(Received for publication, August 10, 1995; and in revised form, October 16, 1995)
From the
We previously reported the ability of protein disulfide
isomerase (PDI) to undergo an ATP-dependent autophosphorylation. Our
efforts to map the modification site have been hindered by the low
abundance and instability of the labeling. Results are presented in
this paper on the nature of phospho-PDI, which appears as an
intermediate with a half-life of 2.5-8.8 min in an ATPase
reaction. ATP binds to PDI with high affinity, K9.66 µM, and the kinetic
parameters K
and k
of the ATPase reaction were measured by using
a pyruvate kinase-lactate dehydrogenase-coupled assay under various
conditions. Strikingly, the ATPase reaction is stimulated in the
presence of denatured polypeptides, while the disulfide oxidization
activity of PDI is not affected by ATP. However, PDI is known to
participate in various unrelated functions in the endoplasmic
reticulum, and ATP could be involved in the regulation of one of these.
The results are discussed in light of recent findings on ATP-chaperone
relationships.
Although there is no direct experimental evidence of the
presence of ATP in the lumen of the endoplasmic reticulum (ER), ()it has been previously established that it is required to
support the correct folding and disulfide bond formation of
proteins(1) . Molecular chaperones of the ER, such as BiP,
grp94, and calnexin, hydrolyze or at least bind ATP in the course of
their activity in vitro(2) , which also strongly
suggests that an ATP pool should exist in the lumen in this
compartment. Finally, the characterization of an ATP translocator in
the ER membranes of mammalian (3) and yeast (4) cells
has settled the question of how ATP accumulates in the ER. Our
laboratory reported that protein disulfide isomerase (PDI), an abundant
microsomal protein, can undergo an ATP-dependent autophosphorylation in vitro and likely represents another physiological target
for ATP in the ER (5) .
PDI was originally characterized as
an enzyme (E.C. 5.3.4.1) involved in the catalysis of the
protein-disulfide formation(6, 7) , but in recent
years, it has been found to be more than just a thioredoxin-like
oxidoreductase. Its role in protein folding and cell physiology are
more complex than initially thought since PDI now appears as a
multifunctional protein (8) and even a
chaperone(9, 10) . These features could reflect the
general ability of this protein to bind various peptides and proteins
apparently regardless of their sequence (7, 11) . PDI
associates with various proteins such as the -subunit of
prolyl-hydroxylase, N-glycosyl transferase, and triglyceride
transfer complex and has been revealed to be identical to the
T3-binding protein, P55 (see (8) for a review).
Interestingly, it has been found by using a genetic approach in
yeast that the essential function of PDI does not reside in the
thioredoxin-like domains(12) . In this context, we previously
suggested that PDI phosphorylation modulates its interaction with
various partners(5) . The precise relationship between ATP and
PDI remains elusive. Preliminary attempts to find the effect of ATP on
PDI catalysis (13) or ATP hydrolysis (14) were
unsuccessful. Using affinity chromatography on various immobilized
denatured proteins, Nigam et al.(15) reported the
Ca-dependent association of several ER proteins,
among them PDI, with these substrates and their elution by ATP.
However, the experiments did not provide a direct association between
PDI and ATP.
The following paper presents the data we obtained on ATP binding by PDI and the use of a sensitive spectrophotometric method to demonstrate the existence of an ATPase activity for this protein. The kinetic parameters of this activity were measured under various conditions. The results suggest a new role for ATP in the ER, which uses the multifunctional PDI as a target.
The ability of PDI to autophosphorylate in the presence of
[-
P]ATP has been shown
previously(5) . However, the phospho-PDI obtained in such
conditions turned out to be very unstable, rendering it very difficult
to handle for further experiments such as mapping of the
phosphorylation site. The transient nature of the phosphorylation was
quantitatively approached, and half-lives of 2.5 and 8.8 min were
measured under ATP chase or without chase conditions, respectively (Fig. 1). The maximal quantity of
P radioactivity
incorporated into PDI indicated that phospho-PDI never accumulates to
more than 0.4%. The substrate specificity of the phosphate donor was
investigated, and we observed that both
[
-
P]dATP and
[
-
S]ATP can be substituted for
[
-
P]ATP without any apparent modification
in the labeling efficiency. On the other hand,
[
-
P]ATP,
[
-
S]ADP, [5`-
P]AMP,
or [
P]PO
did not
give rise to PDI labeling (data not shown). These preliminary data
suggested that phospho-PDI forms by a reaction involving the
-phosphoryl of ATP or dATP and requires cleavage of the bond
between the
- and
-phosphates. The presence of Mg
in the phosphorylation mixture is critical for the formation of
phospho-PDI; therefore, the substrate of the reaction is certainly the
Mg-ATP complex.
Figure 1:
Measurement of the rh-PDI
autophosphorylation turnover. A 5-min labeling with limiting quantity
of [-
P]ATP was performed, and the
autophosphorylated PDI was allowed to incubate either in the absence
(
) or presence of 1 mM chasing ATP (
). Aliquots
were put on a SDS, 10% PAGE, and the decay at the level of the PDI band
was quantitated from the autoradiogram. Data were fitted to an
exponential to obtain the half-life values.
The properties of this ATPase activity of PDI were
assayed in a pyruvate kinase-lactate dehydrogenase-coupled reaction. A
significant deviation of the absorbance at 340 nm was observed in
presence of PDI, which implies a significant consumption of ATP (Fig. 2). Prokaryotic oxidoreductases related to PDI, i.e. thioredoxin and DsbA, are devoid of this activity. This is in good
agreement with our preliminary report that the site of phosphorylation,
and thus probably the ATPase active site, lies somewhere within the
central domain of the protein(5) , which is specific for the
mammalian enzyme. This site is far away from the redox active sites in
the sequence. Furthermore, the measurements of the rates of
PDI-catalyzed refolding of denatured-reduced RNase A in the absence
(0.70 ± 0.02 mmol RNaseAmin
mmol
PDI
) or in the presence of ATP (0.70 ± 0.02
mmol RNaseA
min
mmol
PDI
) show that ATP does not, or only very slightly,
affects this PDI activity (Fig. 3). We are thus in agreement
with a previous report describing the lack of direct effect of ATP on
the disulfide formation catalysis(13) .
Figure 2: Spectrophotometric assay of ATPase activities for proteins of the thioredoxin family. Measurements were made in the pyruvate kinase-lactate dehydrogenase-coupled assay in the presence of 0.1 mM ATP and 1.8 µM rh-PDI (1), thioredoxin (3), or DsbA (4). A control (2) with PDI in the absence of Mg-ATP was carried out to verify the absence of oxidizing effect of PDI on NADH.
Figure 3:
Effect
of ATP on the PDI-catalyzed RNase A refolding. A, the
rh-PDI-catalyzed formation of native RNase A is followed from its
enzymatic activity recovery, i.e. hydrolysis of 2`,3`-cyclic
CMP into 3`-CMP in the absence () or in presence (
) of 0.1
mM Mg-ATP. The amount of active RNase at each time (inset) was calculated from A
versus time as described under ``Experimental
Procedures.'' PDI activity is the slope of the regression in the
linear phase of the reaction and after a 2-3-min lag.
Non-catalyzed RNase renaturation was subtracted in the activity
calculations.
However, when we
measured the kinetic parameters of this ATPase reaction under various
conditions, we saw an unexpected dependence of this parameter on the
assay conditions. While the rate of hydrolysis is 0.057
µMmin
µM monomer
for PDI alone, it increases 6.7-fold
when the hydrolysis is measured under the conditions of the RNase
refolding assay, i.e. in the presence of GSH, GSSG, and RNase
A (Fig. 4). A comparison of various effectors was carried out to
clarify how the PDI-ATPase depends on assay conditions (Table 1).
The redox state of the medium does not seem to be the basis of this
effect since GSH, GSSG, or DTT does not allow the hydrolysis to reach
the optimal value. Nevertheless, both GSH and GSSG lead to a
significant 2-fold increase. The presence of denatured RNase A, either
alone or associated with the redox partners GSH and GSSG, enhances the
ATPase 5-7-fold without significant alteration of the K
. The alkylation of the eight RNase thiol groups
with N-ethylmaleimide did not alter its stimulatory effect on
the hydrolysis. Furthermore, a control experiment performed with
lysozyme displayed similar activity. The redox potential of the medium,
and as a result the redox state of the PDI active site, is not
responsible for the stimulatory effect on the ATPase activity. The
complete chemical modification of the PDI active site with N-ethylmaleimide (verified by using the Ellman reagent) does
not completely abolish the ATPase activity but decreases it by a factor
4 (data not shown). To sum up, the ATPase promotion likely ensues from
the association with a polypeptidic effector in the assay mixture. In
this respect, the difference observed between DTT and GSH could rely on
the peptidic nature of glutathione. The k
determined under optimal conditions, 0.37 min
(Table 1), is in the same range as the rate measured for
the PDI-catalyzed formation of native RNase A from either its
reduced-denatured form (see above and (18) ) or its scrambled
form(19) .
Figure 4:
Kinetic analysis of the PDI ATPase
activity. ATP hydrolysis was estimated from the pyruvate kinase-lactate
dehydrogenase-coupled assay at various ATP concentrations. Kinetic
parameters K and V
were determined from the fitting of the data to a
Michaelis-Menten curve (A) for rh-PDI alone (
) and for
rh-PDI (
) and bovine PDI (
) in the presence of GSH, GSSG,
and Rnase in the assay mixture. The cooperativity under optimal
conditions of ATP hydrolysis by rh-PDI (
) was assessed from a Hill
plot (B).
However, ATPase and redox sites certainly
function independently since various simultaneous measurements of these
activities gave stoichiometric relationships between S-S bridge
formation and ATP consumption ranging from 5 to 10, depending on the
amount of PDI present in the assay (data not shown). When analyzed as a
reciprocal plot 1/v = f(1/S), the
data contained in Fig. 4A depicted an upward curvature
at very low ATP concentrations, which is indicative of a possible
cooperativity in the ATP hydrolysis reaction (data not shown). This
departure from the linearity neither results from the auxiliary system,
since the proportionality curve was linear and intercepted the y axis at zero, nor from the enzyme preparation, since similar
results were obtained for two preparations of rh-PDI and for bovine
liver PDI. A Hill plot (Fig. 4B) gives a cooperativity
number of 1.49 for the PDI dimer, which can be assumed to derive from
the vicinity of two monomeric ATP binding sites within the dimer. We
measured the ATP binding properties of PDI by using
[-
P]dATP as a ligand and in the absence of
Mg
to prevent hydrolysis during the time necessary to
reach equilibrium. The intercept with the x axis of the
Scatchard plot (Fig. 5), from which we calculated 0.7 ATP
binding site per PDI monomer, confirms the presumption anticipated
before from kinetics data. The dissociation constant for ATP is K
= 9.66 ± 0.48 µM.
Under comparable conditions, except for the presence of
Mg
, we measured a K
for Mg-ATP
of 7.1 µM (Table 1), which suggests that ATP
complexed with magnesium would have a better affinity for PDI.
Otherwise, substrate binding will become a very limiting step in the
reaction mechanism.
Figure 5:
ATP binding by rh-PDI at equilibrium.
Measurements were performed by dialysis on rh-PDI and
[-
P]dATP in the absence of Mg
to prevent hydrolysis. The dissociation constant and the number
of ATP binding sites were determined from a linear regression in the
Scatchard secondary plot (inset).
An additional experiment showing the coelution of PDI and ATPase activity on an HPLC column is presented to strengthen previous experiments. When pure rh-PDI is submitted to gel filtration (Fig. 6), three major peaks are detectable that correspond to various oligomeric states of the protein as described previously(20) , namely high molecular weight form (A), tetramer (B), and dimer (C). The ATPase activity profile superimposed nicely with absorbance peaks, indicating that each oligomer certainly has a similar specific activity.
Figure 6: HPLC separation of PDI-associated ATPase. Five runs with 50 µg of pure rh-PDI were performed on a gel filtration column (a typical chromatogram is shown). 25 0.5-ml fractions were collected across the separation and then pooled and concentrated to 50 µl using Centricon 10 devices. ATPase activity was assayed on 25 µl of each fraction in the presence of GSH, GSSG, and RNase. As depicted on the 10% SDS-PAGE, all peaks correspond to the PDI monomer (57 kDa) in various oligomeric states. Lane H represents rh-PDI before injection.
These results describe a new property for the multifunctional
protein PDI. Both ATP binding and hydrolysis properties are clearly
exhibited and were extensively characterized. The data presented here
corroborate the preliminary evidence published in our first paper on
bovine liver PDI(5) . Several soluble and membrane-bound
ATPases have been found in microsomal extracts that could contaminate
our enzyme preparations. We ruled out this possibility for the
following reasons: similar V and K
were measured for both human recombinant PDI expressed in E.
coli and for the bovine PDI extracted from ox liver microsomes (Fig. 4, Table 1), the calculation of the concentration of
ATP binding sites from the Scatchard plot gives a concentration very
close to that of PDI and which cannot be that of a contaminant, and
ATPase copurifies with PDI in HPLC. The rate of ATP hydrolysis could
appear low compared with other kinases, even under the optimal
conditions. However, it is in the same range as those measured for
chaperones, i.e. less than min
, and its
efficiency is quite high when expressed in term of k
/K
, i.e. 85
10
min
M
.
Phospho-PDI appears as a covalent intermediate in the hydrolysis
reaction. Its short half-life, low abundance, and instability partly
explain why we did not succeed in purifying sufficient amounts of
material to identify, at the sequence level, the site of
phosphorylation. The PDI sequence was searched extensively for
homologies with kinase, ATPase, and chaperone subsets from the EMBL
data base as well as for motifs from the Prosite base without any
significant hit. So, the mechanism of ATP hydrolysis might be original.
The presence of peptide substrate in the assay increases the rate of
hydrolysis while ATP by itself does not promote the apparent activity
of PDI on RNase refolding. In some respects, the situation could be
close to that of Hsc70-ATPase, which is stimulated 2-3-fold upon
addition of an unfolded polypeptide(21) . There is no apparent
stoichiometry between ATP hydrolysis and S-S bridge formation.
From a thermodynamic point of view, disulfide bond oxidization is
exergonic and thus, a priori, does not require the energy
input provided by the ATP hydrolysis. In the case of DnaK, it was
clearly demonstrated that the ATPase is not stoichiometrically coupled
to a peptide binding/release cycle(22, 23) . The
requirement for ATP hydrolysis is not even absolute in the chaperone
function of either GroEL (24) or DnaK(25) . Both ATP
and ADP are able to change the conformation of
GroEL(26, 27) , whose protein binding properties are
also regulated by heat shock-induced phosphorylation(28) . The
relationship between PDI and ATP is puzzling and might reveal another
possible likeness with molecular chaperones. However, we regard this
with extreme reservation and we are at work on it to validate this
presumption. The function of PDI, which is possibly under ATP control,
and the role of hydrolysis remain to be determined as well as whether
the conclusions drawn in vitro are also relevant in the
cellular context.