 |
INTRODUCTION |
There are various solute carriers in the mitochondrial inner
membrane to support ATP synthesis by oxidative phosphorylation. The
30-kDa solute carriers, consisting of a three-repeat structure containing a certain consensus sequence, are members of the
mitochondrial solute carrier family (1). Of these, the ADP/ATP carrier
mediating transport of ADP and ATP, the phosphate carrier mediating the symport of orthophosphate (Pi) and H+, and the
type 1 uncoupling protein forming the short circuit of the proton
current (2-4) have received considerable attention. These carriers
take similar topologies of six transmembrane helices with three large
hydrophilic loops facing the matrix, and their homodimers are thought
to be their functional units (2, 3, 5, 6). However, their precise
structural characteristics are not fully understood in relation to
their transport functions.
Because fluorescein derivatives have been thought to have similar
structural features to those of adenine nucleotides (7, 8), they have
been used as fluorescent probes in studies on the kinetics and
conformational changes caused by their interactions with the adenine
nucleotide binding sites of proteins such as ATPases (9-11),
NAD(P)+-dependent dehydrogenases (12, 13), and
kinases (7, 8). In fact, we recently reported that the geometric and
electronic structures of fluorescein analogs are very similar to those
of ADP/ATP (14). In addition, we found that various fluorescein derivatives have high affinities to the ADP/ATP carrier in bovine heart
mitochondria, and the binding leads to inhibition of the transport
activity (4, 14, 15). Of the fluorescein analogs, the SH reagent eosin
5-maleimide (EMA)1 most
significantly interacts with the ADP/ATP carrier; it quickly and
specifically labels Cys159 in the second loop facing the
matrix of the bovine heart mitochondrial carrier in competition with
ADP, showing that the region around Cys159 is a major
recognition and binding site of the adenine nucleotides (14-16).
Accordingly, we studied the effect of the amine/SH-modifying
fluorescein analog of fluorescein 5-isothiocyanate (FITC) on bovine
heart mitochondria and the submitochondrial particles. Unexpectedly, it
specifically labeled the 34-kDa mitochondrial phosphate carrier from
both cytosolic and matrix sides at a physiological pH of 7.2. We
further examined the effects of various fluorescein analogs (for
chemical structures, see Structure I) on
the FITC labeling and Pi uptake of the phosphate carrier
and those of FITC on EMA labeling and ADP uptake of the ADP/ATP
carrier. Based on the results, we discuss the mode of binding of FITC
in relation to the transport activity of the phosphate carrier.
 |
EXPERIMENTAL PROCEDURES |
Reagents--
FITC (isoform I) and EMA were purchased from
Molecular Probes (Eugene, OR). Fluorescein, eosin Y, and
lysylendopeptidase were from Wako Pure Chemical Industries (Osaka,
Japan), erythrosin B was from Fluka (Buchs, Germany), mersalyl was from
Aldrich, and hydroxylapatite and AG 1-X8 were from Bio-Rad. Bongkrekic acid was a gift from Prof. Duine (Delft University of Technology).
Preparations of Bovine Heart Mitochondria and Submitochondrial
Particles--
Bovine heart mitochondria were prepared according to
Smith (17). Submitochondrial particles containing 5 mM
potassium phosphate were prepared by sonication of bovine heart
mitochondria, as described previously (15). Mitochondria and
submitochondrial particles were finally suspended in the standard assay
medium consisting of 250 mM sucrose, 0.2 mM
EDTA, and 10 mM Mops, pH 7.2 (S. E. medium). The
amounts of proteins in mitochondria and submitochondrial particles were
determined with a BCA protein assay kit (Pierce) in the presence of 1%
SDS using bovine serum albumin as a standard.
Labeling with FITC--
For FITC labeling at pH 7.2, freshly
prepared mitochondria and submitochondrial particles (both 10 mg of
protein/ml) suspended in S. E. medium were incubated with 200 µM FITC for 10 min at 0 °C in the dark to avoid
possible damage of membrane proteins caused by singlet oxygen, which
could be generated by fluorescein analogs in the light (14), and the
labeling was terminated by 5-fold dilution of samples with the solution
used for electrophoresis consisting of 1% SDS, 1% dithiothreitol
(DTT), and 25 mM Tris-HCl buffer (pH 6.8). Then, the
samples (25 µg of protein) were subjected to SDS-PAGE on 12%
polyacrylamide gel under reducing conditions according to Laemmli (18).
The fluorescence intensities of protein bands labeled with FITC on the
gel were determined with excitation at 500 nm, as described previously
(15). For study of FITC labeling at various pH values, three Good's
buffers were used; 40 mM Mes-NaOH for pH 5.0 and 6.0, 40 mM Mops-NaOH for pH 7.0 and 8.0, and 40 mM
Ches-NaOH for pH 9.0. For examination of the effects of test compounds
on the labeling, the mitochondria or the particles (10 mg of
protein/ml) were first incubated with a test compound for various
periods, usually 10 min, at 0 or 37 °C and then incubated with FITC
for 5 min at 0 °C in the dark, unless otherwise noted.
Pi Uptake by the Phosphate Carrier--
Freshly
prepared mitochondria (2 mg of protein/ml) that had been incubated with
FITC were suspended in S. E. medium supplemented with inhibitors
of oxidative phosphorylation such as oligomycin (2 µg/ml) and
antimycin A (20 ng/mg protein). Pi uptake was started by
the addition of a final concentration of 1 mM potassium
[32P]phosphate (specific radioactivity, 2.78 kBq/µmol)
at 0 °C. After 30 s, uptake was terminated with 400 µM mersalyl according to Ligeti and Fonyo (19), and the
radioactivity of [32P]phosphate incorporated into
mitochondria was determined in an Aloka liquid scintillation counter,
model LSC-3500, as described previously (15). When the effect of DTT on
the FITC-treated mitochondria was examined, FITC-treated mitochondria
were incubated with 10 mM DTT for 10 min at 0 °C and
then centrifuged. The sedimented mitochondria were washed with S. E. medium and used for the assay of Pitransport. As a
control, mersalyl was added to the mitochondrial suspensions before
examining the Piuptake. The Pi transport
activity of submitochondrial particles was determined in a similar way to that of ADP uptake by the ADP/ATP carrier at 0 °C (15), but Pi was used as a transport substrate with incubation for 3 min. The effects of test compounds on Piuptake by the
mitochondria were examined by incubation with a test compound for 10 min at 0 °C and pH 7.2 and then determining the
Pitransport activity by the addition of a final
concentration of 1 mM potassium
[32P]phosphate.
Effect of FITC on EMA Labeling and Transport Activity of the
ADP/ATP Carrier--
The effect of FITC on EMA labeling of the ADP/ATP
carrier was examined by incubation of the particles (10 mg of
protein/ml) with various concentrations of FITC in S. E. medium at
0 °C for 10 min in the dark. The particles (2 mg protein/ml) were
then incubated with 20 µM EMA for 30 s, and labeling
was terminated with excess DTT (10 µmol/mg of protein). The particles
were subjected to 12% SDS-PAGE, and the fluorescent intensity of the
30-kDa band due to the labeled ADP/ATP carrier was determined in a
Shimadzu chromatoscanner, model CS-9000, with excitation at 530 nm. The effect on ADP uptake was examined as reported previously (15). Briefly,
the particles (10 mg protein/ml) in S. E. medium were incubated
with FITC for 10 min at 0 °C. After a 5-fold dilution, ADP transport
was started with 20 µM [14C]ADP (specific
radioactivity, 0.67 GBq/nmol), and ADP uptake was terminated after
10 s with 20 µM bongkrekic acid. The amount of
[14C]ADP taken up by the particles was determined from
the radioactivity of the incorporated [14C]ADP in an
Aloka liquid scintillation counter, model LSC-3500 (15).
N-terminal Amino Acid Sequence Analysis--
The N-terminal
amino acid sequences of the labeled protein and its peptide samples
were determined with an HP G1005A protein sequencing system
(Hewlett-Packard, Palo Alto, CA).
Determination of FITC-labeling Site--
Mitochondria and
submitochondrial particles (both 10 mg of protein/ml) suspended in
S. E. medium were incubated with 200 µM FITC for 20 min at 0 °C. After removing the free FITC by chromatography on a
Sephadex G-50 column, the fluorescent eluates were solubilized with 5%
Triton X-100 containing 0.5 M NaCl, 0.5 mM
EDTA, and 10 mM Mops (pH 7.2) for 10 min at 0 °C, and
solubilized samples were applied to a hydroxylapatite column
equilibrated with a solution consisting of 0.5% Triton X-100, 0.1 M NaCl, 0.05 mM EDTA, and 10 mM
Mops (pH 7.2). Proteins labeled with FITC in the flow-through fractions
were precipitated with cold acetone at
20 °C, dissolved with 6 M guanidine HCl in 1 mM EDTA and 0.5 M Tris-HCl buffer (pH 8.5), and treated with DTT (0.67 µmol/mg of protein) for 2 h at 37 °C to reduce all the
cysteine residues. The cysteine residues were then
carboxamidomethylated with freshly prepared iodoacetamide (1.41 µmol/mg of protein) for 30 min at room temperature in the dark. The
alkylation of proteins was terminated with DTT (4.2 µmol/mg of
protein), and the sample solution was applied to a column of
G4000SWXL (7.8 × 300 mm, Tosoh, Tokyo) equilibrated with 0.05% trifluoroacetic acid containing 63% acetonitrile and eluted at a flow rate of 0.5 ml/min. The protein labeled with FITC was
detected by monitoring the absorbance at 280 nm and fluorescent intensity at 510 nm with excitation by 450 nm. The fluorescent fractions were pooled and concentrated with a Speed Vac concentrator (Savant, New York). After dilution of the FITC-labeled protein 5-fold
with 200 mM Tris-HCl buffer (pH 8.0), it was digested with lysylendopeptidase (2% w/w) for 20 h at 35 °C. The digest was diluted 4-fold with 7 M guanidine HCl, and peptide
fragments were separated by reversed-phase HPLC on a TSK gel
ODS-120T column (4.6 × 150 mm, Tosoh) with linear gradients of
acetonitrile at 0-12% for 5 min, 12-52% for 80 min, and 52-90%
for 5 min in 0.05% trifluoroacetic acid at a flow rate of 1 ml/min
monitored with absorbance at 210 nm and fluorescence at 510 nm excited
at 450 nm. The eluate was collected at intervals of 1 min.
 |
RESULTS |
Effect of FITC on Mitochondrial Proteins--
First we examined
the labeling of mitochondrial proteins with fluorescein analogs, the SH
reagent EMA, and amine/SH-modifier FITC. Freshly prepared bovine heart
mitochondria and their submitochondrial particles were incubated with
200 µM EMA for 30 s at 0 °C and pH 7.2 in the
dark to avoid possible production of singlet oxygen. Alternatively,
suspensions of mitochondria and submitochondrial particles were
incubated with 200 µM FITC for 10 min at 0 °C in the
dark. Then these samples were subjected to SDS-PAGE. As shown in Fig.
1, EMA predominantly labeled the 30-kDa
ADP/ATP carrier (band AAC) in the submitochondrial particles but not
mitochondria, as we reported previously (15). EMA also labeled the
34-kDa protein slightly. In contrast, FITC did not label the ADP/ATP carrier, but it selectively labeled the 34-kDa protein both in mitochondria and submitochondrial particles (Fig. 1). Because membrane-impermeable EMA labeled the ADP/ATP carrier only from the
matrix side (15, 20), the labeling of the 34-kDa protein by FITC from
both the cytosolic and matrix sides was not due to damage of the
mitochondrial membrane.

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 1.
Labeling with FITC and EMA of membrane
proteins in bovine heart mitochondria and their submitochondrial
particles. Freshly prepared bovine heart mitochondria
(Mito) and their submitochondrial particles (SMP)
at 10 mg of protein/ml were incubated with 200 µM FITC
and 200 µM EMA in assay medium at pH 7.2 and at 0 °C
for 10 min and 30 s, respectively, in the dark. The labeling was
terminated by 5-fold dilution with 50 mM Tris-HCl buffer
(pH 6.8) containing 1% SDS and 1% DTT. Then samples (25 µg of
protein) were subjected to SDS-PAGE on 12% polyacrylamide gel, and the
fluorescent bands were detected by fluorography. AAC, the ADP/ATP
carrier.
|
|
Next we incubated mitochondria and the particles with various
concentrations of FITC for 10 min at 0 °C and pH 7.2. After termination of the labeling, progress of the labeling was assayed from
the intensity of the fluorescent band on SDS-PAGE. As shown in Fig.
2A, the labeling of the 34-kDa
protein by FITC in both membrane systems increased with increase in the
FITC concentration, and the labeling attained a plateau at 600 µM and above in both samples. It is noteworthy that even
800 µM FITC selectively labeled the 34-kDa protein,
giving a single fluorescent band on SDS-PAGE (inset, Fig.
2A). The concentration required for 50% labeling was
determined to be about 120 µM with both mitochondria and
the particles.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 2.
Concentration-dependent
FITClabeling (A) and time course
of labeling (B) of the 34-kDa protein in bovine heart
mitochondria and submitochondrial particles. Mitochondria
(Mito) and the particles (SMP) at 10 mg of
protein/ml were incubated with various concentrations of FITC for 10 min at 0 °C and pH 7.2 in the dark. After termination of the
labeling as described in the legend of Fig. 1, samples were subjected
to SDS-PAGE, and the intensity of the 34-kDa fluorescent band on the
gel was determined by fluorography. The time course of the labeling was
examined with 200 µM FITC under identical conditions.
Values (arbitrary units, ±S.D.) are the means for three separate
experiments. Insets are fluorograms of gels after SDS-PAGE
of mitochondria labeled with 800 µM FITC for 10 min
(A) and 200 µM FITC incubated for 60 min
(B).
|
|
The effect of the incubation period on FITC labeling was examined in
both membrane systems at pH 7.2 and 0 °C. As shown in Fig.
2B, labeling with 200 µM FITC proceeded
rapidly in the first 10 min and then became gradual until it attained a
plateau level. This dye still specifically labeled the 34-kDa protein
by incubation for 60 min (inset, Fig. 2B). In
Figs. 2, A and B, the greater the labeling of the
particles than mitochondria should be due to the higher content of the
34-kDa protein in the particles, which contain only mitochondrial
membrane proteins, showing that FITC specifically labeled the 34-kDa
protein to a similar extent from both the cytosolic and matrix sides at
0 °C at a physiological pH of 7.2.
Identification of the 34-kDa Protein Labeled by FITC--
For
identification of this 34-kDa protein, mitochondria were incubated with
200 µM FITC at pH 7.2 and 0 °C for 20 min, when FITC
labeling almost attained the plateau level (see Fig. 2B). After solubilization of the treated mitochondria with Triton X-100, samples were applied to a column of hydroxylapatite gel. The labeled protein in flow-through fractions was denatured with guanidine HCl, and
the cysteine residues were reduced with DTT and carboxamidomethylated with iodoacetamide. The labeled protein was isolated by gel filtration chromatography. After SDS-PAGE, the fluorescent band was transferred to
a polyvinylidene difluoride membrane and then subjected to amino acid
sequence analysis.
The N-terminal sequence of 51 amino acid residues was determined as
AVEEQYSCDYGRGRFFILCGLGGIISCGTTHTALVPLDLVKCRMQVDPQKY.
Because this sequence was the same as the N-terminal sequence of
the bovine heart mitochondrial phosphate carrier (21-23), the
phosphate carrier was concluded to be specifically labeled with FITC at
0 °C. FITC did not label the N-terminal
-amino group of
Ala1, the
-amino groups of Lys41 and
Lys50, or the SH groups of Cys8,
Cys19, Cys27, and Cys42 in this
N-terminal region of the carrier at pH 7.2. However, it is not certain
whether FITC actually did not label the above cysteine residues because
it is possible that treatment of the phosphate carrier with DTT
released the labeled FITC, as described later.
Effect of FITC on Pi Uptake via the Phosphate
Carrier--
Next, we examined the effect of FITC labeling on
Pi uptake mediated by the phosphate carrier. For this, we
incubated mitochondria with various concentrations of FITC for 10 min
at pH 7.2 and 0 °C and started Pi transport by the
addition of 32Pi. After 30 s,
Pi uptake was terminated with the phosphate transport inhibitor mersalyl, and the amount of Pi incorporated into
the mitochondria was determined. As shown in Fig.
3, FITC inhibited Piuptake
depending on the FITC concentration. The transport in the mitochondria
was almost completely inhibited by 200 µM FITC, and the
concentration necessary for 50% inhibition (IC50) was about 60 µM. A similar inhibitory effect was observed
with the particles (Fig. 3).

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 3.
Concentration-dependent effect of
FITC on Pi uptake by mitochondria and
submitochondrial particles. Mitochondria (Mito,
open circles) and the particles (SMP, open
triangles) at 10 mg of protein/ml in S. E. medium were
incubated with various concentrations of FITC at pH 7.2 and 0 °C for
10 min in the dark. The suspensions were diluted to 2 mg of protein/ml
with S. E. medium containing 2 µg of oligomycin/ml and 20 ng of
antimycin A/mg of protein, and 32Pi was added
(final concentration, 1 mM) to start Pi uptake.
The Pi uptake was terminated with 400 µM
mersalyl after 30 s with mitochondria and 3 min with the
particles, and incorporated radioactivity was determined. The effect of
DTT on the FITC-treated mitochondria was examined (Mito/DTT,
closed circles) by incubation of FITC-treated mitochondria
with 10 mM DTT for 10 min at 0 °C. Then the suspension
was centrifuged, and the sedimented mitochondria were washed with
S. E. medium and used for assay of Pi transport.
Incubation of the FITC-untreated mitochondria with 10 mM
DTT did not affect Pi transport. Values (±S.D.) are means
for three separate experiments. In the absence of FITC, Pi
uptake was 15.3 nmol/min/mg of protein by mitochondria and 9.3 pmol/min/mg of protein by the particles.
|
|
Because FITC labels the deprotonated SH group and amino group (24), it
was necessary to determine whether inhibition of Piuptake
via the phosphate carrier by FITC was due to the labeling of cysteine
and/or lysine residues in the carrier. We examined the effect of DTT on
Piuptake by mitochondria labeled with FITC. If the
Pitransport inhibition was due to labeling of a cysteine reside(s) in the carrier, DTT treatment should cause loss of
Pitransport inhibition (25). We incubated FITC-treated
mitochondria with 10 mM DTT for 10 min at 0 °C, then
removed the added DTT by washing the mitochondria and examined their
Pitransport activity. We confirmed that DTT completely
abolished the inhibited Piuptake caused by SH-modifying
reagents such as mersalyl (data not shown). As shown in Fig. 3, DTT did
not affect Piuptake inhibited by FITC. Therefore, we
concluded that Pitransport inhibition by FITC was due to
its labeling of the lysine residue, and no cysteine residues were associated with the inhibition of Pitransport.
Determination of the FITC-labeling Site--
We tried to identify
the labeling site of FITC in the phosphate carrier. For this, we
incubated bovine heart mitochondria and the submitochondrial particles
suspended in S. E. medium at pH 7.2 with 200 µM FITC
for 20 min at 0 °C. After removal of free FITC by chromatography on
a Sephadex G-50 column, labeled mitochondria and the particles were
solubilized with Triton X-100, and the solubilized preparations were
subjected to chromatography on a hydroxylapatite column. The
flow-through fractions, major components of which were the phosphate
carrier and the ADP/ATP carrier, were treated with cold acetone to
precipitate proteins. Then, the precipitated proteins were dissolved
with 6 M guanidine HCl and treated with DTT to reduce
cysteine residues. Subsequently, cysteine residues were
carboxamidomethylated with iodoacetamide, and the carboxamidomethylated samples were subjected to gel filtration chromatography to isolate the
FITC-labeled phosphate carrier. The fluorescent fractions of the
phosphate carrier were digested with the lysine-specific proteinase
lysylendopeptidase, and the peptide fragments thus obtained were
separated by reversed-phase HPLC.
Fig. 4A shows elution profiles
on reversed-phase HPLC of the peptide fragments of the phosphate
carrier from submitochondrial particles monitored with optical
absorbance at 210 nm. Of the peaks in the chromatogram, two peaks were
found to be fluorescent monitored at 510 nm and excited at 450 nm, as
shown in Fig. 4B (top chromatogram). The same
elution profile was observed with the phosphate carrier from
mitochondria (see Fig. 4B, middle chromatogram). The first
fluorescent peak, referred to as peak 1, eluting at 43 min was
consistently observed both with and without lysylendopeptidase digestion (top and bottom chromatograms). In
contrast, the second fluorescent peak, referred to as peak 2, eluting
at 96 min was not observed when the phosphate carrier was not treated
with lysylendopeptidase. We isolated a fluorescent component of peak 1 by reversed-phase HPLC and performed amino acid sequence analysis of
this component. However, no amino acid derivatized with
phenylthiohydantoin was detected. These results showed that the
fluorescent component of peak 1 was not to be the peptide fragment
labeled with FITC. A similar nonpeptide fluorescent peak was observed
besides the FITC-labeled fluorescent peptide peak in the reversed-phase
HPLC on digestion of the FITC-labeled Ca2+-ATPase with
trypsin (26).

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 4.
Reversed-phase HPLC profiles of peptides from
FITC-labeled protein digested with lysylendopeptidase.
Mitochondria (Mito) or submitochondrial particles
(SMP) at 10 mg of protein/ml were incubated with 200 µM FITC at 0 °C for 20 min at pH 7.2 in the dark.
After isolation, the FITC-labeled protein was incubated with
lysylendopeptidase (LEP) at pH 8.0 for 20 h at
35 °C. The peptide fragments thus obtained were separated by
reversed-phase HPLC on an ODS-120T column in a linear gradient of
acetonitrile at a flow rate of 1 ml/min. For details, see
"Experimental Procedures." The elution profile was monitored as
optical absorbance at 210 nm and fluorescence intensity at 510 nm
excited at 450 nm. Elution profiles A and B (top and
bottom chromatograms) show peptide fragments from the
particles monitored by optical absorbance and fluorescence,
respectively. In B, +LEP and LEP are
elution profiles of peptide fragments with and without treatment with
lysylendopeptidase, respectively.
|
|
The fluorescent fraction producing peak 2 from submitochondrial
particles was subjected to amino acid sequence analysis, and the
results are summarized in Table I. This
peptide consisted of 29 amino acid residues, but the 16th residue could
not be determined due to modification with FITC. According to the
sequence of the bovine heart mitochondrial phosphate carrier (21, 22),
the determined sequence was found to correspond to that from
Gly170 to Lys198. Therefore, the 16th residue
of the peptide was Lys185, located in the putative fourth
transmembrane segment (27). Similarly, Lys185 of the
phosphate carrier in mitochondria was determined to be modified by
FITC. Therefore, the hydrophilic and anionic FITC specifically labeled
the same lysine residue from both the cytosolic and matrix side.
View this table:
[in this window]
[in a new window]
|
Table I
Amino acid sequence analysis of the fluorescent fraction derived from
the phosphate carrier labeled with FITC in submitochondrial particles
from bovine heart mitochondria
|
|
Effect of pH on FITC Labeling--
It is well known that acylation
of amines by isothiocyanates is significantly pH-dependent
and that FITC modification of lysine residues proceeds well at alkaline
pH values, in which lysine residues are deprotonated (28). We examined
the labeling of the phosphate carrier with 200 µM FITC at
0 °C at various pH values between pH 5.0 and 9.0. The results of
SDS-PAGE showed that FITC specifically labeled the phosphate carrier in
mitochondria and the particles at various pH values examined, and the
labeling was significant at pH 6.0 and 7.0 (Fig.
5). It is noteworthy that FITC was not
essentially reactive with the phosphate carrier at pH 9.0. Fig.
6 shows the results of
pH-dependent FITC labeling determined from the intensities
of the fluorescent bands shown in Fig. 5. FITC labeling increased with
increase in pH, attaining a maximum level at pH 7.0, and then decreased
with further increase in pH in both mitochondria and the particles.

View larger version (52K):
[in this window]
[in a new window]
|
Fig. 5.
The effect of pH on the FITC
labeling of proteins in bovine heart mitochondria
(A) and their submitochondrial particles
(B). The suspensions of bovine heart mitochondria
(Mito) and the particles (SMP), each at 10 mg of
protein/ml, were incubated with 200 µM FITC for 10 min at
0 °C at various pH values in the dark. After termination of the
labeling, samples were subjected to SDS-PAGE. Experimental conditions
were essentially as for Fig. 1.
|
|

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 6.
Dependence of FITC labeling of the
phosphate carrier on pH. The fluorescent intensities of the
phosphate carrier labeled with FITC in mitochondria (Mito)
and the particles (SMP) on SDS-PAGE shown in Fig. 5 were
determined as described in the legends of Figs. 1 and 2. The values
(arbitrary units, ±S.D.) are means for three separate
experiments.
|
|
It was surprising that the affinity of FITC to the phosphate carrier at
pH 8.0 was remarkably less than that at pH 7.0 and that FITC was not
reactive at pH 9.0, although FITC labeling was expected to be more
favorable in the alkaline pH region than in the neutral region. When
the labeling was examined at higher temperatures such as 37 °C, FITC
labeled the ADP/ATP carrier in addition to the phosphate carrier
at pH 7.2, and it labeled various mitochondrial proteins
nonspecifically, including these two proteins at pH 9.0 (data not
shown). Because the pH dependence of the FITC labeling was similar to
that of Pi transport via the phosphate carrier (29), the
binding of FITC to the carrier is closely associated with its transport function.
Effects of Pyridoxal 5'-Phosphate and Phenylisothiocyanate on the
Phosphate Carrier--
The phosphate analog pyridoxal 5'-phosphate
(PLP), which modifies lysine residues in proteins by forming a Schiff
base (30), has been used as an inhibitor of the phosphate carrier (31). We examined the effects of various concentrations of PLP on the labeling with 200 µM FITC and the Pitransport
activity of mitochondria at pH 7.2 and 0 °C. As shown in Fig.
7, the FITC labeling of the carrier was
decreased with increases in the PLP concentration, and labeling was
almost completely inhibited by 20 mM PLP.

View larger version (18K):
[in this window]
[in a new window]
|
Fig. 7.
Effects of various concentrations of
pyridoxal 5'-phosphate and phenylisothiocyanate on FITC labeling
and Pi uptake. Mitochondria (10 mg of
protein/ml) were incubated with various concentrations of PLP at
0 °C for 10 min at pH 7.2. An aliquot of the sample
suspension was further treated with 200 µM FITC for 5 min
at 0 °C in the dark and then subjected to SDS-PAGE for determination
of the effect of PLP on FITC labeling. The other part of the sample
suspension was subjected to the assay of Pi uptake after
dilution to 2 mg of protein/ml. Experimental conditions were as for
Fig. 4. In addition, bovine heart mitochondria (10 mg of protein/ml)
were incubated with various concentrations of PITC at 37 °C for 60 min at pH 7.2 and then treated with 200 µM FITC for 5 min
at 0 °C in the dark, and the FITC-treated sample was subjected to
SDS-PAGE. The values (±S.D.) are means for three separate experiments.
In the absence of PLP, Pi uptake was 15.4 nmol/min/mg of
protein by mitochondria.
|
|
PLP inhibited the Piuptake in a similar manner to its
inhibition of FITC labeling. However, the inhibitory effect of PLP was not complete, and about 30% of the transport activity was consistently preserved at 15 mM and above (Fig. 7). This could be due to
reverse reaction of the Schiff base by dilution of PLP in the assay of transport activity (32). The PLP concentration requiring for maximum
inhibition of Pitransport was consistent with the result in
Stappen and Krämer (31). A similar inhibitory effect of PLP on
the FITC labeling was observed with the particles (data not shown).
These results showed that PLP inhibited the Pitransport possibly by binding to Lys185 of the carrier in competition
with FITC, although its affinity was much less than that of FITC.
Accordingly, FITC was suggested to bind to the phosphate binding site
of the carrier more efficiently than PLP.
The lysine-specific reagent phenylisothiocyanate (PITC) does not
contain a fluorescein moiety (for chemical structure, see Structure I),
and it is known to inhibit the Pi transport in bovine heart
mitochondria, but it is effective at the very high concentration of 5 mM (25). In addition, the inhibition is more favorable at
pH 9.0 than at neutral pH (25). Because it is possible that the
hydrophobic PITC preferentially modifies lysine residues in the
membrane region of the phosphate carrier, we examined its effect on
FITC labeling of the phosphate carrier. Mitochondria and
submitochondrial particles that had been preincubated with various
concentrations of PITC at pH 7.2 and 37 °C were incubated with 200 µM FITC at 0 °C, and the effect of PITC on FITC
labeling was determined from the fluorescent band intensity on
SDS-PAGE. As shown in Fig. 7, FITC labeling of the carrier in
mitochondria was not affected at all by pretreatment with PITC at up to
20 mM. Even at 50 mM, PITC was ineffective in
preventing FITC labeling of the carrier of mitochondria and
submitochondrial particles (data not shown). These results clearly
showed that PITC did not modify Lys185, unlike FITC, and
thus, the fluorescein moiety of FITC is responsible for specific
labeling of Lys185.
Effects of Fluorescein and Its Analogs on the Phosphate
Carrier--
We next examined the effects of fluorescein and its
derivatives such as eosin Y and erythrosin B (for chemical structures, see Structure I) on the FITC labeling of the phosphate carrier and
Pi uptake. These analogs all possess a fluorescein moiety like EMA and FITC, and they interact noncovalently with the adenine nucleotide binding site of the ADP/ATP carrier (14). We incubated the
mitochondria with these fluoresceins at pH 7.2 and 0 °C and used
samples for examination of the effects on labeling of the phosphate
carrier with 200 µM FITC and Pi uptake. The
effects on FITC labeling was determined from the intensity of the
fluorescent band labeled with FITC on SDS-PAGE. As shown in Fig.
8A, the fluorescein derivatives inhibited FITC labeling in mitochondria depending on their
concentrations. Erythrosin B was the most effective, fluorescein was
slightly effective, and eosin Y was intermediate. The 50%
inhibitory concentrations (IC50) of erythrosin B and eosin Y were 0.25 and 1.1 mM, respectively. Similar effects were
observed with the particles (data not shown).

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 8.
Effects of fluorescein derivatives on FITC
labeling (A) and Pi
uptake (B). Mitochondria (10 mg of
protein/ml) were incubated with various concentrations of fluorescein
derivatives at 0 °C for 10 min in the dark. An aliquot of the
sample suspension was used for examination on the effect of FITC
labeling, and the other part was subjected to the assay of
Pi uptake after dilution to 2 mg of protein/ml. In
B, the concentrations after dilution of the sample
suspension for Pi uptake are shown on the
abscissa. Experimental conditions were as for Figs. 4 and 7.
The values (±S.D.) are means for three separate experiments. In the
absence of test compounds, Pi uptake was 15.7 nmol/min/mg
of protein by mitochondria.
|
|
These fluoresceins inhibited Pi uptake by mitochondria, as
shown in Fig. 8B. The magnitudes of their inhibitory effects
were in the order erythrosin B, eosin Y, and fluorescein, as observed with their inhibitions of FITC labeling, but their inhibitory concentrations were much less than those observed with FITC labeling due to competition of the noncovalent binding of Pi and
fluoresceins with the phosphate carrier. Namely, the IC50
values of erythrosin B and eosin Y were 37 and 125 µM,
respectively, and that of fluorescein was more than 1 mM.
The inhibitory effects of fluorescein and its analogs on FITC labeling
and Pi uptake depended on their hydrophobicities, as
observed with their effects on the ADP/ATP carrier (14). Because these
fluorescein analogs interact noncovalently with the adenine nucleotide
binding site (14), their inhibitions of FITC labeling and
Pi uptake suggested that there is a region recognizing the
adenine nucleotide moiety in the phosphate carrier and that this moiety
is associated with Pi transport.
Effect of FITC on the ADP/ATP Carrier--
Because various
fluorescein analogs interact with the binding site of adenine
nucleotides of the ADP/ATP carrier (4, 14), we examined the effect of
FITC on the ADP/ATP carrier. The particles preincubated with various
concentrations of FITC at pH 7.2 and 0 °C were further incubated
with 20 µM EMA, which predominantly labels
Cys159 of the bovine heart mitochondrial ADP/ATP carrier
only from the matrix side (15). After incubation, the particles were
subjected to SDS-PAGE, and the effect of FITC was determined from the
intensity of the fluorescent band due to the labeled ADP/ATP carrier
with EMA. As shown in Fig. 9, FITC
inhibited the EMA labeling of the ADP/ATP carrier, and its effect
became greater with increase in its concentration.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 9.
Effects of FITC on EMA labeling and
ADP uptake. Submitochondrial particles (10 mg of
protein/ml) were incubated with various concentrations of FITC at
0 °C and pH 7.2 for 10 min in the dark. After diluting the
suspension to 2 mg of protein/ml, an aliquot of the sample
suspension was treated with 20 µM EMA at 0 °C for
30 s, and the fluorescent intensity of the band labeled with EMA
on SDS-PAGE was determined. The remaining part of the suspension was
subjected to the assay of ADP uptake at 0 °C for 10 s.
Experimental conditions were essentially as for Fig. 4, but 20 µM [14C]ADP was used for the transport
substrate. The values of concentrations of FITC on the
abscissa are those after dilution of the particle
suspensions. The values (±S.D.) are means for three separate
experiments. In the absence of FITC, ADP uptake was 720 pmol/min/mg of
protein by mitochondria.
|
|
ADP transport was started by the addition of [14C]ADP to
the particles preincubated with FITC at 0 °C and pH 7.2, and the
amount of [14C]ADP incorporated into the particles for
10 s was determined. As shown in Fig. 9, FITC inhibited ADP uptake
by the particles in a concentration-dependent manner, and
its effect on ADP uptake was more significant than that on EMA labeling
due to its noncovalent competition with ADP. These results suggested
that the fluorescein moiety of FITC interacted with the nucleotide
binding site of the ADP/ATP carrier.
 |
DISCUSSION |
In this study, we examined the effects of the amine/SH-reactive
fluorescein analog FITC on bovine heart mitochondria and their particles. At a physiological pH and 0 °C, it specifically labeled Lys185 in the putative fourth transmembrane segment of the
phosphate carrier from both the cytosolic and matrix sides, and the
labeling inhibited Pi uptake mediated by the phosphate
carrier. The same Pi transport inhibitions by FITC with
and without DTT treatment clearly showed that the possible labeling of
cysteine residues with FITC (24) was not associated with Pi
transport inhibition by FITC. This conclusion is supported by the
finding that replacement by Ala or Arg of Lys187 of the
yeast carrier, which corresponds to Lys185 of the bovine
carrier, resulted in complete loss of the transport activity (33).
It is noteworthy that besides labeling Lys185 of the
phosphate carrier, FITC inhibited the specific labeling of
Cys159 of the ADP/ATP carrier with the fluorescein SH
reagent EMA and ADP transport across mitochondrial inner membrane.
These results showed that FITC also interacted with the ADP/ATP
carrier. Because the geometric and electronic structural features of
the fluorescein moiety are very similar to those of ADP/ATP (14),
fluorescein and its analogs can be used as efficient probes for
characterization of the adenine nucleotide binding region (7-14).
Accordingly, it is suggested that there is an adenine nucleotide
recognition site in the phosphate carrier, as in the ADP/ATP carrier.
In fact, fluorescein and its analogs such as eosin Y and erythrosin B
prevented the specific FITC labeling of Lys185 and
inhibited Pi transport.
Because the pH dependence of the specific labeling of
Lys185 with FITC was similar to that of Pi
transport via the phosphate carrier (29) and because the phosphate
analog PLP was found to inhibit specific FITC labeling and
Pi transport equally from the cytosolic and matrix sides,
Lys185 should be closely associated with the transport
activity of the phosphate carrier. The importance of this residue is
suggested from the fact that the lysine residue corresponding to
Lys185 is conserved completely in all the nine
mitochondrial phosphate carriers reported to date (1, 34).
Alkaline pH conditions are advantageous for nucleophilic attack of the
isothiocyanate moiety on the deprotonated neutral form of the
-amine
group of lysine residues (28). FITC labels a certain lysine residue of
various proteins such as Na+,K+-ATPase (35, 36)
and H+-ATPase (37) at pH 9.2 and 25 °C, and the labeling
increases at higher pH values (36, 37). We found that FITC
nonspecifically labeled various membrane proteins in bovine heart
mitochondria when labeling was performed at pH 9.0 and 37 °C.
However, FITC specifically labeled Lys185 of the phosphate
carrier at a physiological pH and lower temperature of 0 °C. No
labeling of proteins other than the phosphate carrier was observed
under such conditions, although there are versatile membrane proteins
in the mitochondrial membranes. In addition, it is noteworthy that FITC
specifically recognized Lys185 among 24 lysine residues of
the phosphate carrier under these conditions. This should be mainly due
to the fact that Lys185 in the transmembrane is
deprotonated at a physiological pH, whereas others are protonated or
embedded in the membrane.
Because Lys185 is located in the inner half of the putative
fourth membrane-spanning region, this residue could constitute the path
for the transport of Pi. It is noteworthy that polar FITC accessed to Lys185 in transmembrane segment equally from
both the cytosolic and matrix sides. Possibly, Lys185
functions as an acceptor/donor of H+, which is transported
simultaneously with Pi in their symport process. The
transport of H+ through the membrane has been well studied
with bacteriorhodopsin (38, 39). In this case, H+ is
transported by inter-conversion of protonation/deprotonation of
Lys216/retinal located in the center of the transmembrane
segment. Similarly, ionizable amino acid residues other than
Lys185 are necessary for conducting proton transfer from/to
Lys185 in the phosphate carrier, like Asp96 and
Glu204 in bacteriorhodopsin. In this respect, it is
noteworthy that His32, Glu126, and
Glu137 in the membrane-spanning region of the yeast
phosphate carrier were suggested to be related with proton transfer
(40).
Because both the phosphate carrier and the ADP/ATP carrier are
mitochondrial solute carriers possibly evolving from the same ancestor
protein (1), it is not surprising that there could be an adenine
nucleotide recognition site in the phosphate carrier. The important
role of the nucleotide recognition site in Pi transport is
supported by the finding that nonamine-reactive fluorescein and its
analogs having similar structural features to those of ADP/ATP
inhibited Pi transport efficiently in a manner similar to
their inhibition of adenine nucleotide transport through the ADP/ATP
carrier (14). In addition, it is reported that Pi transport is competitively inhibited by ATP (29).
It is thought that the region around Cys159 in the second
loop facing the matrix space constitutes the nucleotide binding site in
the bovine heart mitochondrial ADP/ATP carrier (14, 16). Therefore, it
is possible that the nucleotide recognition site of the phosphate
carrier is located similarly in the second loop facing the matrix side,
and this region should be located geometrically close to
Lys185 in the putative fourth transmembrane segment. The
fluorescein moiety of FITC should first interact with the putative
nucleotide recognition site of the phosphate carrier, and then the
isothiocyanate moiety of the bound FITC should label
Lys185. In fact, amine-reactive PITC, which does not have a
fluorescein moiety, did not label Lys185 at all, and the
higher concentration of the phosphate analog PLP was necessary for
inhibition of FITC labeling and Pi transport by its binding
with Lys185, showing that the binding of FITC with the
nucleotide binding site of the phosphate carrier before the
modification of Lys185 is necessary for its specific and
efficient labeling of Lys185 and subsequent Pi
transport inhibition.
At present, it is not clear how the nucleotide recognition site takes
part in Pi transport. However, we think that this site functions as a regulator of Pi transport. We found that the
cooperative conformational changes in all the three loops facing the
matrix side of the ADP/ATP carrier take place upon binding of the
transport substrates ADP/ATP to their major recognition site in the
second loop (4, 14, 16). By these conformational changes, the specific
and efficient transport of ADP/ATP through the transport path is
achieved (4, 5). A similar transport mechanism could operate in the
transport activity of the phosphate carrier. Studies on the role of the
adenine nucleotide recognition site in Pi transport are
under way.