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INTRODUCTION |
The ADP/ATP carrier
(AAC)1 is involved in the
last step of the oxidative phosphorylation system by delivering ATP
into the cytosol. Its slow intrinsic turnover is a result of unusually large and highly charged substrates; it is the most strongly expressed member of the mitochondrial carrier family (1). The AAC has been
instrumental in understanding certain principles of transport mechanism, such as the "single binding gated pore mechanism" (2-4) and the "induced transition fit" (5, 6) of transport catalysis. The
drastic conformation changes linked to the transport are well documented (7, 8). Based on its sequence and topological studies, it is
generally assumed that the AAC has six transmembrane helices, with
three repeat domains containing two helices each. However, very little
is known about the three-dimensional structure of the AAC, as is the
situation with all mitochondrial solute transporters.
We have approached the structure-function relationship problem of the
AAC in recent years by mutating residues within the AAC2 from
Saccharomyces cerevisiae. Most mutations involved
neutralizing charged residues and the effects on various functions were
determined (9-11). A beneficial spin-off from the yeast system was the
occurrence of spontaneous revertants by second-site mutations (12-14).
A disadvantage of the yeast expression system is the dependence on
mitochondrial growth and biogenesis on the transport performance of
AAC. The level of AAC expression varied widely among the mutants and
could be drastically suppressed. Therefore, in several mutants that lacked AAC protein, the functional effect could not be accurately determined. Further, it could not be clearly deduced whether the mutated residue was involved primarily in the incorporation or in the
transport function of AAC. In fact, it seemed that functional impairment also decreased the level of AAC expression.
To avoid the dilemma of how to interpret the mutational effect, we
aimed to express AAC heterologously in an organism that does not depend
on AAC such as Escherichia coli. The deposition of the
synthesized AAC into inclusion bodies (IB) instead of the E. coli membrane may prevent a possible devitalizing influence on
E. coli (15). Previously, the expression of the bovine AAC1 in E. coli (16) was reported; however, the very low levels
detected in immunoblots are questionable. In our hands, it was not
possible to express marked amounts of yeast AAC2 in E. coli,
despite the abundant expression reported for other mitochondrial
carriers from yeast (15, 17). This discrepancy might be due to
unfavorable codon usage for yeast AAC2 in E. coli. However,
the AAC from Neurospora crassa could be expressed in high
amounts in E. coli. Here we determined the conditions in
which AAC from N. crassa could be renatured and
reconstituted from IB into phospholipid vesicles by introducing some
additional measures that were not required for the reconstitution of
other mitochondrial carriers from inclusion bodies. Single mutational
neutralizations of 10 positive charges are introduced in the AAC from
N. crassa, and the effects on the transport properties and
the interaction with inhibitors were measured.
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EXPERIMENTAL PROCEDURES |
Materials--
The detergents pentaethylene glycol
monodecylether (C10E5), octaethylene glycol
monododecylether (C12E8), tetraethylene glycol monooctylether (C8E4), phosphatidyl
ethanolamine (PE), and Dowex 1X8 (200-400-mesh) were obtained from
Fluka, Triton X-114 and Triton X-100 from Sigma, the zwitterionic
detergents Empigen BB and Sulfabetaine B12 from Marchon Ltd. (France),
and Amberlite XAD-2 from Aldrich. Octyl polyoxyethylene, a mixture of
C8E2 to C8E12, was
kindly donated by J. Rosenbusch. [14C]ADP and
[14C]ATP were purchased from Amersham Pharmacia Biotech.
Restriction endonucleases and T4-DNA ligase were obtained from Roche
Molecular Biochemicals or New England Biolabs and used as recommended
by the supplier.
Mutagenesis--
The heat shock vector pJLA503 carrying the AAC
gene from N. crassa (18) was obtained from W. Neupert
(Institute of Physiological Chemistry, The University of Munich,
Germany). The shuttle vector pSEYc58 (19) containing the gene
AAC2 from S. cerevisiae (20) was obtained from D. Nelson (Dept. of Biochemistry, University of Tennessee, Memphis, TN).
The human gene AAC1 was supplied by N. Neckelmann (21). The
three wild type (wt) AAC genes were cloned for mutagenesis into the
shuttle vector pT7Blue and for protein expression into the vector
pET-3a (Novagen). DNA isolation, restriction, cloning, and
transformation into the E. coli cell strains DH1 and
BL21(DE3)plysS were performed as described in Ref. 22. All mutants of
the AAC gene from N. crassa were generated by using an
oligonucleotide-directed system (U.S.E. mutagenesis kit, Amersham
Pharmacia Biotech). The AAG codon for the mutant K28A was changed to
GCA, and for the mutant K38A it was changed to GCC. For the arginine
mutants R86A, R143A, R145A, R195A, R243A, R244A, R245A, and R285A, the
codons CGT and CGC were changed to GCA. The mutated AAC gene from
N. crassa contains the restriction sites for
NdeI, BamHI, and EcoRI, which allowed
the recloning of the 1207-base pair DNA fragment from the pT7Blue
shuttle vector into the heat shock pJLA503 vector (23). All the cloned
and mutated AAC genes were sequenced by dideoxy chain termination using
a Thermo Sequenase kit (Amersham Pharmacia Biotech). Expression of the
different AAC proteins (wt and mutants) was carried out in the E. coli strain DH1 with the heat shock vector pJLA503 or in the
strain BL21(DE3)plysS with the pET-3a vector.
Expression of AAC in E. coli--
A total of 500 ml of
Luria-Bertani (LB) medium plus 50 mg of ampicillin was inoculated with
an overnight cell culture of transformed DH1 cells, starting with an
A578 nm of 0.1. Under vigorous shaking at
30 °C (about 3.5 h), cells were grown to an
A578 nm of 1.2-1.4. For the expression vector
pJLA503, AAC expression was induced with the addition of 500 ml of
54 °C LB medium to the cell suspension. Growth was continued for
1 h at 43 °C. For induction by
isopropyl-
-D-thiogalactopyranoside, 600 ml of LB medium
plus ampicillin were inoculated with an overnight cell culture of
transformed BL21(DE3)plysS cells containing the vector pET-3a-AAC.
Under vigorous shaking at 37 °C, cells were grown for 2 h to
A578 nm of 0.6. Expression was induced by the addition of 1 mM
isopropyl-
-D-thiogalactopyranoside, and growth was
continued for 3 h at 37 °C. The cells were harvested and stored at 20 °C.
Purification of AAC-containing Inclusion Bodies--
The cells
were pelleted by centrifugation and resuspended in 10 ml of solution 1 (50 mM Tris base, 25% sucrose, pH 7.0) and incubated with
lysozyme (20 mg/g of cells) for 1 h at 37 °C. All the following
steps were performed on ice. 10 ml of solution 1 was added, with
further additions of 18.5 mM EDTA, 1.5% Triton X-100, and
1 mM phenylmethanesulfonyl fluoride. Cells were disrupted by sonification and centrifuged at 30,000 × g at
4 °C for 30 min. The pellet containing IB was resuspended in 20 ml
of solution 2 (1 M urea, 1% Triton X-100, 0.1%
-mercaptoethanol), sonified, and centrifuged. Further enrichment of
IB was achieved by additional washing steps including centrifugation
and resuspension period, using 20 ml of solution 2 in the first, 20 ml
of solution 3 (20 mM Tris base, 0.5% Triton X-100, 1 mM EDTA, 0.1%
-mercaptoethanol, pH 7.0) in the second,
and 10-20 ml of solution 4 (50 mM Tris base, 1 mM EDTA, 0.1%
-mercaptoethanol) in the final wash. The resulting purified IB pellet (40-50 mg of IB/1 liter of E. coli suspension) was stored in liquid nitrogen.
Phospholipid Preparation--
Phospholipid from turkey egg yolk
(PC) was isolated from fresh turkey eggs as described (24) and was used
as standard PC for developing the standard reconstitution procedure.
The extraction step with diethylether was omitted. To remove
phosphatidylethanolamine (PE), the standard PC was dissolved in a
mixture of chloroform/methanol (2:3) and treated with
Al2O3. To remove residual cholesterol, the
standard PC was dissolved in methanol and centrifuged at 26,000 × g at 4 °C for 10 min. The supernatant was evaporated by a
vigorous stream of nitrogen, resulting in the final PC product.
Solubilization and Reconstitution of AAC from Inclusion
Bodies--
AAC from inclusion bodies (IB-AAC) was solubilized at
0 °C in 100 µl of 1.67% (w/v) N-lauroylsarcosine
(sarkosyl), 0.1 mM EDTA, 1 mM dithioerythrol,
10 mM Tris base, pH 7.0 (15), and 0.05% polyethylene
glycol 4000 (PEG) was added to prevent protein aggregation (25). After
15 min the solution was diluted 3-fold with 10 mM Tris base
to give a final detergent concentration of 0.56% and centrifuged at
12,000 × g for 4 min at 4 °C.
For reconstitution the phospholipid was solubilized by sonicating 200 mg of the PC in 800 µl of 0.1 M PIPES, pH 7.5, until the
suspension became clear. Typically, 100 µl of this PC suspension was
added dropwise to a detergent (D) solution (200 mg/ml
C10E5) to a final volume of 240 µl and stored
on ice. C10E5 was found to be very useful for
the reconstitution of the uncoupling protein (26). The PC-detergent
solution contained 20 mg of PC and 28 mg of
C10E5 (D/PC = 1.4). 80 µl of this
suspension were mixed with 1.6 mg of cardiolipin (CL). To a final
volume of 1 ml, a solution of 1 mM phenylmethanesulfonyl
fluoride, 20 mM potassium gluconate, and 20 mM
ADP or ATP were added. Then, 100 µg of the solubilized IB-AAC protein
were added, followed by 100 mg of Amberlite XAD-2 (Aldrich). 80 µl of
the PC-detergent mixture and 100 mg of Amberlite were added twice in
30-min intervals. In subsequent intervals of 30 min, 2 × 100 mg
and 1 × 200 mg Amberlite were added with gentle shaking
overnight. The Amberlite was separated from the proteoliposomes by
centrifugation, and the external ADP or ATP were removed by passage
over a Sephadex G75 column of 0.6 × 28 cm. The elution buffer
contained 50 mM NaCl and 10 mM PIPES, pH 7.4.
Exchange Measurements with Reconstituted IB-AAC or mt-AAC
Proteoliposomes--
The transport measurements were based on the
counterexchange of external [14C]ADP or
[14C]ATP with internal ADP or ATP. A newly developed
automated sampling and separation device was used, and the time
progress of the exchange was measured by taking samples from 2 to
300 s as described previously (11). Additionally, the equation for
the calculation of the transport rates is given here. For inhibition
studies, 50 µl of IB-AAC proteoliposomes or 35 µl of mt-AAC
proteoliposomes were incubated with 10 µM
carboxyatractylate (CAT), 10 µM bongkrekic acid (BKA), or
a combination of 10 µM CAT and 4 µM BKA.
After 10 min, 100 µM [14C]ADP was added for
5 min. An aliquot of 50 µl of this mixture was injected on a Dowex
column and rapidly processed as described above.
Estimation of Protein Content--
The protein concentration of
the proteoliposomes was estimated using the "Amido Black method"
described in Refs. 27 and 28. Protein was precipitated from 200 µl of
proteoliposomes of IB-AAC or 400 µl of mt-AAC with trichloroacetic
acid (104 g/100 ml) and filtered. (Millipore HAWP 020 00, 0.45-µm
pore size, 24-mm diameter). Filters were stained, dried, and then
eluated. The absorbance of the eluate was read at 630 nm in a 1-ml
cuvette against water. The blank containing all components except
protein was subtracted, and a standard curve was calculated with 2-24 µg of bovine serum albumin.
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RESULTS |
AAC2 (S. cerevisiae) Versus AAC (N. crassa) Expression in E. coli--
To compare AAC mutations expressed in E. coli
with those previously obtained in mitochondria, we first attempted to
express the AAC2 from S. cerevisiae in E. coli. The cDNAs for yeast AAC2 and human AAC1 were
incorporated into the heat shock vector pJLA503 and into the T7 RNA
polymerase pET-3a vector. In both cases, virtually no expression of AAC
protein was observed, as deduced from the lack of IB formation and
documented by immunoblots of extracts from whole cells. We noted that
the codons for several residues are very unfavorable for E. coli expression. In particular, the arginine codons AGG and AGA,
which have a very low expression incidence in E. coli (29)
are present in the AAC2 sequences. We also tried to express human AAC1
in E. coli. Again, no measurable synthesis of human AAC1 was
obtained, probably due to a disparate codon usage. In particular, the
arginine triplet Arg-252/Arg-253/Arg-254 may make translation in
E. coli very difficult. Therefore, the DNA sequence of AAC2
(S. cerevisiae) for this triplet was changed from AGAAGAAGA
to CGTCGTAGA, which has a high translation probability. However, there
was no detectable expression of the modified AAC2 in E. coli. We then turned to the AAC from N. crassa, which
has been shown to be abundantly expressed in E. coli (30).
The codons for arginine in AAC (N. crassa) are mostly CGT or
CGC, which provide a high translation probability.
Expression Systems in E. coli--
Two systems were used to
express AAC (N. crassa) in E. coli: the T7 RNA
polymerase promoter system with the expression vector pET-3a and the
heat shock promoter system with the expression vector pJLA503. The
IB-AAC expressed in the two different systems had different abilities
to reconstitute transport activity. With the pET-3a expression system,
the transport activity reached only about 40% of the activity obtained
with the heat shock promoted vector (data not shown). This difference
was observed throughout the variations of the reconstitution
parameters. For this reason, all of the following solubilization and
renaturation experiments were performed with heat shock-induced IB-AAC
using the pJLA503 vector expression system.
Solubilization-Renaturation-Reconstitution--
The previously
reported procedure for the solubilization and renaturation of the
phosphate and citrate carrier from inclusion bodies did not produce
active vesicles with the IB-AAC. In the pursuit of an active AAC,
several modifications had to be introduced, which are briefly
highlighted here. Among the various solubilizers of IB-AAC tried,
sarkosyl also proved to provide the basis for the reconstitution.
However, some subsequent steps had to be modified, as monitored by the
exchange rate (Table I). These included
addition of PEG during the solubilization of IB and during the
reconstitution. Further, the nonionic detergent required for the
solubilization of the phospholipid and subsequent proteoliposome
formation proved to be quite critical. The best reconstitution was
obtained with C8E4 and
C10E5, whereas the use of Triton X-100 or X-114
resulted in low exchange rates, probably due to leaky proteoliposomes. Several other factors were tested to improve the reconstitution yield
(data not shown). For example, the addition of salts such as KCl,
ammonium acetate, or Na2SO4 decreased the
activity of reconstituted E. coli IB-AAC (see also Fig. 3).
This is in contrast to the conditions applied to reconstitute AAC from
mitochondria in which salts increased the activity (31).
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Table I
Dependence of reconstitution on various detergents, phospholipid
composition, and detergent adsorbers
Different solubilization and reconstitution conditions were used to
develop the standard reconstitution procedure. Phospholipid from fresh
turkey egg yolk (PC) was isolated as described in Ref. 24 and was used
as standard PC. The estimated D/D exchange activity of these exchange
measurements were related to the exchange activity of the basic D/D
exchange VT = 1260 (µmol/min g protein) of the
heat shock-induced, reconstituted wt IB-AAC.
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Phospholipids--
The choice of appropriate phospholipids was an
important factor for reconstitution. (Table I). Phospholipids from egg
yolk (PC) have been shown to be superior for reconstitution of yeast AAC, as compared with phospholipids from other sources such as asolectin from soybean or from E. coli (32). The additional extraction step of the standard PC with methanol, which removed residual cholesterol improved activity more than 3-fold. In contrast, purification using Al2O3, which removes PE,
resulted in less active proteoliposomes. On the other hand, further
addition of PE to the standard PC did not enhance activity.
The slow addition of the detergent phospholipid mixture to the
sarkosyl-solubilized IB-AAC was essential for successful
reconstitution. Three equal portions of the detergent phospholipid
mixture were added every 30 min. The formation of the proteoliposomes
was initiated by the stepwise removal of the detergent by polystyrene
beads. As shown in Table I, removal of detergent for the reconstitution with Biobeads was less effective than with Amberlite XAD-2. This procedure is similar to those used for the reconstitution of uncoupling protein from brown adipose tissue (33) and of AAC from yeast and bovine
heart mitochondria (34).
Cardiolipin--
The dependence of AAC activity on CL has been
documented for the AAC from bovine heart and yeast (32, 35, 36).
Typically, AAC isolated from mitochondria carries some CL; however,
this may not be sufficient for full activity. CL is not expected to be
present in IB-AAC and this was experimentally confirmed (data not
shown). Therefore, it was not surprising to find an absolute dependence
on CL addition. As shown in Fig. 1, this
dependence is nonlinear. Marked translocation activity is obtained only
when the phospholipids contain more than 4% CL and maximum is reached at 12%. The nonlinear dependence on the CL content in the present system differs from the linear dependence observed with reconstituted AAC2 isolated from yeast mitochondria (36).

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Fig. 1.
Dependence of the exchange activity
VT (µmol/min g
protein) on the cardiolipin content. The content of CL (0, 0.2, 0.4, 0.8, 1.6, 2.4, and 4 mg) is given in percentages related to PC (20 mg). Reconstitution and D/D exchange measurements of the heat
shock-induced wt IB-AAC from N. crassa were performed using
the standard reconstitution procedure (see "Experimental
Procedures") without internal and external addition of potassium
gluconate and valinomycin.
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Protein/Phospholipid Ratio--
One problem when comparing
reported transport activity is the widely different
protein/phospholipid ratio used (see "Discussion"). We therefore
examined the relationship of transport activity to the
protein/phospholipid ratio to find optimum conditions for accurately
measuring the transport rates in the reconstituted proteoliposomes
(Fig. 2). While the 14C
uptake measured at 20 s decreased with the protein amount, the exchange rate VT related to the protein content
increased surprisingly 5-fold when changing the phospholipid/protein
ratio from 10
2 to
10
3. In a compromise for accurate
measurements, which are required for mutants with decreased activity, a
PC/protein ratio of 200 was employed.

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Fig. 2.
Influence of protein-phospholipid ratio on
the exchange activity. A, the protein-related exchange
activity VT (µmol/min g protein).
B, the directly measured [14C]ADP uptake of
20-s exchange. Results from n = 5 different
reconstitution experiments. Solubilization and reconstitution
conditions of heat shock-induced wt IB-AAC were different from the
standard reconstitution procedure as follows: IB-AAC solubilization
with 1.67% sarkosyl without PEG addition; use of standard PC without
methanol extraction step; D (Triton X-114), D/PC of 1.1; protein/PC of
1/100 to 1/1000; CL (6-9.9%); D was removed by stepwise addition of
Biobeads (220-350 mg) at 20-min intervals.
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Charge Compensation by K+--
Transport by AAC is
electrical in the case of an exchange between ADP3
and
ATP4
(37). To compensate for these charge differences,
K+ and valinomycin were added to the proteoliposomes.
K+ ions were usually added in the form of KCl for
reconstitution of mitochondrial AAC. To assess the effect of
K+ ions on the transport activity, the two electroneutral
(homo) exchange modes D/D and T/T and the electrical (hetero) exchanges D/T and T/D were measured. To better characterize the influence on the
exchange modes of IB-AAC, AAC from N. crassa mitochondria (mt-AAC) was also isolated and reconstituted. With mt-AAC (N. crassa), high activity was observed for the two hetero exchange modes in the presence of KCl, whereas potassium gluconate at high concentrations reduced the homo exchange modes to about 80% without enhancing the hetero exchange modes (Fig.
3A). This is in agreement with
results previously obtained with AAC from bovine heart and S. cerevisiae (37, 11). Surprisingly, when IB-AAC was reconstituted in the presence of 150 mM KCl, the transport activity was
suppressed to a few percentage of that observed in the absence of KCl
(Fig. 3B), whereas with the
mt-AAC (N. crassa), high activity was observed in the
presence of KCl. Potassium gluconate instead of KCl was much less
inhibitory. At 20 mM decreased the D/D exchange to about 28%, but actually increased the activity of the hetero exchange modes
of IB-AAC. The exchange mode pattern, i.e. the relative distribution of activity in the four exchange modes, differed in the
IB-AAC from that of the mt-AAC. K+ at 150 mM
either as KCl or potassium gluconate caused a decrease of the homo
exchange modes D/D and T/T, whereas at 20 mM K+
the hetero exchange modes D/T and T/D were increased. With IB-AAC the
highest activity was observed in the D/D exchange, whereas with mt-AAC
the T/T exchange was the highest.

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Fig. 3.
The influence of KCl and potassium gluconate
on the four exchange modes of AAC reconstituted from inclusion bodies
(IB-AAC) and from mitochondria (mt-AAC). Isolation,
reconstitution, and exchange measurements of the wt mt-AAC were
performed as described in Ref. 11. For reconstitution of the heat
shock-induced wt IB-AAC, the newly developed standard reconstitution
procedure was used. Reconstitution of the mt-AAC and IB-AAC into
liposomes was performed without or with internal loading of KCl or
potassium gluconate. External KCl respective potassium gluconate and 2 µM valinomycin were added prior to the start of the four
various exchange measurements. D/D, [14C]ADP
external/ADP internal exchange; D/T,
[14C]ADP/ATP exchange; T/D,
[14C]ATP/ADP exchange; T/T,
[14C]ATP/ATP exchange.
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Fig. 4.
Folding diagram of the AAC from N. crassa and localization of mutated residues. Figure
shows the three-repeat structure of the AAC with transmembrane helices
in shaded blocks. The mutated residues are
represented in black boxes. The frames around the
residue symbols signify acidic ( ), basic ( ), and neutral ( )
residues.
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AAC Mutants--
To elucidate structure-function relationships in
the AAC by mutagenesis, we concentrated here as previously for the
yeast AAC2, on the mutagenesis of charged residues. In the N. crassa AAC as in most mitochondrial carriers a frequent repeat
positioning of charged residues occurs in the three repeat structure.
In this paper we target only positively charged residues. The triad of intrahelical arginines located in the second helix of each repeat domain, Arg-86, Arg-195, and Arg-285 (Fig. 4), were converted by
site-directed mutagenesis into neutral residues, R86A, R195A, and
R285A. The motif (+X+) on the matrix side close to the first helix also forms a triad via conservation in the three repeats. It is
adjacent to a characteristic signature of the mitochondrial carriers
(PX-XX+). Within this motif, the following
mutations were introduced in the first domain K38A, in the second
domain R143A and R145A, and in the third domain all members of the
arginine triplet R243A, R244A, and R245A. This triplet is a
characteristic of all known AACs. Another target residue, Lys-28, is
located only in the first helix and found in all AACs.
Transport Activity--
The results of these mutations were
assayed after reconstitution of mutant IB-AAC (N. crassa)
following the standard reconstitution procedure described above. A
survey of the mutational influence on the "basic" exchange rate D/D
is given in Table II. The intrahelical arginine mutants show generally low activity reaching 21% or less of
the wt activity. The mutations of the (+X+) motif in the
three repeats on the matrix side resulted in widely varying activities. Although K38A and R145A nearly lost their ability to exchange D/D,
R143A still retained 34% of wt activity and R245A had even higher
activity than wt. Thus, the involvement of the two positive residues of
the (+X+) motif varied between the domains. Although in the
second domain the downstream residue was more important, in the
arginine triplet of the third domain, only the first two residues were
essential.
Inhibition--
The degree to which the exchange is affected by
the specific AAC inhibitors, CAT and BKA is of interest concerning the
possible involvement of the residues in inhibitor binding and the
orientation of the AAC incorporation into the liposomes. CAT is
membrane-impermeable and binds to the cytosolic-oriented state
(c-state), whereas bongkrekate is membrane-permeable and binds to the
matrix-oriented state (m-state) of the AAC. Therefore, CAT should
inhibit transport only of the right side-outside AAC, whereas BKA may
affect both types of incorporated AAC. The inhibition by CAT was 55%
in the wt IB-AAC but could reach up to 80% in some of the mutants
(Table III). In two mutants the
inhibition by CAT was decreased to 19% (R143A) and 38% (R245A). BKA
caused very strong inhibition in all mutants with the exception of
R243A. The combined addition of CAT and BKA resulted in 100% inhibition in all mutants. To exclude the possibility that the mutant
R143A protein is incorporated largely inverted, and thus the binding
site for CAT is masked, vesicles with the mutant and wt protein were
internally loaded with CAT. Also here the inhibition with R143A was
lower (24%) than in the wt (57%). In the combined external and
internal inhibition by CAT, also the mutant inhibition was lower than
in wt, i.e. 76% versus 98%.
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Table III
Inhibition of the ADP/ADP exchange by CAT and BKA in isolated AAC
reconstituted liposomes
Inhibitors were added to the proteoliposomes 10 min before the start of
the reaction with [14C]ADP for 5 min. Three types of addition
were made: 10 µM CAT, 10 µM BKA, and 10 µM CAT plus 4 µM BKA combined. Exchange
measurements were performed with heat shock-induced wt and mutant
IB-AAC from N. crassa using the standard reconstitution
procedure and with the mt-AAC from N. crassa as described
previously in Ref. 11. For calculation of the mean value and S.D. for
the wt mt-AAC (N. crassa), IB-AAC (N. crassa),
and for the mutant IB-AAC (N. crassa), n = 4 experiments were performed.
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Exchange Modes--
The distribution of the exchange activities
into the four different transport modes of AAC (see above) is of
importance in analyzing a more specific influence of the mutations.
Previous results with mutants of AAC2 from yeast showed that the
elimination of positive groups may affect the transport of ADP and ATP
differently. As discussed above, K+ and valinomycin were
added to compensate charge differences generated by the hetero
exchanges T/D and D/T. Fig. 5A
shows a general decrease of the four exchange activities of the
intrahelical mutants and a change of the exchange mode pattern compared
with wild type. ATP-linked modes were more affected by the elimination
of the positive charges than the purely ADP-linked basic D/D exchange, with the exception of the R195A mutant. It is also to be noted that the
mutations changed the relative activity between the two hetero modes;
whereas in the wt the D/T exchange was lower than the T/D exchange, the
reverse was true for mutants.

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Fig. 5.
The exchange mode patterns in mutant AAC from
N. crassa. Exchange measurements
(n = 5) were performed with heat shock-induced wt and
mutant IB-AAC (K28A, R86A, R195A, and R285A (A); and K38A,
R143A, R145A, R243A, R244A, and R245A (B)) using the
standard reconstitution procedure. The error bars
represent the S.D.
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The second group of mutations dealt with the (+X+) motif
found on the matrix side in the three domains. Fig. 5B shows
that here as well the three modes involving ATP were more strongly affected by the removal of the positive charge than the D/D mode with
the exception of mutant R245A. The K38A mutant produced an inactive AAC
in all exchange modes. The relative decrease of the T-linked modes were
particularly drastic in R143A; although the D/D exchange was still
largely retained, the T-linked modes were suppressed more than 90%.
The mutations in the arginine triplet in the last domain diminished the
T-linked modes in the first two arginine mutants (R243A, R244A). The
surprising increase of D/D transport activity on eliminating the last
arginine, R245A, was not translated into the T-linked modes. The
activity of the T/D mode was still as high as in wt, whereas the modes
with internal ATP (D/T, T/T) were decreased.
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DISCUSSION |
Expression of AAC in E. coli--
The expression of recombinant
AAC in E. coli in IB and its renaturation combined with
reconstitution into liposomes should facilitate the mutagenetic
approach to the functional role of amino acids. An advantage of the
heterologous expression in E. coli is the independence of
the expression level on functional AAC, whereas the expression level in
yeast of some functionally impaired mutants is virtually zero.
Unfortunately, the E. coli system does not express yeast
AAC2 and mammalian AAC, such as human AAC1, due to unfavorable codon
usage. Similarly, Fiermonte et al. (15) noted only spurious
expression of bovine AAC in E. coli but retarded cell
growth. They speculated that this is due to incorporation of AAC into
the cell membrane. In our hands, however, no incorporation of AAC2
(S. cerevisiae) or human AAC1 into E. coli cell
membranes was observed. Even conversion of the unfavorable codons for
arginine in the RRR triplet of the yeast AAC2 failed to increase
expression in E. coli. Obviously, the unfavorable codons
remaining for other arginines in AAC are still a barrier. It is
noteworthy that among the mitochondrial carriers from yeast only the
AAC has unfavorable codons, whereas those used by the phosphate,
citrate, and ketoglutarate carriers are favorable for expression in
E. coli (15, 38, 39). Fortunately, the cDNA of the AAC
(N. crassa) has an equivalent codon usage for E. coli and therefore can be expressed in sufficient amounts.
Reconstitution of AAC from Inclusion Bodies--
The
renaturation/reconstitution of the AAC from inclusion bodies posed a
challenge. The various steps described for reconstitution of other
carriers (14, 38, 39) had to be partially modified to obtain AAC with
transport activity. It seems that the AAC is more sensitive toward the
environmental conditions, because it must undergo much larger
conformation changes due to its large substrates. The extensive energy
changes involved may require stronger interaction with the lipids, as
exemplified by the unusually stringent requirement for cardiolipin (see
below). Among three anionic detergents tried, all containing the
dodecyl group, renaturation was achieved only with sarkosyl. It appears
that sarkosyl can be more easily removed from the protein than SDS or
lauryl taurine. Only sarkosyl can readily lose its negative charge by
protonation at neutral pH in a micellar environment. Sarkosyl was
introduced to solubilize actin expressed in E. coli IB to
prevent coaggregation with bacterial membrane proteins (40). At
mitochondrial carriers it was first applied for the solubilization of
the bovine ketoglutarate/malate carrier from E. coli IB
(15). Nonionic detergents are also required to mediate the
incorporation of the AAC into the phospholipids. The renaturation was
further improved by PEG 4000 in stoichiometric amounts to the AAC. PEG
may prevent aggregation of the AAC during the transfer into the
nonionic detergent-phospholipid mixture. During this transfer a gradual
exchange of the sarkosyl is important. It seems that a sudden addition
of Amberlite sequesters sarkosyl from the AAC too rapidly for the slow
renaturation process and thus causes aggregation. For reasons yet
unknown, detergents with higher critical micelle concentration
such as C8E4 or C10E5
seem to be important for the reconstitution of the AAC. Previously C10E5 has been found to be optimal for the
reconstitution of the uncoupling protein from mitochondria (26).
The renaturation/reconstitution of the AAC from IB is sensitive to high
ionic strength. In the presence of 150 mM KCl, there is
virtually no transport activity whereas reconstitution from mitochondria profits from KCl. Potassium gluconate is well tolerated, possibly due to the larger anion. The drastic inhibition of transport activity by KCl may be caused by an inhibitory effect of KCl on renaturation, which is a critical step in the reconstitution procedure of IB-AAC, unlike in the reconstitution of mt-AAC. It can be reasoned that a high Cl
concentration effectively shields the
numerous positive groups in the IB-AAC and thus prevents formation of
the ionic bonds necessary for folding. The smaller Cl
should be more effective than gluconate in binding to the large excess
of positive charges in AAC.
The strong increase of the protein-related transport rates with the
"dilution" by the phospholipids, i.e. with a higher
PC/protein ratio, renders the comparison of reported rates difficult,
where the ratios vary between 200 and 10,000 (15, 38, 39). The reason
for this observation must be related to larger vesicle size at high
PC/protein ratios. Here we used a lower PC/protein ratio,
i.e. 200-500. Using a low PC/protein, we could measure the
higher absolute rates with improved accuracy. This enabled us to cover
a broad range including the low rates with the mutant AAC, although the
specific transport rates were lower. The kinetic measurements were
facilitated, employing an automated mixing and sampling apparatus.
When estimating the success of renaturation/reconstitution by a
comparison with reconstitution of native AAC from mitochondria as shown
in Fig. 3, the lower dilution factor of protein to phospholipid (PC/protein = 200 with IB-AAC versus 800 with mt-AAC)
must be accounted for, which increases the specific activity more than 2-fold, as shown in Fig. 2. An additional 1.4-fold increase would have
been possible by saturating with 12% instead of the suboptimal 8% CL
present in the reconstituted IB-AAC. Whereas mt-AAC activity is
saturated at this level of CL, IB-AAC activity reaches only two thirds
of the maximum activity (Fig. 1). Taking these factors into account,
the activity of reconstituted IB-AAC reaches more than 70% of the
mt-AAC.
Cardiolipin--
The AAC is distinguished by its unusually high
content of bound CL. Up to 6 molecules of CL are tightly bound to
bovine and yeast AAC, and at least another 2 molecules of CL are bound
more loosely (41, 42). The apparent loss of only 3 CL molecules on
isolation of the yeast AAC2 causes the transport to become fully
dependent on addition of CL (36). Recently we reported the transport
activity of reconstituted AAC isolated from mitochondria, obtained from
CL-deficient yeast cells (43). The transport activity was virtually
only 1% of that measured on CL addition as compared with about 10% of
the AAC from wt yeast. The nonlinear dependence of the transport
activity of reconstituted IB-AAC (N. crassa) on CL adds a
new facet to the interaction of CL with the AAC. Whereas for AAC2
reconstituted from yeast mitochondria the transport activity increases
linearly with the content of CL (36), for IB-AAC (N. crassa)
a minimum of 4% CL is required, before further CL addition starts to
activate transport. It appears that the difference between IB-AAC
(N. crassa) and mt-AAC2 (S. cerevisiae) is due to
the complete absence of CL in IB-AAC (results not shown). This
indicates that a required minimum of CL molecules must be bound to
permit activation by further CL additions. This required minimum amount
of CL is still present in the isolated AAC2 from yeast mitochondria
(36). CL is visualized to bind to positive groups of AAC at the
membrane interface. For this purpose AAC is equipped with a large
excess of positive charges, in particular lysines (44).
Exchange Mode Pattern--
One of the striking features of the
single neutralization of these positive residues is the alteration of
the exchange mode pattern. In general, the modes involving ATP,
particularly the T/T mode, were more strongly decreased than the D/D
mode. Additionally, the T/D mode and to a lesser extent the D/T mode
were more reduced than the "basic" D/D mode. The explanation for
the difference between the mt-AAC and IB-AAC may reside in the
renaturation process, which takes place in the presence of high
concentrations (20 mM) of ADP or ATP. ADP may facilitate
renaturation more than ATP, since the binding of ADP to the AAC is
known to induce more flexibility in AAC and a more rapid change between
the c- and m-conformations than ATP (7). As a rule, the translocation
of ATP is more sensitive to the removal of a positive charge because of
its additional negative charge (ATP4
versus
ADP3
).
The particular sensitivity of ATP translocation, as observed in all 10 mutations distributed over a wide range in the protein, points to an
interdependent charge network in the AAC. Wherever a positive charge is
eliminated, its effect is propagated to the translocation channel and
sensed more strongly in the ATP4
than ADP3
translocation. Obviously positive charges are involved in the interaction with those highly charged solutes, and the binding of
ATP4
requires more positive charges than the binding of
ADP3
. The fact that positive charge defects, widely
spread over the AAC, may inhibit transport suggests that they act
within charge network rather than in punctual ion bonds. In this way
high mobility is guaranteed and deep energy traps are avoided.
The inhibitors CAT and BKA are believed to bind to sites overlapping
with those for ADP and ATP since they displace these substrates. Thus,
the removal of a positive charge can be expected to lower the binding
affinity also of these ligands. The inhibition by CAT is generally
lower than by BKA, due to the partially inverted incorporation of AAC
in the vesicles, which does not affect BKA binding (1). Among the
present mutants, CAT binding varies much more than BKA binding and is
only 19% in R143A. After loading the internal binding site with CAT,
the inhibition reaches 66%, as compared with 91% for wt. It further
increases to 77% as compared with 88% with external CAT. These
results provide evidence for a decreased affinity for CAT rather than a
decreased right side location of the R143A-AAC. Additionally, the R245A
mutant has a decreased affinity for CAT, although it is less pronounced.
Comparison to Yeast AAC--
In all the mutated IB-AAC from
N. crassa, with the exception of R143A and R245A, the
transport activity is reduced to one fourth or less. The general
sensitivity to the elimination of positive charges in these positions
was also noted with the AAC2 from yeast mitochondria mt-AAC2 (S. cerevisiae). However, there are several significant differences
between the mutational effects on IB-AAC (N. crassa) and
mt-AAC2 (S. cerevisiae), which can be attributed to the
involvement of the respective residue in import into the mitochondria.
In yeast the observed activity decrease is a composite of low AAC
content due to an impaired protein import and decreased activity, which
are difficult to segregate. Since expression in E. coli is
immune to the import problem, a comparison of the results with the
corresponding residues obtained with IB-AAC (N. crassa)
expressed in E. coli and with mt-AAC2 (S. cerevisiae) in yeast should indicate the roles of the mutated
residues in these alternative functions. The ratio of the exchange
rates (VT (N. crassa)/VT (S. cerevisiae)) is
useful for differentiation of the two effects (Table
IV). According to these criteria, the dominant role of the intrahelical arginines in the first and second domain (N. crassa Arg-86 versus S. cerevisiae Arg-96 and N. crassa Arg-195
versus S. cerevisiae Arg-204), is the import of
AAC2 into mitochondria since they have nearly zero activity in yeast
but are active in the E. coli system. The transport activity
of N. crassa-R86A, which is only 14% of wt, also indicates
a role in D/D transport. Interestingly, the mutation of the
intrahelical arginine (N. crassa R285A versus
S. cerevisiae R294A) in the last domain retains activity in
the yeast, although activity in E. coli is decreased by
80%. This puzzling result might suggest that the intrahelical arginine
in the last domain is more important for refolding from the IB and does
not play a role in the AAC import into mitochondria. In yeast AAC2, the
three arginines in the triplet were replaced with Ile, which caused a
much greater loss of activity than the substitution with Ala in the
IB-AAC. In view of the steric difference between Ile and Ala, it is
difficult to decide to what extent this is due to impairment of the AAC import.
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Table IV
Comparison of the ADP/ADP exchange activity (%) of different mutants
between N. crassa AAC and S. cerevisiae AAC2 in reconstituted
proteoliposomes
The relative exchange activities (%) are related to the exchange
activity of the wt VT (N. crassa) = 1260 (µmol/min g protein) and VT (S. cerevisiae) = 4270 (µmol/min g protein). The exchange rates
of the wt and mutant mt-AAC2 Sc were published in Refs. 10 and 11.
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