©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification and Purification of a 10-Kilodalton Protein Associated with Mitochondrial Benzodiazepine Receptors (*)

(Received for publication, November 16, 1994; and in revised form, May 15, 1995 )

Jaroslav Blahos II Michael E. Whalin Karl E. Krueger (§)

From the Fidia-Georgetown Institute for the Neurosciences, and the Department of Cell Biology, Georgetown University School of Medicine, Washington, D. C. 20007

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The isoquinoline carboxamide photoaffinity probe PK14105, a ligand with selectivity for mitochondrial benzodiazepine receptors, has been established to photolabel an 18-kDa protein. When this radioactive probe is used to photolabel rat mitochondrial preparations, a protein of 10 kDa, in addition to the 18-kDa protein, is identified following electrophoretic separation and extended autoradiography. These proteins are referred to herein as pk10 and pk18, respectively. Both proteins exhibited the same specificity to a series of ligands used in competition photolabeling studies and are mutually present at apparently similar ratios across multiple tissues. Subcellular fractionation of rat adrenals indicated that pk10 and pk18 comigrated with the mitochondrial marker enzyme cytochrome c oxidase. In numerous paradigms examining specificity, photolabeling of pk18 invariably coincided with photolabeling of pk10. In detergent-solubilized extracts of rat adrenal mitochondria, pk18 and pk10 coimmunoprecipitated when using antisera raised against pk18. Furthermore, purification of the photolabeled proteins using nondenaturing conditions demonstrated that pk18 and pk10 copurify substantiating their intimate association. A set of three antisera, specific to different regions of pk18, did not recognize pk10 on Western blots. Likewise, partial amino acid sequence of peptide fragments indicate that pk10 is not derived from proteolytic cleavage of pk18. These data suggest that pk10 represents another component of mitochondrial benzodiazepine receptors whose identity is not apparent with any known protein.


INTRODUCTION

The principal pharmacological actions of benzodiazepines, a class of widely prescribed anxiolytic drugs, is believed to be mediated primarily through binding to a specific allosteric modulatory site located on ionotropic -aminobutyric acid receptors. In addition to binding to this class of inhibitory neurotransmitter receptors, there is another class of high affinity binding sites for benzodiazepines and other drugs found in virtually all tissues, primarily localized on mitochondria(1, 2, 3) , hence they are referred to as peripheral-type or mitochondrial benzodiazepine receptors (MBR)(^1)(4, 5) .

Despite the prevalent therapeutic use of benzodiazepines, the potential secondary drug effects mediated through MBR are not understood, but recent progress has been made to understand the function of this protein complex(5) . In steroidogenic tissues MBR were found to be coupled to the regulation of steroid biosynthesis(6, 7, 8, 9) . This activity appears to involve a specific interaction with a cytosolic protein known as diazepam binding inhibitor (10, 11, 12, 13) to facilitate intramitochondrial cholesterol transport, the rate-limiting step of steroidogenesis(14) . What remains to be elucidated, however, is the precise molecular mechanism of MBR in this process and what role MBR plays in cells which do not synthesize steroids. It seems probable that MBR might play a fundamental role in the activity of the discreet mitochondrial populations that contain these receptors. With relevance to understanding the relationship of MBR to mitochondrial function several proteins which comprise this complex have tentatively been identified(15) .

In attempts to elucidate MBR function a number of other pharmacological agents have been discovered such as aryl indoleacetamides(16) , imidazopyridines(17) , quinoline propanamides(18) , and isoquinoline carboxamides(19) , along with several other more recently reported classes of compounds(20) . Isoquinoline carboxamides include the highly selective fluoro-nitro photoaffinity probe PK14105 (21) which has been widely reported to photolabel an 18-kDa protein almost exclusively. This mitochondrial protein has been purified (22, 23) and the corresponding cDNA cloned(24, 25, 26, 27) . Transfection experiments with the cDNA demonstrate the expression of binding sites for benzodiazepines and isoquinoline carboxamides with characteristic MBR specificity(24, 25, 28) .

To gain further insight into other proteins associated with MBR, we have made further use of PK14105 as a selective photoaffinity probe. The studies reported here present data showing the existence of a second protein specifically photolabeled by PK14105 which is associated with the 18-kDa protein described above.


EXPERIMENTAL PROCEDURES

Subcellular Fractionation

Tissues from adult Sprague-Dawley rats or bovine adrenal cortices were homogenized in 20 volumes of ice-cold 10 mM HEPES (pH 7.4), 0.32 M sucrose using 12 strokes in a tight fitting Potter-Elvehjem glass tissue grinder. The HEPES/sucrose buffer ordinarily contained a protease inhibitor mixture consisting of 0.1 mM 4-(2-aminoethyl)-benzenesulfonylfluoride, 0.1 µM Pepstatin A, 1 µg/ml leupeptin, and 1 mM EDTA, although experimental results were found to be identical when performed in the absence of these inhibitors. The homogenates were then centrifuged for 5 min at 750 g. The resulting supernatants were recovered and centrifuged a second time at 750 g. The supernatants were removed again, then centrifuged at 5,200 g for 10 min yielding membrane pellets that were resuspended in 5 volumes of HEPES/sucrose buffer and centrifuged for 10 min at 9,000 g resulting in mitochondrial-enriched pellets.

In some experiments, rat adrenals were subfractionated into four subcellular compartments. A nuclear-enriched pellet was obtained from the centrifugation at 750 g which was resuspended in HEPES/sucrose buffer and centrifuged again at 750 g yielding the nuclear pellet. The mitochondrial-enriched fractions in these experiments were obtained by centrifugation of the 750 g supernatants for 10 min at 8,000 g, wherein the pellets were washed by resuspension and centrifugation. A final centrifugation of the 8,000 g supernatant at 100,000 g for 30 min produced a pellet consisting of microsomal membranes and a supernatant representing the cytosolic fraction.

Protein of all subcellular fractions was quantified by the method of Bradford (29) using bovine -globulin as a standard. All fractions were stored in HEPES/sucrose buffer at -70 °C until ready for use.

Generation of Antibodies

The peptides YGSYIIWKELGGFTE and LNYYVWRDNSGRRGGSRL, corresponding to an internal sequence and the carboxyl-terminal region, respectively, of the 18-kDa MBR protein (24) were synthesized. Each synthetic peptide was coupled to keyhole limpet hemocyanin at a concentration of 2 mg/ml of peptide and protein, each dissolved in Dulbecco's phosphate-buffered saline. To these mixtures 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide was added at 2 mg/ml and allowed to react for 1 h at room temperature followed by an overnight incubation at 4 °C. The peptide-protein conjugates were dialyzed twice against 1 liter of Dulbecco's phosphate-buffered saline and stored frozen.

For immunizations, the peptide conjugates were emulsified with an equal volume of complete Freund's adjuvant and 800 µl, corresponding to about 800 µg of antigen, was injected intradermally into New Zealand White rabbits. For boostings, the conjugates were emulsified in Freund's incomplete adjuvant and injected subcutaneously 2 months and 8 months following the initial injections. The quantities of antigen used for these subsequent injections were 250 and 100 µg, respectively. One week following boosting injections, rabbits were bled from ear veins and antisera were prepared.

Photoaffinity Labeling

Photolabeling of mitochondrial or subcellular fractions were performed as described in earlier reports (8, 22) with several modifications. Membranes were suspended in 10 mM HEPES (pH 7.4), 0.32 M sucrose at concentrations from 50 to 1000 µg of protein per ml in the presence of 10 nM [^3H]PK14105, unless indicated otherwise. After 90 min at 4 °C, when binding of the radioligand had reached equilibrium, the 1-ml samples were placed in separate wells of 24-well tissue culture dishes and irradiated for 15 min from a distance of 2 cm with a 15 watt ultraviolet light having maximum emission at 366 nm. The samples were then centrifuged at 15,000 g and the pellets were solubilized in sample loading buffer for electrophoresis.

SDS-PAGE and Transblot Analysis

Electrophoresis of proteins was performed using the Tris/Tricine/SDS-PAGE system described by Schägger and von Jagow(30) . As according to this reference, the polyacrylamide gels of 1.5-mm thickness were 12% T/3% C (interpreted as a 12% total monomer concentration of which 3% of the original monomer concentration was bisacrylamide) with a stacking gel of 4% polyacrylamide. Electrophoresis was performed at 10 V/cm in a Bio-Rad Mini Protean II electrophoresis unit. Protein was transferred from gels to nitrocellulose membranes using a Bio-Rad Mini Protean II transblot apparatus at 75 V for 60 min at 4 °C in 25 mM Tris, 200 mM glycine, 20% methanol. Autoradiography of photolabeled proteins transferred to nitrocellulose was performed by spraying the membranes with En^3Hance (Du Pont) and exposing them to x-ray film at -70 °C.

For immunoblot analysis the nitrocellulose membranes were blocked with Dulbecco's phosphate-buffered saline containing 0.1% Tween 20 and 3% bovine serum albumin for 2 h, incubated with antisera (1:1000) in blocking buffer for 2 h, then washed three times (10 min each) with phosphate buffer. The membranes were incubated for 60 min with a 1:1000 dilution of secondary antibody (alkaline phosphatase-linked goat anti-rabbit IgG) in blocking buffer, washed three times for 10 min with phosphate buffer and developed in 0.1 M Tris-HCl (pH 9.5), 0.1 M NaCl, 0.05 M MgCl(2) with nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolylphosphate.

Radioactive Quantitation of Photolabeled Protein Species

To quantify the extent of photolabeling in distinct protein species each sample was loaded onto polyacrylamide gels with a small amount of Rainbow Protein Molecular Weight markers from Amersham. After transfer to nitrocellulose the migration of the molecular weight markers in each lane was clearly visible and served as a reference to facilitate excising the small patches of membrane containing isolated photolabeled protein bands. These pieces of nitrocellulose membrane were placed in scintillation vials and incubated overnight with liquid scintillation fluid prior to radioactive determination. Quantitative variation using this method was <10% comparing identical photolabeled samples electrophoresed in several lanes.

Immunoprecipitation

Photolabeled rat adrenal mitochondrial fractions were solubilized at 4 °C in 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate at a final protein concentration of 1 mg/ml. After 30 min, the mixtures were centrifuged at 20,000 g for 10 min and the supernatants were collected. To 250-µl aliquots of supernatant, 10 µl of antiserum were added and the samples were incubated overnight at 4 °C. Immunoprecipitates were collected by adding 25 µl of a 1:1 slurry of Protein A-Sepharose/Dulbecco's phosphate-buffered saline, gently mixed for 2 h at 4 °C, and centrifuged at 12,000 g for 30 s. The pelleted beads were washed 3 times with 0.5 ml of phosphate-buffered saline, once with 10 mM Tris-HCl (pH 7.4), and bound protein was dissociated by heating to 95 °C in 100 ml of 10 mM Tris-HCl (pH 7.4), 0.5% SDS for 5 min. The supernatants were recovered and combined with a subsequent wash of the beads accomplished with 50 µl of 10 mM Tris-HCl (pH 7.4).

Purification of MBR Proteins

All buffers used in these procedures included the protease inhibitor mixture described under ``Subcellular Fractionation.'' Pellets of photolabeled rat adrenal mitochondria were resuspended at a protein concentration of 5 mg of protein/ml in 4 M urea, Buffer A (50 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, pH 8.0) and incubated on ice for 60 min. The samples were then diluted 2-fold with ice-cold Buffer A and centrifuged at 27,000 g for 45 min. The supernatant was discarded and the pellets were resuspended to a final concentration of 10 mg of protein/ml with 2% digitonin (Lot PTP 9176, Wako Chemicals, Richmond, VA) in Buffer A and kept on ice for 60 min with constant stirring. The samples were then centrifuged for 20 min at 12,000 g after which the digitonin-solubilized supernatant was transferred to 12-ml Sorvall polyallomer tubes (14.7 88.9 mm). To the bottom of each tube a 1-ml layer of 30% sucrose in Buffer A with 2% digitonin was underlaid. Overnight centrifugation at 150,000 g resulted in concentration of photolabeled protein into the sucrose layer with a small residual insoluble pellet at the bottom. The upper layer from each tube was removed carefully and the sucrose cushion was diluted 3-fold with 2% digitonin in Buffer A.

Partially purified receptor fractions were next applied to Q-Sepharose columns previously equilibrated with 0.1% digitonin in Buffer A at a ratio of 4 ml of column bed volume per 50 mg of initial mitochondrial protein used in each preparation. All the columns were packed to a diameter:length ratio of 1:50 and a flow rate of 0.2 ml/min was used where 3-ml fractions were collected. Under such conditions the photolabeled protein did not bind. The flow-through fractions were thus pooled, supplemented with additional digitonin to 1%, and applied to another Q-Sepharose column equilibrated in 0.1 M glycine (pH 9.5), 0.1% digitonin. Again, with this second buffer system, the photolabeled protein was found predominantly in the flow-through fractions.

The pooled MBR-containing fractions from the second column were concentrated to about 2 ml with an Amicon Centriprep-30 unit and applied to a 20-40% sucrose gradient prepared with 0.025% digitonin in Buffer A. The gradient was centrifuged overnight at 40,000 rpm in a Sorvall TH-641 rotor. Fractions of 1 ml were collected where the radioactive peak was found near the middle of the gradient. These fractions were pooled, diluted to 9 ml in Buffer A (without digitonin) and placed in the centrifuge tube underlaid with 25% sucrose in Buffer A. After another overnight centrifugation at 40,000 rpm the photolabeled protein was concentrated at the bottom. The upper portion of the discontinuous gradient was carefully removed. The sucrose fraction was collected, diluted with an equal volume of ethanol, and kept on ice. This caused the protein to precipitate within 10 min where centrifugation at 15,000 g for 10 min produced a protein pellet relatively free of digitonin, suitable for electrophoretic separation after dissolution in SDS-PAGE loading buffer.


RESULTS

Identification of Multiple Proteins Specifically Photolabeled by PK14105

Previous studies by this and other laboratories had shown that the isoquinoline carboxamide PK14105 specifically photolabels an 18-kDa mitochondrial protein(21, 22, 23, 25) . This protein has been clearly demonstrated to be associated with high affinity recognition sites for isoquinoline carboxamides and benzodiazepines(24, 25, 28) . When [^3H]PK14105 photolabeled mitochondrial preparations from nearly any mammalian tissue source are subjected to electrophoresis, autoradiography of the corresponding gels for relatively short times typically reveals only one specifically labeled protein. We have noticed, however, that when autoradiography is extended for much longer times (5-10-fold longer) other specifically labeled proteins may be detected.

The autoradiograms in Fig. 1show photolabeling of mitochondrial fractions from 10 different rat organs. Adrenals exhibit specific photolabeling of three proteins having apparent molecular masses of about 36, 15, and 10 kDa. In order to simplify referring to these specifically photolabeled proteins in the context of this report they are signified as pk36, pk18, and pk10, respectively. Because a protein of 10 kDa was identified, all studies described here were performed using a Tris/Tricine/SDS-PAGE system (30) which affords excellent resolution of polypeptides in the range of 100 to 1 kDa. With this buffer system pk18 migrates with an apparent mass of 15 kDa despite the fact that the corresponding cDNA has been cloned and sequenced, from which a mass of about 18 kDa is predicted for this protein(24) . Furthermore, earlier studies using the SDS-PAGE protocol of Laemmli (31) give a relative migration more consistent with 18 kDa (21, 22) .


Figure 1: Specific [^3H]PK14105 photolabeling in mitochondrial fractions from various rat tissues. Mitochondrial-enriched membrane fractions from different rat organs (Ad, adrenal; Ov, ovary; Te, testis; He, heart; Ki, kidney; Lu, lung; Sp, spleen; SM, diaphragm skeletal muscle; Pa, pancreas; SC, spinal cord) at concentrations of 1 mg of protein/ml were photolabeled in the presence of 10 nM [^3H]PK14105 as described under ``Experimental Procedures.'' Nonspecific photolabeling (N), performed with 10 µM PK11195 included in the incubation buffer, is compared with total photolabeling (T) where no competitor was introduced. After solubilization in electrophoresis sample buffer the following amounts of protein were electrophoresed in their respective lanes: A-SM, 100 µg; P-SC, 150 µg. Following transfer to nitrocellulose, the membranes were autoradiographed for either 7 days (A-Sp) or 21 days (SM-SC). Migration of molecular weight markers is indicated and the three specifically photolabeled protein bands of adrenals are denoted.



All of the rat mitochondrial preparations shown in Fig. 1are known to have MBR, but their levels vary by 2 orders of magnitude. For example, mitochondrial fractions from rat adrenals contain densities of >100 pmol/mg of protein, whereas those from the skeletal muscle or neuronal tissue have 1-3 pmol/mg of protein(2) . In all of these mitochondrial preparations pk18 and pk10 were detected as proteins specifically photolabeled by [^3H]PK14105. Note also that the relative ratio of intensity between these two photolabeled adducts remained fairly constant despite the fact that MBR density is highly variable among the different tissue sources.

In contrast to the similar pattern observed for these two proteins, pk36 was detected only in adrenals. Although MBR was shown to be functionally coupled to steroid biosynthesis (6, 7, 8, 9) the presence of pk36 does not seem to be a feature common to all steroidogenic tissues because this protein was not identified in mitochondrial fractions from testes or ovaries (Fig. 1). In several instances, other photolabeled proteins were observed in different tissues. Many of these are nonspecifically labeled as discriminated using 10 µM PK11195 as a competitor, but in certain tissues such as kidney or skeletal muscle other specifically labeled proteins are faintly detected. The remaining experiments were performed with adrenals because this tissue contains the highest MBR levels.

Ligand-specific Competition of Photolabeling

To examine whether the nature of these proteins to be photoaffinity labeled by PK14105 exhibits specificity of MBR several discriminatory ligands were used as competitive displacers at concentrations of 1 µM in the photolabeling experiments. Rat adrenal and bovine adrenocortical mitochondrial preparations were compared for this purpose (Fig. 2).


Figure 2: Specificity of [^3H]PK14105 photolabeling in rat and bovine adrenal mitochondrial fractions. Either rat adrenal (100 µg of protein/ml) or bovine adrenocortical (300 µg of protein/ml) mitochondrial fractions were photolabeled with 10 nM [^3H]PK14105 in the presence of 1 µM of the following competing ligands: Co, control (no competitor); PK, PK11195; Q-,(-)-PK14067; Q+, (+)-PK14068; A, alpidem; Z, zolpidem; Ro, Ro5-4864; FM, flumazenil; FG, FGIN-2. The quantities of photolabeled mitochondrial protein electrophoresed on each lane were 20 µg of protein for rat samples and 60 µg of protein for bovine samples. Autoradiograms of the corresponding transblots were exposed for 14 days. Beneath each autoradiogram are listed values in µM of the IC each competing ligand displayed in competing for specific [^3H]PK14105 binding. These binding experiments were performed as described previously (33) in an incubation volume of 0.2 ml containing mitochondrial protein and [^3H]PK14105 at concentrations equal to those used for photolabeling in this experiment. Five concentrations of each competing ligand were tested to displace specific [^3H]PK14105 binding for estimation of IC values by nonlinear regression analysis.



The competitor ligands tested are derived from five classes of aromatic compounds, each having a well characterized specificity for MBR. In understanding the results obtained with rat adrenal mitochondria it is best to consider each class of compounds individually. 1) The isoquinoline carboxamide PK11195, a high-affinity congener of PK14105 (19) , effectively competed against photolabeling of all three proteins. 2) A pair of quinoline propanamide stereoisomers,(-)-PK14067 and (+)-PK14068, display stereoselective specificity of MBR. The (-)-isomer exhibits a low nanomolar affinity being over 2 orders of magnitude more potent than the (+)-isomer(18) . This stereoselective specificity was observed in the competition photolabeling of all three proteins. 3) Alpidem and zolpidem are imidazopyridines showing subnanomolar and micromolar affinities for MBR, respectively(17) , a pattern which is again reflected in the photolabeling paradigm. 4) The benzodiazepines Ro5-4864 and flumazenil show reciprocal selectivity for MBR and type A -aminobutyric acid receptors(20) . Consistent with the specificity of mitochondrial benzodiazepine recognition sites, Ro5-4864 competed against the photolabeling of all three proteins whereas flumazenil had no apparent effect. 5) FGIN-2, an aryl indoleacetamide displaying high affinity for MBR(16) , also proved to be an effective competitor. In summary, with rat adrenal mitochondria, photolabeling of pk36 and pk10 exhibited the same sensitivity to all competitors as the established pk18 MBR protein.

An equivalent scenario can be drawn with the photolabeling of bovine adrenocortical mitochondria (Fig. 2, lower panel). There are several important differences, however, with respect to the results from rat adrenals. Several other proteins with masses of 30-60 kDa are found to be photolabeled by PK14105, but these represent nonspecifically labeled adducts as neither PK11195 nor any of the other compounds competed against their photolabeling. Therefore, a bovine counterpart of pk36 is not observed, but it may be masked by the nonspecifically labeled proteins evident here. The photolabeling of pk18 and pk10 is easily detected in bovine samples and these show the same sensitivity to the different ligands as was found in the rat except for one major difference; Ro5-4864 did not appreciably compete against their photolabeling. This is an important point because it is well documented that Ro5-4864, while having nanomolar affinity for rodent MBR, exhibits micromolar affinity in bovine tissues(25) . As this result was anticipated with pk18, a conspicuous relationship is evident in that photolabeling of pk10 not only shows the chemical and stereoselective specificities of MBR, but it also displays this characteristic species-specific difference. These results alone provide strong evidence that pk10 may be another component of MBR in close association with pk18.

Subcellular Fractionation Studies

To corroborate whether pk36 and pk10 are directly associated with MBR, subcellular fractionation of rat adrenals was performed to examine whether these proteins are found predominantly with mitochondrial fractions. Four subfractions were obtained corresponding to preparations enriched in nuclear, mitochondrial, microsomal, and cytosolic compartments(2) . Photolabeling of all four subfractions was performed in addition to measuring in each the specific activity of the mitochondrial marker enzyme cytochrome c oxidase (Fig. 3). The levels of photolabeled pk18 and pk10 showed distributions parallel with that of the mitochondrial marker. In contrast to this profile, pk36 cofractionated partly with mitochondria and also with the cytosolic fraction. Subsequent experiments have therefore focussed on the mutual relationship that pk10 with pk18 consistently manifest because the relevance of pk36 to MBR is dubious based on several observations discussed to this point.


Figure 3: Subcellular distribution of photolabeled proteins. Rat adrenal homogenates were subfractionated to yield pellets at 750, 8,000, and 100,000 g, indicated as P-750, P-8K, and P-100K, respectively. These membrane pellets and the supernatant remaining following the 100,000 g centrifugation (Supe) were photolabeled at a concentration of 50 µg of protein/ml. Membranes were pelleted whereas the supernatant samples were concentrated to 0.1 ml on Centricon-30 units and 10 µg of protein was processed for Western transfer and autoradiography. The lower panel of the figure shows quantitation of radioactivity migrating with each protein species. The open bars represent specific activity of cytochrome c oxidase measured as described in earlier studies(2) . Quantitation of enzyme activity and radioactivity associated with each of the three protein species are normalized relative to the values determined in P-8K which were: cytochrome c oxidase (open bars), 381 nmol/min/mg of protein; pk36 (hatched bars), 5.2 10^2 dpm; pk18 (solid bars), 2.0 10^4 dpm; pk10 (cross-hatched bars), 1.2 10^3 dpm. Means ± S.D. of at least three photolabeling experiments are shown.



Comparison of pk18 and pk10 Photolabeling Properties

The dissociation constant of MBR for PK14105 is typically about 5 nM. Photolabeling of pk18 and pk10 at [^3H]PK14105 concentrations in the range of 1-100 nM show equivalent patterns which approach saturation at 100 nM (Fig. 4A). Photolabeling competition studies with nonradioactive PK14105 and(-)-PK14067 at concentrations from 10 nM to 10 µM were also performed. The IC values for each ligand to compete against photolabeling by 10 nM [^3H]PK14105 was about 20 nM for both pk18 and pk10 (Fig. 4B), consistent with their reported affinities for MBR. These findings continue to highlight the close correlations between photolabeling of pk18 and pk10.


Figure 4: Comparison of photolabeling properties for pk18 and pk10. All photolabeling experiments included rat adrenal mitochondrial fractions at a concentration of 100 µg of protein/ml. In Panel A the concentration of [^3H]PK14105 was varied and radioactivity migrating with pk18 (bullet) and pk10 (Delta) was quantified following electrophoresis and transfer to nitrocellulose. Means ± S.E. of duplicate experiments are shown. Note that both axes are plotted on log scales. In Panel B 10 nM [^3H]PK14105 was used in the presence of different concentrations of either nonradioactive PK14105 or(-)-PK14067 and radioactivity was determined for both proteins. Symbols indicate mean values of duplicate determinations; the S.E. was <5% in competition photolabeling experiments with (-)-PK14067. Data using nonradioactive PK14105 are virtually superimposable with the data plotted for(-)-PK14067. The competition curve was fitted by nonlinear regression analysis (assuming a Hill coefficient of 1) from the data for pk18 using nonradioactive PK14105 as a displacer.



Another criterion that was exploited in this comparison was the fact that MBR affinity for Ro5-4864 at 4 °C is about 1 order of magnitude higher than it is at 37 °C(32) . This property seems particular to benzodiazepines as binding of isoquinoline carboxamides is not appreciably temperature dependent. Given these parameters it would be expected that Ro5-4864 should be a potent inhibitor of photolabeling at 4 °C and not as potent at 37 °C, whereas competition by PK11195 should be independent of temperature. Competition photolabeling experiments with these two ligands at 100 nM demonstrate this expected behavior for both pk18 and pk10 (Fig. 5). This criterion again reveals the cognate features that pk18 and pk10 have in their photolabeling properties. It is worthy to note that photolabeling of pk36 does not show a similar temperature dependence in photolabeling (Fig. 5, upper panel). This finding places further skepticism on the possibility that pk36 is associated with MBR.


Figure 5: Temperature dependence of photolabeling competition by Ro5-4864. Rat adrenal mitochondrial fractions at 100 µg of protein/ml were incubated and photolabeled with 10 nM [^3H]PK14105 at either 4 or 37 °C in the absence (Co) or presence of 100 nM PK11195 (PK) or Ro5-4864 (Ro). Autoradiography (upper panel) and quantitation of radioactivity (lower panel) in the pk18 (solid bars) and pk10 (hatched bars) bands are shown.



pk10 Is Not a Proteolytic Fragment of pk18

To rule out the possibility that pk10 is a proteolytic fragment derived from pk18 several antisera specific for different regions of pk18 were used. The first antiserum (aNI) was generated against synthetic peptides corresponding to the sequences from Ser^4 to Met and Gly to Leu(26) giving specificity for internal and amino-terminal segments of pk18. Antiserum aI was specific to another internal sequence from Tyr to Glu and the third antiserum aC was directed to the carboxyl-terminal region of pk18 from Leu to Leu.

A photolabeled preparation of rat adrenal mitochondria was determined to have 17-fold higher level of radioactivity incorporated into pk18 relative to pk10. This preparation was subjected to immunoblot analysis wherein a 17-fold difference in the amounts of protein were loaded into pairs of neighboring lanes during SDS-PAGE. With this experimental design, if the antisera detect pk18 in the lanes having 17-fold less protein, then the neighboring lane would have an equivalent level of pk10 (based on radioactivity) which should be detected if it is a fragment of pk18. All three antisera vividly revealed pk18 in the lanes loaded with less protein, however, pk10 was not recognized by any of the three antisera (Fig. 6). The largest peptide segment of pk18 spanning a region not recognized by any of the three antisera is about 6.5 kDa, hence, it is unlikely that a 10-kDa fragment of pk18 could escape detection by the battery of antisera. This immunochemical evidence suggests that pk10 is a protein immunologically distinct from pk18. Additional studies to rule against proteolytic cleavage of pk18 are that extended incubations (1-2 h) at 37 °C either before and during (Fig. 5) or after photolabeling (data not shown) do not change the proportion of photolabeled pk10 relative to pk18. Furthermore, the presence of a diversity of protease inhibitors (see ``Experimental Procedures'') did not affect the photolabeling pattern when compared with experiments omitting these inhibitors.


Figure 6: Immunoblot analysis with antisera against different regions of pk18. A preparation of photolabeled rat adrenal mitochondria containing 17-fold higher radioactivity associated with pk18 compared to pk10 was electrophoresed in four sets of double lanes, one lane (17 ) having 17 times more protein than its neighboring lane (1 ). The amount of protein signified as 1 corresponds to 9 µg except for the lanes under aC where this level is 20 µg. After transfer to nitrocellulose one pair of lanes was autoradiographed ([^3H]PK) whereas the other lanes where processed for immunoblot analysis using three antisera generated to different segments of pk18. Antiserum from a rabbit immunized with keyhole limpet hemocyanin alone (NS) is used as an indicator of nonspecificity arising from immunizations using this carrier protein conjugated with peptides corresponding to amino-terminal/internal (aNI), internal (aI), or carboxyl-terminal (aC) sequences of pk18 as described in the text.



Molecular Association and Purification of pk18 and pk10

By all methods used up to this point, photolabeling of pk10 displays precisely the same ligand specificity and physicochemical sensitivity as that of pk18 suggesting that these two proteins are closely associated with each other. To investigate this possibility more directly immunoprecipitation experiments were performed with antiserum against pk18. From detergent-solubilized preparations of photolabeled rat adrenal mitochondria, antiserum aI coprecipitated pk18 and pk10, whereas, preimmune serum or an appropriate control antiserum to hemocyanin did not precipitate either protein (Fig. 7). Note also that pk36 did not precipitate with this complex. Identical results were obtained with antiserum aNI (data not shown). These data, therefore, provide more conclusive evidence that pk18 and pk10 are likely to be tightly associated in a heteromeric complex.


Figure 7: Immunoprecipitation with antiserum against pk18. Rat adrenal mitochondria were photolabeled at a concentration of 500 µg of protein/ml in the presence of 10 nM [^3H]PK14105 and processed through an immunoprecipitation scheme as described under ``Experimental Procedures.'' The protein recovered from this procedure was lyophilized, dissolved in 20 µl of water, and prepared for SDS-PAGE, transfer to nitrocellulose and autoradiography. Shown are immunoprecipitates obtained using antiserum aI (see Fig. 6) against pk18, rabbit antiserum against keyhole limpet hemocyanin as a nonspecific control (Co) for antiserum aI, and preimmune serum (Pre).



Further support for this possibility was obtained by developing a purification scheme to isolate the photolabeled proteins under nondenaturing conditions. The protocol devised for this was adapted from our previous studies (22, 33) using a detergent different than those used for immunoprecipitation. In all purification methods we tested, pk18 and pk10 were always found to cofractionate as exemplified by the purified preparation shown in Fig. 8. These purified fractions typically show several protein species of which pk18 and pk10 are major components. Several proteins with a mass of 30-34 kDa were verified to be the voltage-dependent anion channel and ADP/ATP carrier using specific antibodies. These proteins were reported earlier to copurify with pk18 in a similar purification scheme with rat kidney mitochondria(15) . The relative abundance of pk18 is much greater, however, using the methods described here, probably because adrenal mitochondria were used which contain much higher MBR levels. The presence of additional proteins at 80, 38, and 5 kDa is under investigation concerning their possible relationship with MBR. Of particular concern with these findings is that the voltage-dependent anion channel and ADP/ATP carrier are known to be among the most abundant mitochondrial proteins (apparently with the 5-kDa protein as well, as seen in Fig. 8) and their cofractionation with pk18 may simply be an unavoidable consequence of their contamination in detergent micelles where pk18 was solubilized. Nevertheless, the photolabeling experiments provide strong support that pk10 is closely tied with MBR and therefore its copurification with pk18 most likely reflects this intrinsic association. The staining intensities seen in Fig. 8suggest that the molar ratios of pk18 and pk10 are more comparable than the relative intensities by which both proteins are photolabeled.


Figure 8: Purification of pk18 and pk10. Rat adrenal mitochondria photolabeled with [^3H]PK14105 were processed through the purification scheme detailed under ``Experimental Procedures.'' The left panel shows the electrophoretic pattern of proteins stained with silver according to the procedure of Morrissey(34) . From a preparation starting with 50 mg of mitochondrial protein the lanes represent specific proportions of different fractions as follows: A, 0.5% of total mitochondrial protein; B, 0.5% of the urea-washed membrane pellet; C, 3% of the final purified preparation. The right panel shows an autoradiogram of fraction C.



From preparations as shown in Fig. 8, pk10 is easily resolved from other proteins by SDS-PAGE. This has enabled us to purify pk10 for generation of various peptide fragments. Among these, a tryptic digest of pk10 gave a peptide bearing the sequence XLADK demonstrating that pk10 is not a fragment of pk18. Additional sequence not reported here indicates that pk10 is not currently represented in all sequence data bases of the National Center for Biotechnology Information. Further studies are thus necessary to clone a cDNA for pk10 and reveal the chemical nature of this newly discovered MBR protein.


DISCUSSION

The development of PK14105 has proven to be a crucial step in unveiling proteins associated with MBR. The protein referred to as pk18 in this report has been unequivocally shown to be an essential component for expressing the specific drug recognition sites of this complex(24, 25, 26, 27, 28) . Other proteins have been reported to be photolabeled specifically by PK14105 (15, 23, 25, 35) but a salient association of these proteins with MBR has not been demonstrated. Of concern in some of these reports is that very long periods of photolabeling are necessary to observe the additional proteins, in which case a broader photolabeling selectivity and damage through extensive ultraviolet irradiation may be occurring. By comparison, all photolabeling studies described here involved only 15 min of irradiation and were scrutinized for specificity with a series of competitor ligands.

The identification of pk36 and pk10 is a subject which had gone unnoticed in many previous studies. The primary reason for this is that the level of photolabeling of these two proteins is much lower than that of pk18. Much longer times of autoradiography are required to detect these less apparent adducts and thus are normally undetected in the time it takes to clearly visualize pk18. An earlier study reported observing pk36(35) , where it was also indicated that in mitochondrial fractions from several rat tissues, this adduct was only found in adrenals as is confirmed here. The disclosure of pk10 is a novel finding and, for the case of MBR structure, is the most relevant discovery in these studies. This photolabeled species had been observed by us previously (see (2) and (13) ), however, it was inconsistently detected and initially believed to result from a breakdown of pk18. SDS-PAGE performed by the procedure of Laemmli (31) gives poor resolution and reproducibility in this molecular weight range accounting primarily for the inconsistency we initially observed in detecting pk10. Changing to a different SDS-PAGE system which permitted superb resolution as low as 1 kDa proved to greatly facilitate the analysis of pk10. As a result of this improvement in methodology, pk10 is invariably detected when pk18 is present.

Because several tests showed discrepancies in the photolabeling of pk36 compared to pk18, it is difficult to validate an association of pk36 with MBR. In the attempt to correlate other proteins with MBR, the revelation of pk10 fulfills many stringent criteria. 1) pk10 and pk18 are mutually present in all tissues examined containing MBR. The apparent ratio of PK14105 photoincorporation into these two proteins also seems invariable despite the fact that MBR levels vary by 2 orders of magnitude among different tissues. 2) Photolabeling of pk10 and pk18 exhibit identical responses when testing chemical specificity, stereoselectivity, and species-dependent variation in benzodiazepine recognition. 3) Equivalent patterns in photolabeling of both proteins are observed at PK14105 concentrations between 1 and 100 nM and the same IC values are obtained in competition photolabeling studies with nonradioactive PK14105 or(-)-PK14067. 4) Photolabeling of pk10 displays the characteristic MBR behavior that affinity for Ro5-4864 is markedly temperature dependent, whereas the binding of PK11195 is insensitive to temperature. 5) pk10 cofractionates with mitochondrial markers plus, with two different detergent solubilization schemes, it immunoprecipitates and copurifies with pk18 suggesting a tight physical association between the two proteins. Furthermore, partial amino sequence and the use of three separate antisera recognizing defined regions of pk18 argues against the possibility that pk10 is a proteolytic fragment of, or is antigenically related to pk18.

The most plausible explanation to account for this long list of coincident photolabeling patterns between pk18 and pk10 is that these two proteins are associated together in a heteromeric complex. A number of laboratories have verified that pk18 is essential for expression of MBR drug binding sites(15, 24, 25, 26, 27, 28) . The fact that PK14105 intensely labels pk18 upholds the proposal that this protein constitutes the binding domain for isoquinoline carboxamides. It is feasible that pk10 lies in close proximity to this domain such that in only 5% of instances pk10 is covalently modified by PK14105, whereas pk18 is labeled in the remaining 95% of successful photolabeling events. This ratio can account for the 15-20-fold difference in photolabeling efficiency consistently observed between these two proteins. Such a possibility can account for the observation that the molar ratios of pk10 and pk18 are not as disparate as judged by silver staining following SDS-PAGE of subfractionated receptor preparations. pk10 may actually comprise a small facet of the binding domain, or be labeled by virtue of being a near neighbor, where photoactivated ligand may dissociate from its binding site and diffuse a short distance in this excited state to encounter pk10. In studies not shown here, the use of a scavenger such as diethylamine(21) , does not inhibit the photolabeling of pk18 or pk10, implying that PK14105 may not need to leave its binding domain in order to label pk10.

The partial amino acid sequence derived from purified pk10 establishes that this protein is not a fragment of pk18, nor is it the 10-kDa cytosolic protein diazepam binding inhibitor which is suspected of interacting with MBR(10, 11, 12, 13) . An identical protein containing this and other sequence obtained is not represented in any of the current sequence data bases. Because pk10 appears to show a distinct association with pk18 it seems credible that this protein has not been identified in another context outside of MBR.

Evidence is mounting that MBR exists as a hetero-oligomeric complex (15, 33, 36, 37) and identification of all proteins in this entity are essential to understand its biochemical role. Strong data have been reported for the function of MBR in steroidogenesis (5) and mitochondrial respiratory control (38) but the molecular mechanisms underlying these findings remains a mystery. Current research goals include revealing the identity of pk10 to hopefully obtain novel insight into MBR function and the pharmacological consequences of drugs which bind to these sites.


FOOTNOTES

*
This work was funded by National Institutes of Health Grant MH44284 (to K. E. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Cell Biology, Georgetown University School of Medicine, 3900 Reservoir Rd., Washington, D.C. 20007. Tel.: 202-687-1094; Fax: 202-687-1823.

(^1)
The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; Ro5-4864, 4`-chlorodiazepam; PK11195, 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide; PK14105, 1-(2fluoro-5-nitrophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide; PK1406(7/8), (-/+)-N,N-diethyl-2-methyl-3-[4-(2-phenyl)quinolinyl]propanamide; FGIN-2, N,N-di-n-propyl-2-phenylindole-3-acetamide; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.


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