled methionine was
carried out at 23 °C. After 60 min (indicated by an arrow)
cycloheximide and RNase A were added. The LuxA and LuxB translation
mixtures were divided into nine aliquots (A, C, and E). Then, two aliquots were supplemented with water (
with solid line in A, C, and E), three
aliquots were supplemented with apyrase (final concentrations: 0.5
units/ml (
with solid line in A), 16 units/ml
(
with broken line in A), or 20 units/ml
(
in C), one aliquot was supplemented with apyrase
(final concentration: 20 units/ml) plus ATP (final concentration: 8
mM) (
in C), one aliquot was supplemented with
apyrase (final concentration: 20 units/ml) plus ATP
S (final
concentration: 8 mM) (
in C), one aliquot was
supplemented with ATP (final concentration: 8 mM) (
in E), one aliquot was supplemented with ATP
S (final
concentration: 8 mM) (
in E). Equal aliquots of
the LuxA and LuxB translation mixtures were combined. After incubation
for the indicated times aliquots were withdrawn and analyzed for
luciferase activity. Plasmid pLX709fab2 was used to synthesize Fab2 in
a coupled transcription/translation system in the presence of unlabeled
methionine at 23 °C. After 35 min (indicated by arrow),
cycloheximide was added to stop the translation reaction. The Fab2
translation mixture was divided into nine aliquots (B, D, and F). Then, the various aliquots were supplemented as described
above, with the exceptions that the final concentrations of apyrase
were 0.5 (solid line) or 2 (broken line) units/ml in B and 0.5 units/ml in D, and that the final
concentration of ATP and ATP
S were 4 mM in D and F. After incubation for the indicated times aliquots were
withdrawn and analyzed for luciferase
activity.
Figure 4: Folding of LuxAB and Fab2 is ATP-dependent during early stages of folding. LuxA, LuxB, and Fab2 were synthesized for a given time, supplemented with water or apyrase (20 and 2 units/ml, respectively), and incubated further as described in the legend to Fig. 3. The translation time was varied between 45 and 65 min for LuxA/LuxB and between 28 and 60 min for Fab2. The inhibitory effect of ATP depletion on LuxAB or Fab2 folding was determined and was plotted against the translation time (i.e. the time of cycloheximide and RNase A addition).
We conclude from this set
of data that folding of the heterodimeric luciferase occurs after
separate synthesis of the two subunits in reticulocyte lysates. Under
these conditions the assembly-competent subunits lose their competence
in a time-dependent fashion, and no enzymatically active homodimers are
observed when the separately synthesized subunits are separately
incubated further (data not shown). Furthermore, on the basis of these
results we conclude that folding of both the monomeric and the
heterodimeric luciferases involves the hydrolysis of ATP and,
therefore, the folding is drastically reduced either by ATP depletion
or by competition with ATPS. We note that these inhibitory effects
are found to be most pronounced when the inhibitor is added after short
translation (Fig. 4) and that under all conditions they can be
prevented by the addition of ATP (data not shown). Furthermore, we note
that the effect of ATP depletion or ATP
S after coexpression of
LuxA and LuxB is much less pronounced when compared to the separate
expression experiments (data not shown), suggesting that the
ATP-dependent step occurs early in the folding of the two enzymes, i.e. prior to the assembly reaction.
Figure 5:
Folding of LuxAB and Fab2 is sensitive to
CsA and FK506. Plasmids pLX203ab, pLX203-b, and pLX709fab2 were
separately transcribed with T7 polymerase. Translation in reticulocyte
lysates in the presence of transcripts synthesized in vitro and coding for LuxA, LuxB (A, C, E, and F), or
Fab2 (B and D), was carried out at 23 °C. After
60 or 120 min (indicated by arrow) cycloheximide (100
µg/ml) and RNase A (80 µg/ml) were added. A, B, C, and D, equal aliquots of the LuxA and LuxB translation mixtures
were combined and the LuxAB and Fab2 translation mixtures were divided
into two aliquots. One aliquot was supplemented with MeSO
(1%) (
) and one aliquot was supplemented with CsA (40
µg/ml; stock solution: 4 mg/ml in Me
SO) (
) or
with FK506 (100 µg/ml, stock solution 10 mg/ml in Me
SO)
(
). E, equal aliquots of the LuxA and LuxB translation
mixtures were combined and the LuxAB translation mixture was divided
into four aliquots. The aliquots were adjusted to 1% Me
SO
with Me
SO (
), with CsA in Me
SO (40
µg/ml) (
), with FK506 in Me
SO (100 µg/ml)
(
), or with CsA plus FK506 (
). After incubation for the
indicated times aliquots were withdrawn and analyzed for luciferase
activity. F, translation in rabbit reticulocyte lysates in the
presence of transcripts, coding for LuxA and LuxB was carried out at 23
°C. After 120 min cycloheximide and RNase A were added to stop the
translation reaction and the translation mixture was divided into 12
aliquots. Then, the aliquots were adjusted to final concentrations of
1% Me
SO with Me
SO, CsA in Me
SO, or
FK506 in Me
SO at the indicated concentrations. After
incubation for given times, aliquots were withdrawn and analyzed for
luciferase activity. The inhibitory effect of the two drugs at the
different concentrations was calculated after incubation for 360
min.
We therefore
reasoned that simultaneous inhibition of both types of PPIases should
result in a more pronounced inhibition of luciferase folding. Thus the
folding kinetics of the heterodimeric enzyme were studied after
supplementing the translation mixture with the two inhibitors at the
same time. LuxA and LuxB were synthesized at 23 °C. After the
translation reaction was inhibited, the combined translation mixture
(containing similar concentrations of LuxA and LuxB) was divided into
four aliquots. The first aliquot was supplemented with
MeSO, the second and third aliquots were supplemented with,
respectively, CsA and FK506, and the fourth aliquot was supplemented
with CsA plus FK506. Then the incubation was allowed to continue at 23
°C and the enzyme activities were measured. Folding of LuxAB under
these conditions (Fig. 5E) was found to be more
sensitive to a combined addition of CsA and FK506 than to the addition
of CsA or FK506 alone, therefore corroborating a possible role of
PPIases in the folding of newly synthesized luciferases.
In order to
demonstrate the inhibitory effect of CsA and FK506 on PPIases directly,
the translation mixture was supplemented with purified cyclophilin (i.e. a CsA-sensitive PPIase) and the folding kinetics of the
heterodimeric enzyme were studied in the absence or presence of FK506.
The two subunits of the heterodimeric enzyme were synthesized
separately, combined subsequently (resulting in similar concentrations
of LuxA and LuxB), and incubated further under various conditions. LuxA
and LuxB were synthesized at 23 °C. After inhibition of the
translation process, the combined translation mixture was divided into
four aliquots. The first aliquot was supplemented with
MeSO, the second aliquot was supplemented with
Me
SO plus human cyclophilin, the third aliquot was
supplemented with FK506, and the fourth aliquot was supplemented with
FK506 plus cyclophilin. The incubation was allowed to continue at 23
°C and the enzyme activities were monitored by luminometry. After
analyzing these data we found that the inhibitory effect of FK506 on
luciferase folding was partially suppressed by addition of cyclophilin (Fig. 6, A and B). In a follow up experiment
we asked whether the ability of cyclophilin to suppress the inhibitory
effect of FK506 depends on the native state of the enzyme (Fig. 6B). Both, heat and CsA pretreatment of
cyclophilin inhibited its stimulatory effect on luciferase folding in
the presence of FK506.
Figure 6:
Folding
of LuxAB is stimulated by addition of active cyclophilin in the
presence of FK506. Plasmids pLX203ab and pLX203-b were transcribed with
T7 polymerase. Translation in reticulocyte lysates in the presence of
transcripts, coding for LuxA or LuxB was carried out at 23 °C.
After 60 min (indicated by arrow) cycloheximide and RNase A
were added to stop the translation reaction. A, equal aliquots
of the LuxA and LuxB translation mixtures were combined and the LuxAB
translation mixture was divided into four aliquots. Then, one aliquot
was supplemented with MeSO (
), one aliquot was
supplemented with Me
SO and purified cyclophilin (human
CyP-A, recombinant, 270 µg/ml, stock solution: 3.4 mg/ml) (
),
one aliquot was supplemented with FK506 (100 µg/ml) (
), and
one aliquot was supplemented with FK506 and purified cyclophilin (270
µg/ml) (
). B, equal aliquots of the LuxA and LuxB
translation mixtures were combined and the LuxAB translation mixture
was divided into eight aliquots. Then, four aliquots were supplemented
with Me
SO (lightly dotted) and four aliquots were
supplemented with FK506 (50 µg/ml) (densely dotted). One
aliquot of each set of four was supplemented with water (1),
one with purified cyclophilin (270 µg/ml) (4), one with
purified cyclophilin which had been denatured by incubation for 10 min
at 95 °C (2), and one with purified cyclophilin which had
been pretreated with CsA (10 µg/ml) for 10 min at 23 °C (3). After incubation for the indicated times (in B 430 min) aliquots were withdrawn and analyzed for luciferase
activity.
Therefore, we conclude that at least a significant proportion of the inhibitory effect of FK506 on luciferase folding is due to an inhibition of FKBPs which are present in the reticulocyte lysate and not due to either direct or indirect effects of this drug on LuxA and/or LuxB. We also suggest that the same is true for cyclosporin A. Furthermore, we conclude that proline isomerization may indeed be involved in folding of LuxAB and that PPIases which are present in reticulocyte lysates may become limiting under conditions of partial inhibition of PPIase activity. On the basis of these results we propose that one of the slow folding steps which was observed for the folding of the bacterial luciferase in denaturation/renaturation studies (Ziegler et al., 1993) is related to proline isomerization. However, it is possible that luciferase folding involved the general chaperoning activity of the immunophilins (Freskgard et al., 1992) rather than their PPIase activity. In any case, sensitivity of luciferase folding to CsA and FK506 and the partial suppression of the inhibitory effect of FK506 by the addition of cyclophilin provide the first direct evidence for the involvement of immunophilins in protein biogenesis in a eukaryotic cytosolic system.
Relatively little is known about protein folding and subunit
assembly following de novo synthesis of polypeptides in the
eukaryotic cytosol (Ellis and van der Vies, 1991; Gething and Sambrook,
1992; Georgopoulos and Welch, 1993; Hartl et al., 1994). The
best studied example for the latter folding conditions is the folding
of -tubulin and its subsequent assembly with pre-existent
-tubulin (Yaffe et al., 1992). Here, we describe studies
on protein folding using two enzymes catalyzing light emiting
reactions, by employing rabbit reticulocyte lysate as an environment
similar to the eukaryotic cytosol. The two bacterial luciferases, LuxAB
and Fab2, were chosen as model enzymes because their enzymatic activity
is easily detected (high sensitivity, no background) and due to the
available information about their folding characteristics after
denaturation followed by renaturation in vitro as well as
after their expression in E. coli cells (Waddle et
al., 1987; Olsson et al., 1988; Escher et al.,
1989; Flynn et al., 1993; Ziegler et al., 1993;
Escher and Szalay, 1993). Reticulocyte lysate appeared to us as the
eukaryotic cytosol of choice since it allows high yields in expression.
Furthermore, it has been shown in denaturation/renaturation studies
that the system contains various molecular chaperones (such as
t-complex polypeptide 1 ring complex/TriC, and Hsc70) (Gao et
al., 1992; Rommelaere et al., 1993; Nimmesgern and Hartl,
1993; Schumacher et al., 1994).
Here, we demonstrate that
folding and assembly of the two luciferases does occur after synthesis
in the cell-free translation system. We also show that the extent of
folding and assembly can be quantified under these conditions.
Furthermore, bacterial luciferases proved to be a unique system for
direct demonstration of the effects of molecular chaperones in protein
biogenesis. The following folding characteristics were observed for the
two enzymes. (i) Folding of LuxAB and Fab2 can clearly be separated in
time from synthesis at temperatures of 23 and 25 °C. (ii) Folding
of LuxAB and Fab2 shows cooperativity. (iii) Following separate
synthesis, the two subunits of the heterodimeric enzyme stay assembly
competent for a limited time. (iv) Preservation of the assembly
competent state of LuxA and/or LuxB and folding of Fab2 are dependent
on the hydrolysis of ATP. Therefore, it appears that folding of these
luciferases involves ATP-dependent molecular chaperones. The most
likely explanation for the observed ATP effect is that TriC may be
involved in the folding of the two luciferases in the reticulocyte
lysate. This was demonstrated earlier to be the case in -tubulin
folding (Yaffe et al., 1992). Furthermore, the bacterial
homolog of TriC, GroEL, was previously shown to be able to preserve the
assembly competent state of the two subunits of the heterodimeric
enzyme after separate expression in E. coli cells and (in
collaboration with GroES) to reverse the temperature-sensitive folding
phenotype of the hybrid luciferase (Escher and Szalay, 1993; Flynn et al., 1993). At this time Hsc70 cannot be excluded as an
alternative or additional contributor to the ATP-dependent folding.
Hsc70 is assumed to be associated with newly synthesized polypeptide
chains (Beckman et al., 1990; Nelson et al., 1992)
and has been observed to play a role in protein topogenesis in an
ATP-dependent manner (Chirico et al., 1988; Deshaies et
al., 1988; Zimmermann et al., 1988).
PPIases catalyze conversion between cis- and trans-isomers of proline-containing amide bonds of peptides and proteins in vitro. PPIases are abundant proteins and belong to either one of two related protein families which can be distinguished by their sensitivity to immunosuppressant drugs, such as CsA and FK506 (Lang et al., 1987; Takahashi et al., 1989; Fischer et al., 1989; Tropschug et al., 1989, 1990). However, the cellular functions of the PPIases have not yet been established. Three observations pointed to a role of PPIases in protein folding following de novo synthesis of polypeptides in vivo. CsA inhibits the folding of the triple helix of type I collagen in the endoplasmic reticulum of fibroblasts (Davis et al., 1989; Steinmann et al., 1991). In Drosophila melanogaster the cyclophilin homolog ninaA is essential for transport of rhodopsin through the secretory pathway (Colley et al., 1991). In addition, some of the cyclophilins have been shown to be involved in the heat shock response in yeast (Sykes et al., 1993).
Here, we asked if the folding of two luciferases after de novo synthesis in rabbit reticulocyte lysates involves endogeneous immunophilins. The following observations were made: (i) folding of Fab2 and LuxAB, respectively, is partially sensitive to CsA and FK506. (ii) A combination of CsA and FK506 leads to almost complete inhibition of folding. (iii) The inhibitory effect of FK506 on folding can be suppressed by exogeneously added cyclophilin, i.e. a CsA-sensitive PPIase. Therefore, it appears that folding of both luciferases involves PPIases, cyclophilins as well as FKBPs presumably present in the reticulocyte lysate. Thus, these two luciferases provide the first direct evidence documenting the involvement of immunophilins in protein biogenesis in a eukaryotic cytosol.