(Received for publication, October 8, 1996, and in revised form, February 6, 1997)
From the Institut für Biotechnologie 1, Forschungszentrum Jülich GmbH, 52425 Jülich, Germany
and the ¶ Boston Biomedical Research Institute and Department
of Biological Chemistry and Molecular Pharmacology, Harvard Medical
School, Boston, Massachusetts 02114
Wild type and mutant phosphate carriers (PIC)
from Saccharomyces cerevisiae mitochondria were expressed
in Escherichia coli as inclusion bodies, solubilized,
purified, and optimally reconstituted into liposomal membranes. This
PIC can function as coupled antiport (Pi/Pi
antiport and
Pi
net transport, i.e.
Pi
/OH
antiport) and uncoupled
uniport (mercuric chloride-induced Pi
efflux). The basic kinetic properties of these three transport modes
were analyzed. The kinetic properties closely resemble those of the
reconstituted PIC from beef heart mitochondria. A competitive inhibitor
of phosphate transport by the PIC, phosphonoformic acid, was used to
establish functional overlap between the the physiological transport
modes and the induced efflux mode. Replacement mutants were used to
relate the reversible switch from antiport to uniport to a specific
residue of the carrier. There are only three cysteines in the yeast
PIC. They are at positions 28, 134, and 300 and were replaced by
serine, both individually and in combinations. Cysteine 300 near the
C-terminal loop and cysteine 134 located within the third transmembrane
segment are accessible to bulky hydrophilic reagents from the cytosolic
side, whereas cysteine 28 within the first transmembrane segment is
not. None of the three cysteines is relevant to the two antiport modes.
Cysteine 134 was identified to be the major target of bulky SH
reagents, that lead to complete inactivation of the physiological
transport modes. The reversible conversion between coupled antiport and
uncoupled uniport of the PIC depends on the presence of one single
cysteine (cysteine 28) in the PIC monomer, i.e. two
cysteines in the functionally active dimer. The consequences of this
result with respect to a functional model of the carrier protein are
discussed.
The mitochondrial phosphate carrier
(PIC)1 catalyzes transport of inorganic
phosphate into the mitochondrial matrix where the phosphate is utilized
for phosphorylating ADP to ATP (LaNoue and Schoolwerth, 1984; Wohlrab,
1986
; Wehrle and Pedersen, 1989
; Krämer and Palmieri, 1989
,
1992
). The function of the PIC was described as
Pi
/H+ symport, respectively,
Pi
/OH
antiport. The primary
structure of the beef heart PIC was elucidated by protein (Aquila
et al., 1987) and DNA sequencing (Runswick et al.,
1987
), and the PIC gene was cloned and sequenced from Saccharomyces cerevisiae (Phelps and Wohlrab, 1991
). The PIC
is a typical member of the structural family of mitochondrial carriers with six transmembrane segments (Aquila et al., 1985
; Kuan
and Saier, 1993
). Recently the yeast PIC has been expressed as
inclusion bodies in Escherichia coli (Murakami et
al., 1993
; Wohlrab and Briggs, 1994
). Procedures have been
described to solubilize the PIC from inclusion bodies in a functionally
active state (Wohlrab and Briggs, 1994
).
The function of the PIC was studied after purification from various
kinds of mitochondria (for reviews see Wohlrab (1986), Wehrle and
Pedersen (1989)
, and Krämer and Palmieri (1989)
) and reconstitution into proteoliposomes (Wohlrab, 1980
; De Pinto et al., 1982
; Wehrle and Pedersen, 1982
). PIC catalyzes both
homologous Pi
/Pi
as
well as heterologous Pi
/OH
antiport with high activity (Wohlrab and Flowers, 1982
; Stappen and
Krämer, 1993
, 1994
). Transport kinetics using bireactant initial
velocity studies identify the PIC as a member of the mitochondrial carrier family also in functional terms. Its mechanism is of the simultaneous (sequential) type, involving a ternary complex in transport catalysis that requires the binding of two ligands at the
same time (Stappen and Krämer, 1994
). An additional property that
places the PIC into this functional family is its ability to switch to
uniport (efflux) activity after chemical modification with some
mercurial reagents (Stappen and Krämer, 1993
). It was previously
shown that the aspartate/glutamate carrier (Dierks et al.,
1990a), the ADP/ATP carrier (Dierks et al., 1990b
), and the
carnitine carrier (Indiveri et al., 1991
) can reversibly be converted by mercurial reagents from coupled antiport to uncoupled uniport, a function that comprises both carrier-like and channel-like properties. An analysis of this conversion, specifically with the
aspartate/glutamate carrier, identified the involvement of at least two
cysteines and was interpreted to reveal an intrinsic preformed channel
structure as a common element in those carriers (Dierks et
al., 1990b
). These kinds of structural domains had already been
postulated on the basis of functional considerations (Klingenberg,
1981
) as well as of transport experiments (Brustovetsky and
Klingenberg, 1996
).
The aim of the present work was to relate the reversible switch from
coupled antiport to uncoupled uniport to a specific residue of the
carrier protein by using replacement mutagenesis of the yeast PIC
expressed in E. coli. This technique has already been used
to identify a number of other residues, which are important for the
carrier's physiological function with respect to substrate recognition
and inhibitor interaction (Wohlrab and Briggs, 1994; Phelps et
al., 1996
). We demonstrate now that the conversion between antiport and uniport of the PIC exclusively depends on the presence of
cysteine 28 in the PIC monomer, i.e. two cysteine 28 residues in the functionally active dimer.
[33P]Phosphate was obtained from Amersham-Buchler. Sigma supplied Triton X-114, mersalylic acid, pCMBS, DTT, phosphonoformic acid, HEPES, PIPES, and turkey egg yolk phosphatidylcholine. Dowex 2-X10 and SLS were from Fluka, Bio-Beads SM-2 from Bio-Rad, Sephadex G-75 from Pharmacia, and pyridoxalphosphate and HgCl2 from Merck. All SH reagents (HgCl2, pCMBS, mersalylic acid) were prepared from frozen stock solutions and were diluted with water or the respective gel filtration buffer. Pyridoxalphosphate was dissolved in 1 M imidazole (pH 6.5). All other chemicals were of analytical grade.
Generation of Mutants, Expression, Isolation, and Purification of the PICThe gene coding for the PIC was cloned from a yeast
genomic library as described earlier (Phelps and Wohlrab, 1991) and the PIC was expressed in the E. coli strain BL21 (DE3) (Murakami
et al., 1993
; Wohlrab and Briggs, 1994
). Mutants were
generated as described earlier (Phelps and Wohlrab, 1993
; Wohlrab and
Briggs, 1994
). The expression strain Bl21 (DE3) was transformed with
the plasmid pNYHM131 either coding for the wild type PIC or a mutant. A
total of 1 liter of 2 × YT medium (plus 100 mg of carbenicillin) was inoculated with an overnight colony of transformed BL21 (DE3) and
grown to an A600 of 0.6 (about 5 h) under
vigorous shaking at 37 °C. PIC expression was initiated by adding 1 mM isopropyl-1-thio-
-D-galactopyranoside plus 100 mg of carbenicillin. Growth was continued for 3 h, cells were harvested and stored at
20 °C, not longer than 48 h
before continuing the procedure. All the following steps were carried out at 0-4 °C. The pellet from 125 ml of culture was suspended in
TE (10 mM Tris base, 0.1 mM EDTA, 1 mM DTT, pH 7.0) and passed twice through a French pressure
cell, followed by 10 min of centrifugation at 12,000 × g. The pellet was homogenized with 10 ml of TE and centrifuged at 1100 × g for 5 min. 8.8 ml of the
supernatant was centrifuged at 12,000 × g for 3 min,
and the pellet was stored at
70 °C. Isolation of the PIC from
inclusion bodies was carried out applying a previously described
procedure for the oxoglutarate carrier (Fiermonte et al.,
1993
) with some modifications. The pellet was washed three times in
TE-buffer containing 2% Triton X-114, followed by centrifugation at
12,000 × g for 2.5 min. The pellet was solubilized in
400 µl of TE containing 1.67% SLS, followed by addition of 800 µl
of water. The resulting solution was used for reconstitution.
Fig. 1 shows a silver-stained SDS-PAGE of the PIC after
purification.
Reconstitution Procedure
The solubilized PIC was
reconstituted into preformed liposomes by the Amberlite method as
described for the bovine heart PIC (Stappen and Krämer, 1993),
including addition of Triton X-114 in excess over SLS. The
reconstitution procedure was modified with respect to the
phospholipid/protein and phospholipid/detergent ratio. Optimal
transport activity was obtained at a phospholipid concentration of 16 mg/ml, a phospholipid/protein ratio of 140 mg/mg, and a
detergent/phospholipid ratio of 0.62 mg/mg. This means that 70 µl of
Triton X-114 (10%, w/w), 112 µl of liposomes (10% EYPC (w/w) in 50 mM KCl, 20 mM HEPES, 20 mM
potassium Pi, pH 6.5), and 20 µl of protein solution were
mixed with HEPES/potassium Pi buffer (50 mM
HEPES, 30 mM potassium Pi, pH 6.5) to yield a final volume of 700 µl. A detergent/Amberlite ratio of 12 mg/g and 15 column passages were used for detergent removal. The amount of PIC
recovered in the proteoliposomes after reconstitution was found to vary
between 27 and 43% of the protein in the SLS-solubilized fraction.
The reconstituted
transport activities (Pi/Pi antiport,
Pi net transport, and Pi efflux) were
determined by measuring the flux of [33P]phosphate. The
applied methods resemble those described previously for the analysis of
the aspartate/glutamate carrier (Dierks and Krämer, 1988; Dierks
et al., 1990a
). In most experiments, antiport activity was
determined by the forward exchange procedure. The assay was started by
adding labeled phosphate to the proteoliposomes containing unlabeled
substrate inside and the increase in internal label was followed. In
some experiments, the Pi/Pi antiport mode was
measured by the backward exchange method, which is similar to the
procedure used for determination of the net transport mode (Pi
/OH
) or the
HgCl2-induced efflux mode. For this, the internal pool was
prelabeled by incubating the proteoliposomes with
[33P]phosphate of high specific radioactivity at 21 °C
for 10 min. The external substrate (and label) was then removed by size
exclusion chromatography on Sephadex G-75 columns at 4 °C. Since the
PIC also catalyzes net transport, the use of this method depends on the
availability of reversible inhibition during chromatography. Routinely,
200 µM mersalylic acid was used for this purpose. After removal of external phosphate, transport was started by adding 10 mM DTT (net transport) or 10 mM DTT together
with external phosphate (Pi/Pi antiport). The
mercurial-induced uniport was assayed by adding 0.5 mM
HgCl2 to proteoliposomes after the size exclusion
chromatography. After the desired period of time carrier-mediated transport was stopped by adding 25 mM pyridoxal phosphate
with or without 10 mM DTT. After application of the stop
mix, each sample was passed through an anion exchange column (Dowex
1-X10, Cl
form) to remove the external label. Further
details were as described previously (Dierks and Krämer, 1988
;
Dierks et al., 1990a
).
For PIC mutants lacking Cys-134, an alternative inhibition technique had to be developed since these mutants cannot be inhibited by mersalyl. In this case, the internal pH of the proteoliposomes was adjusted to pH 6.8 during formation by high internal buffer (HEPES, 50 mM). The external pH in the size exclusion chromatography was set to 5.5 (PIPES, 5 mM). Due to the H+-coupled transport mechanism, the pH gradient prevented loss of internal substrate. Before starting net transport, the external pH was adjusted to 6.8 by adding 50 mM HEPES, pH 7.0. The data of Table I indicate that retention of internal phosphate by this method was comparable with the generally applied procedure involving inhibitor addition.
|
Forward exchange rates were determined by fitting the time course of
isotope equilibration to a single exponential y = A · (1 e
kt) + B,
leading to the first order rate constant k (in
min
1). The specific activity (µmol/min · mgProt) was calculated from k
(min
1), the final value of isotope equilibration (dpm),
the specific radioactivity (dmp/nmol), the volume of the proteoliposome
fraction (ml), and the protein concentration (µg/ml) as published
previously (Dierks and Krämer, 1988
). Backward exchange rates
were calculated by the equation v
= k · Sin · Vin in which
k (min
1) is the apparent first order rate
constant calculated by the equation
= 100 (1
e
kt) (
, percentage of isotope
equilibration; Sin, internal substrate concentration; Vin, internal volume of liposomes
catalyzing transport) (Dierks and Krämer, 1988
).
Protein concentrations were
determined by the modified Lowry method after precipitation with
deoxycholate and trichloroacetic acid and extraction of detergent and
lipid by organic solvents (Peterson, 1977; Dulley and Grieve,
1975
).
So far, for functional
characterization of the mitochondrial PIC, mainly the protein from beef
heart or pig heart was studied after isolation using Triton X-100 or
X-114 (Wohlrab, 1986; Krämer and Palmieri, 1989
). A detailed
functional analysis after reconstitution by chromatography on Amberlite
was carried out with the beef heart protein (Stappen and Krämer,
1993
, 1994
). In the present work, we use a different protein (yeast
instead of beef heart PIC), a different source (bacterial inclusion
bodies instead of intact mitochondria), a different detergent (SLS
instead of Triton X-114), and an altered reconstitution procedure
(optimized for SLS-solubilized protein). Consequently, it was essential
to compare the properties of the yeast PIC isolated from E. coli inclusion bodies with the data previously obtained in
experiments using the beef heart protein.
Since for the PIC, in contrast to the ADP/ATP carrier, no side-specific
inhibitors are available, the orientation of the carrier protein in
proteoliposomes was previously established by analyzing side-specific
substrate interaction (Stappen and Krämer, 1993). Such a kinetic
analysis of the
Pi
/Pi
antiport of
the yeast PIC, both for the wild type and the Cys-28
Ser mutant as
representative examples for the recombinant proteins, is shown in Fig.
2. For the
Pi
/Pi
and the
Pi
/OH
antiport (net transport),
the Km values (transport affinity) at both sides of
the proteoliposomes are shown for the wild type and several mutants
(Table II). The following conclusions could be drawn.
(i) Only one single kinetic component is observed for interaction of
phosphate at the inside and the outside, respectively. (ii) The
Km values for phosphate in the wild type and the
Cys-28
Ser mutant are identical. (iii) A comparison shows that the
corresponding values of the beef heart and yeast PIC for interaction
with phosphate at the two sides of the protein are more or less
identical. (iv) These data also prove that the PIC of beef heart and
yeast are oriented in the same direction after reconstitution,
i.e. right side out (Stappen and Krämer, 1993
). These
results indicate that the methods previously derived for the PIC from
beef heart mitochondria can be applied to the reconstituted yeast
carrier protein.
|
The reconstituted beef heart PIC was shown to catalyze three different
functions, i.e. homologous
Pi/Pi
antiport,
heterologous Pi
/OH
antiport
(net transport), as well as substrate-unspecific uniport after
treatment with HgCl2 (Stappen and Krämer, 1993
). The
same was observed for the PIC in intact yeast mitochondria (Stappen, 1994
), as well as for the wild type yeast PIC isolated from inclusion bodies after expression in E. coli (Table II, see also Fig.
5). The transport affinities (Km) turned out to be
very similar, whereas the Vmax values were in
general higher for the yeast PIC. Since the efficiency of
reconstitution (the share of successfully incorporated protein) was not
quantitated in these experiments in detail, the somewhat different
Vmax values do not necessarily mean that the
molecular activity of the PIC from the different sources is in fact
different. Irrespective of this restriction, it is interesting to note
that the ratio of
Pi
/Pi
antiport to
Pi
/OH
antiport (net transport)
is also very similar for the two proteins (Stappen and Krämer,
1994
).
Characterization of Uniport Activity of the Yeast PIC Expressed in E. coli Cells
To unequivocally correlate the observed uniport
activity to the reconstituted yeast PIC and to define its properties in
the mutants studied, we used various inhibitors. The unspecific
inhibitors pyridoxal phosphate and mersalylic acid were previously
described as effective reagents stopping transport (Stappen and
Krämer, 1993). The data in Table III show that
pyridoxal phosphate works in all cases. Mersalylic acid, on the other
hand, is ineffective when applied to mutants in which Cys-134 has been
replaced by Ser. Consequently, we had to develop another method for
reversibly inhibiting net transport during size exclusion
chromatography, namely application of an inverse pH gradient (see
"Experimental Procedures"). The SH reagent pCMBS also reacts with
Cys-134 and/or Cys-300 (Table III), as shown above for mersalyl.
Besides interacting with Cys-28, HgCl2 blocks the transport
reaction, at least in part, by reacting also with Cys-134 and/or
Cys-300.
|
The availability of phosphonoformic acid (PFA) (Kempson, 1988),
originally discovered to block the kidney phosphate carrier, prompted
us to characterize the properties of the mercurial-induced efflux by
the yeast PIC in more detail. The only evidence that the
HgCl2-induced uniport activity of the PIC involves the
phosphate binding site is its inhibition by external phosphate (Stappen and Krämer, 1993
). PFA significantly inhibits
Pi
/Pi
antiport, but
has no effect on Pi
net transport
(Pi
/OH
antiport) (Table III).
PFA blocks the mercuric chloride-induced phosphate efflux (Fig.
3A) to about the same extent as phosphate (Stappen and Krämer, 1993
). Similar concentrations of sulfate, which is not a ligand of the PIC, shown no effect. We analyzed the
effect of PFA on
Pi
/Pi
antiport in
more detail. Fig. 3B reveals its competitive inhibition of
Pi
/Pi
antiport with
a Ki of 0.75 mM.
Antiport and Uniport Activity of Wild type and Mutant Yeast PIC
We measured the Vmax for all three
transport modes of several reconstituted mutant PICs. The results were
compared with those obtained with proteoliposomes reconstituted either
with the PIC from beef heart or that from S. cerevisiae
mitochondria. It should be noted that the results for Cys-134 Ser
mutants were obtained using an inverse pH gradient during size
exclusion chromatography (see "Experimental Procedures"). In Fig.
4, the
Pi
/Pi
antiport
activities of the various mutants are compared with that of the wild
type. In all cases the transport rates were lower than that of the
parent strain. The transport rates, however, are still significant,
i.e. more than 20% of that the wild type. Most importantly,
the ratio of Pi
/Pi
antiport to Pi
/OH
antiport (net
transport) remained more or less constant, irrespective of the altered
absolute values of the two physiological transport modes.
Whereas both the homologous and the heterologous antiport activities
were retained in the PIC proteins, this was not the case with the
mercuric chloride-induced uniport (Fig. 4). This induced phosphate
efflux catalyzed by the wild type as well as the Cys-28 Ser and
Cys-300
Ser mutant proteins is shown in Fig. 5. The relative efflux rate by the Cys-300
Ser mutant is similar to that
of the wild type, while the Cys-28
Ser mutant, even when treated
with high mercurial concentrations, is not able to undergo the
functional switch from coupled antiport to uncoupled uniport (efflux).
The induced uniport activities of all PICs are compared in Fig. 4. In
all mutants in which Cys-28 was replaced by Ser, independent of a
replacement of the other two cysteine residues, Cys-234 and Cys-300),
the functional switch to uniport could not be induced.
The mitochondrial PIC can reversibly be switched from coupled antiport to uncoupled uniport. We have used replacement mutants of the yeast PIC expressed in E. coli to relate this switch to specific residues of this protein. The involvement of cysteine residues in this switch can most appropriately be studied with the yeast PIC, since 1) it has only three cysteines versus eight in the beef heart PIC used in earlier studies, and 2) its preparation is facilitated with E. coli inclusion bodies. We have shown that the yeast PIC, isolated and purified from inclusion bodies, solubilized by SLS and reconstituted into liposomes resembles the beef heart PIC in all the relevant functional aspects.
The mercuric chloride-induced uniport has now been definitely
identified with the reconstituted PIC. Contaminating mitochondrial channel proteins cannot be responsible for this activity since the PICs
are heterologously expressed. Furthermore, we found that PFA, a
competitive inhibitor of
Pi/Pi
antiport,
effectively inhibits also the induced uniport mode. Since PFA
stimulates Pi
/OH
(Pi
net transport), we conclude that it is a
transport substrate of the PIC. PFA is not available in labeled form,
and thus its true substrate properties could not be directly
investigated. Nevertheless, its effect on the mercuric chloride-induced
Pi
efflux can be taken as a further
indication that the external binding site of the reconstituted PIC has
retained its properties. This site, as shown earlier, is the cytosolic
site of the PIC (Stappen and Krämer, 1993
).
It has been shown for many other carrier proteins that cysteines are
not essential for basic transport functions (van Iwaarden et
al., 1991, 1992
). The present results permit us to characterize the functional significance of the yeast PIC cysteines (Fig.
6). Cys-300 does not seem to be relevant for transport
activity or for uniport induction. We did, however, detect a 50%
reduction of transport after its replacement by serine. No additional
changes were observed in combination with the Cys-134
Ser
replacement. Cys-134 is responsible for inhibition of all transport
modes by mersalylic acid and also by pCMBS. A comparison with published effects on the PIC (Stappen and Krämer, 1993
) makes it likely that Cys-134 is also responsible for inhibition by
5,5-dithiobis(2-nitrobenzoic acid). Although a reaction of mersalyl
with Cys-300 seems to be responsible for a slight reduction in antiport
activity by the Cys-134
Ser mutant relative to the Cys-134
Ser/Cys-300
Ser and the Cys-28
Ser/Cys-134
Ser/Cys-300
Ser mutants, it is obvious that the major target of mersalyl is
Cys-134. The reactivities of the cysteines indicate that Cys-134 (and
presumably also Cys-300) is accessible to large, hydrophilic ligands,
whereas Cys-28 is accessible only to the small Hg2+ ion.
This was documented by the observation that mercurials other than
HgCl2 did not affect the reaction of Hg2+ with
Cys-28. Whereas the accessibility of Cys-300 from the external (cytosolic) side seems obvious, this is not so for Cys-134. Our results
suggest that Cys-134 is in an aqueous environment with connected to the
cytosolic side of the protein, whereas Cys-28 is not. Interestingly,
Cys-28 has previously been found to be the target for inhibition of
phosphate transport by oxygen (Phelps and Wohlrab, 1993
). In a recent
paper, furthermore, His-32, Glu-126, and Glu-137 were found to be
essential for a coupled phosphate/H+ pathway in the PIC
(Phelps et al., 1996
). In fact, these residues line up at
the same side of helices A and C, in which Cys-28 and Cys-134 are
located. It may be questioned whether a reaction of HgCl2
with other residues besides cysteine should be considered. This has in
fact been analyzed in detail with respect to the aspartate/glutamate carrier, where a functionally significant reaction was found to be
confined to cysteine residues (Dierks et al., 1990a
, 1990b
). In any case, the specific action on Cys-28 and Cys-134 of the PIC was
proven here by the absence of these effects in the mutants lacking
these cysteines.
Most interesting, however, is the observation that the reversible
switch from the physiological transport modes to the unphysiological and mercuric chloride-induced uniport depends on the presence of
Cys-28. This functional shift from antiport to uniport, which is
correlated with the appearance of some channel-type functions, has been
observed in several mitochondrial carriers, namely the aspartate/glutamate, the ADP/ATP (Dierks et al., 1990a;
Dierks et al., 1990b
), and the carnitine carrier (Indiveri
et al., 1991
). The present results, however, for the first
time correlate a specific residue with this phenomenon. The observation
that mitochondrial carriers retain their original activation energy of
transport after this functional switch was interpreted to indicate that a similar conformational change occurs during solute transfer both in
the antiport and the mercuric chloride-induced uniport (Herick and
Krämer, 1995
). Modification of Cys-28 by HgCl2 yields a loss of specificity of ligand interaction at the internal PIC binding
site. No such loss of the external binding site is observed (Dierks
et al., 1990b
; Stappen and Krämer, 1993
). In
conclusion, our findings suggest that Cys-28 may possess a gating
function on the matrix side of the PIC.
There is evidence from a large number of investigations that the
structural basis for differences in the mechanisms of coupling among
antiport, symport and uniport in secondary systems and differences between carrier and channel-type of functions may be very subtle (Nikaido and Saier, 1992; Krämer, 1994
). Thus, for example, a single amino acid replacement in the bacterial lactose carrier (Lac-permease) shifts its transport from a coupled to an uncoupled mechanism (Eelkema et al., 1991
; King and Wilson, 1990
;
Kaback, 1992
). Furthermore, a number of studies using
electrophysiological techniques provide evidence for channel properties
in carrier proteins, e.g. neurotransmitter transporters
(Cammack and Schwartz, 1996
; DeFelice and Blakely, 1996
), the
chloroplast triose phosphate carrier (Schwarz et al., 1994
)
as well as the mitochondrial ADP/ATP carrier (Tikhonova et
al., 1994
; Brustovetsky and Klingenberg, 1996
). This concept is
particularly attractive in view of the fact that conditions are known
where large pores appear in the inner mitochondrial membrane
(permeability transition pore, "megachannel") (Zoratti and Szabo,
1995
; Bernardi and Petronilli, 1996
). Taken together, the distinction
between "carrier-type" and "channel-type" of transport
mechanisms seems to become minimal, when analyzed in terms of
functional elements in both types of solute transport systems (Nikaido
and Saier, 1992
; Krämer, 1994
; DeFelice and Blakely, 1996
).
The data presented here prove that the reversible shift between the
coupled and the uncoupled transport mode of the mitochondrial PIC only
depends on the modification of a single cysteine. It has to be pointed
out, however, that mitochondrial carrier proteins are functional
dimers, i.e. there are two cysteines at position 28. Interestingly, earlier investigations of the aspartate/glutamate carrier indicated that the modification of two cysteine residues is
necessary for the reversible shift from antiport to uniport (Dierks
et al., 1990a; Dierks et al., 1990b
). Although
the primary structure of the aspartate/glutamate carrier is not yet
known and thus the two cysteines have not been identified, it is
obvious that, on the basis of the apparently common mechanism of this antiport/uniport conversion in mitochondrial carriers, the two results
are related. Consequently, studying the functional involvement of the
two cysteines located in the two homologous monomers of mitochondrial
carrier proteins may be an interesting clue for understanding the
crosstalk of the individual subunits during transport catalysis.
We are grateful to D. Pain for providing us the plasmid pNHYM131, to K. Herick for intensive collaboration in the optimization of the purification and reconstitution procedure, and to H. Sahm for continuous and generous support. We also thank A. Phelps and C. Briggs for constructing the PIC mutants in the bacterial expression vector.