(Received for publication, September 14, 1995)
From the
Mammalian mitochondria possess an inner membrane channel, the
permeability transition pore (MTP), which can be inhibited by nanomolar
concentrations of cyclosporin (CS) A. The molecular basis for MTP
inhibition by CSA remains unclear. Mitochondria also possess a matrix
cyclophilin (CyP) with a unique N-terminal sequence (CyP-M). To test
the hypothesis that it interacts with the MTP, we have studied the
interactions of CyP-M with rat liver mitochondria by Western blotting
with a specific antibody against its unique N terminus. Although
sonication in isotonic sucrose at pH 7.4 releases a large proportion of
CyP-M, a sizeable CyP-M fraction sediments with submitochondrial
particles at 150,000 g. We show that the interactions
of this CyP-M pool with submitochondrial particles are disrupted (i) by
the addition of CSA, which inhibits the pore, but not of CSH, which
does not, and (ii) by acidic pH condition, which also leads to
selective inhibition of the MTP; furthermore, we show that the effect
of acidic pH on CyP-M binding is prevented by diethylpyrocarbonate,
which fully prevents the inhibitory effect of H
on the
MTP (Nicolli, A., Petronilli, V., and Bernardi, P.(1993) Biochemistry 32, 4461-4465). These data suggest that
CyP-M binding is involved in opening of the MTP and that pore
inhibition by CSA and protons may be due to unbinding of CyP-M from its
putative binding site on the MTP. A role for CyP-M in MTP regulation is
also supported by a study with a series of CSA derivatives with graded
affinity for CyP. We show that with each derivative the potency at
inhibition of the peptidylprolyl cis-trans-isomerase activity
of CyP-M purified to homogeneity is similar to that displayed at
inhibition of MTP opening, relative to that displayed by CSA. Decreased
binding to CyP-M (but not CyP-A) and decreased efficiency at MTP
inhibition is obtained by substitutions in position 8 while a
4-substituted, nonimmunosuppressive derivative is as effective as the
native CSA molecule, indicating that calcineurin is not involved in MTP
inhibition by CSA.
Mitochondria from a variety of sources possess a regulated inner
membrane channel, the permeability transition pore (MTP). ()Pore opening is dependent on both the transmembrane
potential difference (1) and on matrix pH (2) and is
modulated by a variety of effectors acting at multiple sites (for
recent reviews, see (3) and (4) ). Among pore
inhibitors, CSA stands out for its potency (apparent I
is
in the submicromolar range) and for its selectivity (no other
mitochondrial functions appear to be affected by this drug) ( (3) and (4) and references therein). In fact, it is
because of inhibition by CSA (5, 6, 7) that
the nature of the ``permeability transition'' (a
Ca
-dependent increase of inner membrane permeability
to solutes with molecular mass
1500 Da) was recently recognized as
being due to opening of a channel(8, 9, 10) ,
as first proposed by Hunter and Haworth in
1979(11, 12) , rather than to a permeability change of
the membrane lipid phase (for review, see (13) ).
Inappropriate MTP opening is becoming increasingly recognized as a
causative event in cell injury by a variety of conditions, including
ischemia(14, 15, 16) . On the other hand, and
although a role of the pore in cellular Ca homeostasis appears
likely(17, 18, 19) , most questions regarding
its physiological function, regulation, and molecular structure await
an answer.
A soluble mitochondrial extract displays PPiase activity, which can be inhibited by CSA and CSG but not by CSH or FK506(20) . Since this pattern is shared by the permeability transition, it has been suggested that a mitochondrial CyP mediates CSA inhibition of the pore(20) . Although a 20-kDa CyP isoform (CyP-M) with a unique N-terminal sequence has later been isolated from mitochondria(21) , its role in the permeability transition is unclear.
A study of radiolabeled CSA binding to mitochondria defined two classes of high affinity CSA-binding sites, suggesting the possible existence of further CS-binding proteins besides CyP-M(22) . In keeping with this hypothesis, a 10-kDa protein could be labeled with a photoactive CSA derivative in a membrane-associated fraction obtained after sonication of mitochondria, and it was suggested that the labeled protein was part of the membrane CSA receptor on the pore(23) .
The question of whether CSA interacts directly with the pore, or rather whether its inhibitory effects on the pore are mediated by CyP-M is a fundamental one. Besides the intrinsic importance for the mechanistic aspects of MTP function, assessing this point has a specific relevance for any strategy aimed at pore isolation. Indeed, if CyP-M is required for pore opening, any protocol designed for its reconstitution must include this protein.
In this study, we have
isolated CSA-binding proteins from a subcellular fraction enriched in
mitochondria by affinity chromatography on immobilized CSA. We show
that CyP-M is the main and probably the only mitochondrial receptor for
CSA and address the question of its role in MTP function with two
approaches. In the first, we have studied the interactions of CyP-M
with intact rat liver mitochondria by Western blotting with a specific
antibody against its unique N terminus. We show that CyP-M is a matrix
protein that is engaged in interactions with the mitochondrial inner
membrane, and that these interactions are partially retained by SMP.
Interactions of CyP-M with SMP are disrupted by the addition of CSA and
by mildly acidic pH values, conditions that also lead to selective
inhibition of the MTP. On the other hand, CyP-M interactions with the
inner membrane are unaffected by CSH, which does not inhibit the MTP,
while the effects of acidic pH can be prevented by DPC, which also
prevents MTP block by H(2) . These
observations have been exploited to devise a novel protocol for CyP-M
purification to homogeneity. In the second approach, we have then
studied a series of CSA derivatives with graded affinity for CyP-A for
their inhibition of CyP-M and of MTP. We find that with each derivative
the potency at inhibition of the PPiase activity of purified CyP-M,
relative to that of CSA, is similar to that displayed at inhibition of
MTP opening. Structure-function analysis of CS derivatives reveals that
decreased binding to CyP-M (but not CyP-A) and decreased efficiency at
MTP inhibition is obtained by substitutions in position 8, while a
4-substituted, nonimmunosuppressive derivative, which does not bind
calcineurin, is as effective as the native molecule at inhibition of
the MTP and of CyP-M. Taken together, these data (i) suggest that CyP-M
binding is involved in MTP function and that pore inhibition by CSA and
H
may be due to unbinding of CyP-M from its putative
binding site on the MTP and (ii) show that inhibition of calcineurin
activity is not required for the inhibitory effects of CSA on MTP.
Figure 1:
Identification of CSA-binding proteins
in a mitochondrial preparation by affinity chromatography on D-Ala-(3-amino)-8-CSA. A Triton X-100 mitochondrial extract
was incubated with 0.5 ml of the D-Ala-(3-amino)-8-CSA
affinity matrix as described under ``Materials and Methods,''
washed with column buffer and sequentially eluted with 0.42 mM CSA and 6 M urea. Half-milliliter fractions were
collected, and 15 µl were analyzed by SDS-PAGE and silver staining. Lane 1, last wash with column buffer prior to CSA elution; lanes 2-7, elution with CSA; lane 8, last wash
with column buffer prior to urea elution; lanes 9-13,
elution with 6 M urea. Molecular weight standards (bars on the right) were bovine serum albumin (66,000 Da), ovalbumin
(45,000 Da), glyceraldehyde-3-phosphate dehydrogenase (36,000 Da),
bovine carbonic anhydrase (29,000 Da), bovine pancrease trypsinogen
(24,000 Da), soybean trypsin inhibitor (20,000 Da), and bovine milk
-lactalbumin (14,200 Da).
Figure 4:
Purification of CyP-M. A SMP preparation
(corresponding to lanes 3, panels A and B of Fig. 3) was resuspended in isotonic sucrose, pH 5.0, and spun at
150,000 g for 1 h. The supernatants were brought to pH
8.2, and CyP-M was purified to homogeneity by sequential
chromatographic steps on Q-Sepharose, S-Sepharose, and HR 5/5 mono-S
HPLC columns as described in detail under ``Materials and
Methods.'' Panel A, SDS-PAGE (15% acrylamide-0.4%
bisacrylamide, Coomassie Blue staining) and Panel B, Western
blot analysis with anti-CyP-M antibody of 150,000
g supernatant, pH 5.0 (lanes 1); flow-through of
Q-Sepharose column (lanes 2); flow-through of S-Sepharose
column (lanes 3); elution of S-Sepharose column (peak
fraction) (lanes 4); elution of HR 5/5 mono-S column (CyP-M
peak fraction eluting at about 0.1 M NaCl) (lanes 5).
The position of CyP-M is indicated by the horizontal arrow.
For details see ``Materials and
Methods.''
Figure 3:
Distribution of CyP-M between SMP soluble
and membrane fractions, the effect of NaCl, CSA, acidic pH, and DPC on
CyP-M-membrane interactions. SMP were prepared by sonication of
isolated mitochondria as described under ``Materials and
Methods'' in isotonic sucrose, pH 7.4. After centrifugation at
8,000 g to sediment unbroken mitochondria, the SMP
suspension (approximately 30 mg of protein
ml
) was spun at 150,000
g for 1 h
to separate a membrane from a soluble fraction. Panel A, SDS-PAGE (15% acrylamide, 0.4% bisacrylamide, Coomassie Blue
staining); panel B, Western blot analysis with anti-CyP-M
antiserum of intact mitochondria (lanes 1), SMP suspension
after the low speed centrifugation (lanes 2), and SMP pellet
and soluble fractions (lanes 3 and 4, respectively).
It can be seen that a substantial fraction of CyP-M (arrow)
sediments with the SMP membranes. Panel C, the SMP pellet
(corresponding to lanes 3 of panels A and B)
was resuspended in identical volumes of isotonic sucrose, pH 7.4
(controls), or as detailed below and spun again at 150,000
g for 1 h. CyP-M distribution between the membrane (pellets, upper row) and soluble (snts, lower row) fractions was then analyzed by Western blotting
with anti-CyP-M antiserum after protein separation by SDS-PAGE and
transfer to nitrocellulose. The resuspension medium was isotonic
sucrose, pH 7.4 (controls, lanes 1 and 7), isotonic
NaCl, pH 7.4 (lanes 2), isotonic sucrose, pH 7.4, supplemented
with 50 µg
ml
CSA (lanes 3) or CSH (lanes 4); isotonic sucrose, pH 5.0 (lanes 5), and
isotonic sucrose, pH 5, with DPC present at 10 mM during
sonication and at 1 mM thereafter (lanes 6). It must
be stressed that after decanting the supernatants, the pellets were
gently rinsed and carefully resuspended in the same initial volume of
buffer prior to treatment of both supernatants and resuspended pellets
with Laemmli gel sample buffer. Identical aliquots were then separated,
so that the sum (pellet + supernatant) of each lane represents the
total CyP-M initially present in that sample. Note that data from two
different SMP preparations are collected (lanes 1-4 and 5-7, respectively).
Figure 2:
Characterization of a monospecific rabbit
antiserum against the N terminus of CyP-M. Mitochondria-associated CyPs
were purified by affinity chromatography on the D-Ala-(3-amino)-8-CSA affinity matrix followed by elution with
CSA as described in the legend to Fig. 1. Identical aliquots of
the CSA-eluted proteins (lanes 2-5) and an aliquot of
unfractionated mitochondrial proteins (lanes 1) were treated
with Laemmli's gel sample buffer, and separated onto a 15%
acrylamide, 0.4% bisacrylamide slab gel. The gel was transferred to
nitrocellulose, stained with Ponceau red, and photographed (panel
A). B, the individual lanes were then cut, destained, and
reacted with anti-CyP-M antiserum (lanes 1 and 3),
preimmune serum (lane 2), anti-CyP-M antiserum in the presence
of 0.14 µg ml
ASDGGARGANSSSQC (lane
4), or KVEKIGEGTYGVVYK peptide (lane 5). Antiserum
dilution was 1:200 in all cases, and the position of CyP-M is denoted
by a horizontal arrow.
PPiase activity was determined with the spectrophotometric method of Fischer et al.(27) and analyzed according to Harrison and Stein(28) . The concentration of CyP-M or of recombinant human CyP-A (a generous gift of Dr. Mauro Zurini, Sandoz Pharma AG) was adjusted to give an observed first-order rate constant for the catalyzed reaction about 5-fold larger than that of the uncatalyzed reaction.
MTP opening was followed as the rate of
absorbance or 90 °C light scattering change at 540 nm in 0.20 M sucrose, 10 mM Tris-MOPS, pH 7.4, 5 mM succinate-Tris, 1 mM P, 20 µM EGTA-Tris, and 2 µM rotenone. Mitochondria (0.5 mg
ml
) were incubated in thermostatted,
magnetically stirred cuvettes (final volume, 2 ml, 25 °C). After
accumulation of 40 µM Ca
, 0.5 mM EGTA-Tris was added to prevent opening of the Ca
channel, and MTP opening was then triggered by the addition of
0.2 µM carbonyl cyanide p-trifluoromethoxyphenyl
hydrazone (see (29) for further details and representative
traces).
Liposomes reconstituted with mitochondrial proteins were prepared, and their permeability to solutes was measured as described in (30) , and measurements of SMP membrane potential with oxonol VI (Molecular Probes, Eugene, OR) were performed as described previously(31) .
In order to identify mitochondrial CSA-binding proteins, a Triton X-100 extract of a subcellular fraction enriched in mitochondria was subjected to affinity chromatography on a D-Ala-(3-amino)-8-CSA Affi-Gel 10 affinity matrix. CSA specifically and reproducibly eluted three major proteins with apparent molecular weights of 18, 20, and 22 kDa (lanes 2-7, arrows). The minor component of about 50 kDa could not always be observed in repeats of this experiment. Elution with 6 M urea reproducibly released two proteins of 30 and 32 kDa, and occasionally higher molecular weight proteins (lanes 9-13).
The major CSA-eluted proteins were identified by amino-terminal sequencing. The 18-kDa species had sequence VNPTVFFDI, which is identical to the amino-terminal sequence of cytosolic rat brain CyP-A. The 20-kDa species had the sequence ASDGGARGANSSSQNPLV, which matches that obtained by Connern and Halestrap (21) on a matrix PPiase isolated by ion exchange chromatography. The 22-kDa species had sequence NDKKKGPKVTVKVYFDF, which is identical to residues 26-42 of rat kidney CyP-B (for a review on CyPs, see (35) ). Thus, as suggested earlier (25) , only the 20-kDa species can be considered a selective mitochondrial CyP (termed CyP-M in this paper), while the 18- and 22-kDa species, which can be purified from endoplasmic reticular fractions(25) , are most likely localized in mitochondria-associated endoplasmic reticulum.
The urea-eluted proteins of 30 and 32 kDa revealed a blocked N terminus. The 32-kDa species was identified as porin (experiments not shown) by Western blotting with a specific antibody against the rat liver protein (a generous gift of Professor Vito De Pinto, University of Catania, Italy), while the identity of the 30-kDa species remains unknown. Since under no circumstances could these proteins be eluted with CSA, the nature of their binding to the affinity matrix remains unclear, and was not investigated further in this paper.
Identical
affinity purification protocols were carried out in the presence of
Ca (which promotes opening of the MTP) or of ADP and
Mg
(which inhibit it synergistically with CSA, (36) and (37) ), and no major differences emerged
relative to the results presented in Fig. 1(not shown). We then
focused on CyP-M, the only obvious candidate as a mitochondrial CSA
receptor and therefore as the target for MTP inhibition by CSA.
In order to study the interactions of CyP-M with mitochondria, we prepared rabbit antisera against a synthetic peptide modelled on the unique rat CyP-M N-terminal sequence. The experiments depicted in Fig. 2document the properties of one such antiserum. It can be seen that a single 20 kDa band was recognized in Western blots of total mitochondrial proteins (lanes 1) and in the CSA eluate of the D-Ala-(3-amino)-8-CSA affinity matrix (lanes 3). The reaction was specific, in that it was not detected with an identical dilution of preimmune serum (lanes 2), and it was selective for the N terminus of CyP-M in that (i) no reactivity was detected with either CyP-A or CyP-B (lanes 1 and 3, compare panels A and B) and (ii) antibody binding could be prevented by the synthetic N-terminal-like peptide ASDGGARGANSSSQC (lanes 4) but not by an irrelevant KVEKIGEGTYGVVYK peptide (lanes 5). Thus, this antibody is a useful tool to probe interactions of CyP-M with mitochondria.
In the experiments reported in Fig. 3, we have studied the distribution of CyP-M between a membrane and a soluble fraction obtained by ultracentrifugation of a suspension of sonicated mitochondria in a sucrose-based medium (panels A and B). CyP-M was clearly detectable both in the membrane-associated (lanes 3) and in the soluble fractions (lanes 4), showing that a subpopulation of CyP-M molecules interacts with the mitochondrial inner membrane, and that this interaction is retained after sonication and despite the large dilution of the matrix following disruption of mitochondria.
The nature of these interactions was investigated further by exposing this SMP preparation to a variety of conditions (Fig. 3, panel C). CyP-M could be released by isotonic NaCl (lanes 2, compare with lanes 1), suggesting that CyP-M membrane interactions are influenced by the ionic strength and demonstrating that all CyP-M is bound to the outer surface of our SMP preparation and accessible to the external milieu. Importantly, a large fraction of CyP-M could be released by CSA, which inhibits the pore, but not by CSH, which does not (lanes 3 and 4, respectively, compare with lanes 1), providing a first indication that CyP-M (un)binding to the inner membrane may be instrumental in MTP operation.
Release
of CyP-M could also be detected after exposure of the SMP preparation
to an acidic medium (lanes 5, compare with lanes 7),
and this effect could be prevented by treatment of SMP with DPC prior
to acidification (lanes 6, compare with lanes 7). We
have previously shown that matrix acidification is accompanied by MTP
closure, due to reversible protonation of histidyl residues, and that
the inhibitory effects of matrix H on the MTP can be
prevented by carbethoxylation of critical histidyl residues with
DPC(2) . Thus, these experiments further support our hypothesis
on MTP
CyP-M interactions. It should be noted that a small
proportion of CyP-M could be detected in the soluble fraction of the
controls (SMP resedimented in sucrose medium at pH 7.4, panel
C, lanes 1 and 7), most likely a dilution
effect. With no exception, however, exposure to CSA, acidic pH, or
isotonic NaCl resulted in an increase of the CyP-M released in the
soluble fraction relative to the controls.
To investigate the role
of CyP-M interactions in MTP regulation directly, we tried to detect
MTP activity in SMP by a variety of methods. These included (i) a study
of the permeability properties of liposomes reconstituted with SMP
proteins (30) (these liposomes were permeable to sucrose and
raffinose as are intact mitochondria after MTP opening; however,
sucrose permeation was insensitive to CSA and was unaffected by
Ca or EGTA, and therefore could not be unequivocally
related to MTP (not shown)) and (ii) measurements of SMP membrane
potential with the probe oxonol-VI (31) under a variety of
conditions that should promote or inhibit MTP opening. We detected a
decrease of membrane potential after treatment with Ca
and phenylarsine oxide (two powerful MTP agonists), but again
this depolarization was insensitive to CSA (experiments not shown).
Thus, from these experiments we could not obtain clear evidence for MTP
operation in SMP. As a result, we could not assess a role for CyP-M
associations in MTP regulation directly. While our efforts in this
direction are continuing, we decided to further probe the potential
role of CyP-M in MTP modulation with an independent approach.
Besides giving clues about the interactions of CyP-M with mitochondria, the experiments of Fig. 3allowed us to devise an efficient protocol for purification of CyP-M in enzymatically active form, which is illustrated in Fig. 4. SMP were prepared from isolated mitochondria in sucrose medium and sedimented by ultracentrifugation. Resuspension of SMP in acidic medium released CyP-M and relatively few other proteins (lanes 1), resulting in a substantial one-step CyP-M enrichment. This soluble fraction was adjusted to pH 8.2, sequentially passed through an anion- and a cation-exchange matrix (lanes 2 and 3, respectively), and eluted from the latter with 0.1 M NaCl (lanes 4). After dialysis, CyP-M was purified with one passage on a HPLC cation-exchange column, wherefrom it eluted at about 0.1 M NaCl (lanes 5). CyP-M purity was further checked by silver staining of the HPLC eluate, which confirmed the absence of any other proteins (not shown).
CyP-M purified with this protocol exhibits PPiase activity, which could be inhibited by CSA but not by CSH (see the following paragraphs). We therefore decided to study the inhibitory profile of CyP-M PPiase activity and of MTP opening with a panel of CSA derivatives possessing different affinities for CyP-A. Besides giving information on the properties of CyP-M itself, a correlation between the relative potencies at inhibition of CyP-M and of MTP would be a good indication that a functional link between them indeed exists. The CSA derivatives we studied and their structural modifications relative to the native CSA molecule are illustrated in Table 1.
Fig. 5shows typical experiments where the
concentration dependence of inhibition of the MTP (panels A and B), of PPiase activity of purified CyP-M (panels
C and D), and of PPiase activity of human recombinant
CyP-A (panels E and F) was studied, allowing accurate
determinations of the apparent I for the tested inhibitor.
In these representative experiments, the inhibitory compounds were CSA (panels A, C, and E) and MeVal-4-CS (panels B, D, and F). The results of a
series of experiments carried out with identical protocols with all
derivatives are summarized in Table 2, where the indicated
I
values for PPiase and MTP inhibition were obtained from
triplicates of full titration curves. In the case of MTP inhibition, it
can be seen that modification of residue 3 was without effect, while a
small decrease of affinity followed modification of residues 4, 1, and
2. The most relevant change, with a clear decrease in affinity, was
displayed by the CSA derivative modified in position 8. In the case of
inhibition of PPiase activity with both CyP-M and CyP-A, the relative
affinity was increased for the three (cf. (38) ) and
four substitutions. Interestingly, the least efficient inhibitor of
CyP-M was the 8-substituted derivative, which was nearly as effective
at inhibiting CyP-A as CSA itself. On the other hand, CSH was
ineffective in all cases.
Figure 5: Inhibition of the MTP and of the PPiase activity of CyP-M and CyP-A by CSA and MeVal-4-CS. Determinations of MTP activity (panels A and B) and of the PPiase activity of CyP-M (panels C and D) and of CyP-A (panels E and F) were carried out as described under ``Materials and Methods'' in the presence of the indicated concentrations of CSA (closed symbols) or MeVal-4-CS (open symbols). CyP-A was the human recombinant species. Vertical bars denote the standard error as obtained from at least three determinations.
The absolute I values of the
CSA derivatives for MTP inhibition cannot be directly compared with
those obtained for inhibition of PPiase activity, because the former
values were derived from studies on whole mitochondria and may
therefore be influenced by membrane partitioning. It is more
instructive to compare the potency of the inhibitor under study
relative to that of CSA. Table 3illustrates the results of this
analysis, where the IC
of all CSA derivatives has been
divided by that displayed by CSA. The data have been arranged so that
the series reflects decreased affinity for inhibition of the MTP. It
can be appreciated that within each group the derivatives appear in the
same order for inhibition of PPiase activity, both with CyP-M and
CyP-A. The only exception is for substitution in positions 1 and 2,
where the difference is, however, negligible. When the absolute value
of the ratios is considered, on the other hand, a striking difference
emerges for the CSA derivative substituted in position 8. Indeed, the
affinity is decreased over 10-fold for MTP, over 5-fold for CyP-M, and
less than 2-fold for CyP-A. In other words, for this derivative the fit
is extremely good between inhibition of MTP and CyP-M but not of CyP-A.
In this paper we have (i) characterized CSA-binding proteins associated with isolated mitochondria, showing that the 20-kDa CyP-M is the main and possibly the only specific mitochondrial CSA binding protein; (ii) studied the interactions of CyP-M with SMP and their modulation by potent inhibitors of the MTP, a CSA-sensitive mitochondrial channel; (iii) devised a new protocol for CyP-M purification; and (iv) studied the inhibitor profile of highly purified CyP-M and of MTP with a series of CSA derivatives with graded affinity for CyP-A. Our results support the idea that CyP-M association with the MTP is essential for channel opening, suggest that MTP inhibition by CSA and protons may be due to CyP-M unbinding from its putative site on the MTP, and show that calcineurin inhibition is not involved in MTP inhibition by CSA.
Recently, Andreeva
and Crompton (23) have exploited the synergistic effects of ADP
and CSA at MTP inhibition (36, 37) to identify a
10-kDa protein in a rat liver mitochondrial fraction that could be
photolabeled with a tritiated, 8-substituted CSA derivative in the
presence of ADP after soluble components had been removed by sonication
at pH 8.1 and centrifugation(23) . It was proposed that the
10-kDa species is an integral membrane protein, and that it most likely
represents the target for CSA on the pore(23) . Subsequently,
the same group has reported photolabeling also of a 22-kDa protein
possessing CSA-sensitive PPiase activity (K for
CSA 5 nM) from rat heart mitochondria, and of a 18-kDa protein
from rat liver mitochondria (39) and suggested that the 10-kDa
liver species may in fact be a proteolytic product of the 18-kDa
protein rather than the CSA membrane receptor(39) . In the
absence of structural information on these proteins, it is hard to make
predictions about their possible relationships with CyP-M, and with the
MTP regulatory mechanism proposed here. However, we note that (i) due
to the tight association of the endoplasmic reticulum with
mitochondria, both the 22-kDa CyP-B and the 18-kDa CyP-A copurify with
the 20-kDa CyP-M in CSA affinity-based protocols (Fig. 1); (ii)
the protocol used for membrane preparation by Andreeva and co-workers
(sonication at pH 8.1; (23) and (39) ) is expected to
remove a large fraction of CyP-M (Fig. 3); (iii) the endoplasmic
reticulum is expected to retain its integrity in sonication protocols
designed for disruption of mitochondria and thus to represent a major
component of the vesicular fraction sedimented by ultracentrifugation
after sonication; and (iv) the ion-exchange chromatography protocol
used by Andreeva et al.(39) for partial purification
of the 22-kDa PPiase is virtually identical to that used to purify
several CyP isoforms (e.g.(21) and Fig. 3).
Based on these considerations, on their apparent molecular weights, and
on their physico-chemical properties, we suspect that the larger
proteins labeled by Andreeva et al.(39) in heart and
liver subcellular fractions enriched in mitochondria are the 22- and
18-kDa endoplasmic reticulum-associated CyP-B and CyP-A, respectively.
Yu and Fesik (40) have shown that His-126 of CyP-A, which is
in close proximity to the CSA binding site, has a pK of 6.34 in the uncomplexed protein, while in the CSA-complexed
form, the pK
is shifted to 4.65. His-126 has been
implied in ligand-substrate interactions and appears to contribute to
the hydrophobic pocket, which is essential for substrate binding (41) and for enzyme function(42) . A similar situation
occurs with FKBP, the intracellular target for the immunosuppressant
drugs FK506 and rapamycin, which is structurally unrelated to CyPs (43, 44) yet possesses PPiase activity and shares
calcineurin as the target for
immunosuppression(45, 46) . Binding of the FK506
analogue ascomycin to FKBP shifted the pK
of
His-87, which is again located close to the ligand binding pocket, from
5.92 to 4.86(40) .
It must be stressed that although His-126
of CyP-A appears not to be involved in catalysis(40) , it is
essential for enzyme function since a His-126 Gln mutant of
CyP-A displayed less than 1% of the activity of the
wild-type(42) . It appears likely that a critical histidyl
residue with similar properties will be found in the substrate-binding
pocket of CyP-M, and based on the results of Fig. 3, it seems
plausible that CSA binding, or protonation of this residue, may cause
similar conformational effects on CyP-M, leading to its dissociation
from the putative binding site on the MTP.
A number of analogies link the MTP to
these observations. Single-channel recordings of MTP in rat liver
mitoplasts show a number of subconductance states(53) , which
are reminescent of those of FKBP-stripped SR-CRC(51) . It is
tempting to speculate that this MTP behavior depends largely on
variable degrees of CyP-M extraction because CyP-M is largely removed
by the hypotonic shock procedure needed for mitoplast preparation. ()A second analogy between modulation of MTP and SR-CRC by
CSA and FK506, respectively, is that in neither case does the
immunosuppressant need inhibition of calcineurin activity. Indeed,
MeVal-4-CS is as effective as CSA itself at pore inhibition ( (25) and Table 2), while the effects of FK506 on the
SR-CRC can be mimicked by rapamycin, which likewise does not inhibit
calcineurin(51) .
The submitochondrial localization of CyP-M
has not been studied in detail, but its copurification with matrix
components (21) ()indicates that it is a soluble
matrix enzyme. Yet, as shown here, CyP-M can associate with SMP. These
observations suggest that only a fraction of CyP-M may participate in
MTP regulation at any given time and that MTP opening-closure may be
dependent on modulation of CyP-M binding to the membrane. Consistent
with this idea, it has been shown that phenylarsine oxide, perhaps the
most potent MTP agonist(1, 9, 54) , increases
the affinity of binding of CyP-M to mitochondrial
membranes(55) . Based on the findings of the present work and
because of the striking analogies with SR-CRC and IP
R
mentioned above (see (19) for a general discussion), we
predict that the MTP
CyP-M complex will prove to be the
mitochondrial homologue of the SR-CRC
FKBP12 and
IP
R
FKBP12 complexes.
The match between the structural requirements for inhibition of MTP and of CyP-M PPiase catalytic activity by CSA is of particular relevance when the mechanism of CyP inhibition by CSA in aqueous solution is considered. In an apparent paradox, a CSA derivative with marginal affinity for CyP-A retained a considerable immunosuppressive activity (57) , and the x-ray crystallographic maps at 1.86 Å resolution of CyP-A complexed with CSA or with a 4-substituted CSA derivative were virtually identical despite a 4-fold higher affinity for CyP-A of the latter(58) . This led to the proposal that CSA conformer equilibria in aqueous solution rather than that differences in three-dimensional architecture govern binding to CSA(57) . This hypothesis has recently been confirmed by direct measurements showing that CyP-A only recognizes a well defined conformation of CSA that exists in water prior to binding and that the rate-limiting step in complex formation between CSA and CyP-A is the rate of cis-trans isomerization of CSA in position 9,10(38) . These observations imply that kinetic factors play a major role in the determination of apparent affinity constants for CSA derivatives. Indeed, the time required for equilibrium binding of CSA to CyP-A was as high as 1 h, and it dropped to less than 5 min (the earlier time point that could be taken) with two 3-substituted CSA analogs mimicking the conformation of CyP-Acomplexed CSA(38) .
Based on these findings, it appears likely that the match between inhibition of CyP-M and MTP would be even better with the purified channel, where the complexities arising from CSA partitioning between membrane and aqueous mitochondrial phases can be overcome.