Phosphate Transport in Rat Liver Mitochondria MEMBRANE COMPONENTS LABELED BY N-ETHYLMALEIMIDE DURING INHIBITION OF TRANSPORT*

N-ethylmaleimide (NEM) inhibits the transport of phosphate in mitochondria but is without effect on permeation of other metabolites. In spite of its specificity for inhibition of phosphate transport, NEM reacts in an unspecific manner with inner membrane proteins in general. Treatment of mitochondria with [3H]NEM just sufficient to produce inhibition of phosphate transport results in labeling of at least 10 polypeptide components of the inner membrane. A marked increase in the specificity of reaction of NEM for components of the phosphate transport system is attained by first protecting the transport system with p-mercuribenzoate (p-MB) and then by irreversibly blocking reactive sulfhydryl groups unassociated with transport by the addition of unlabeled NEM. Subsequent addition of dithiothreitol removes p-MB and restores 65 to 75% of the original phosphate transport activity. Reinhibition of transport with [SH]NEM results in both a 6-fold decrease in the amount of [SH]NEM bound by purified inner membrane vesicles and a substantial reduction in the number of labeled polypeptide components. Five distinct labeled species are detected by this method, one of which is a 32,000 molecular weight protein containing 40% of the bound radioactivity,

x mg-' at 0" (Coty, W. A., and Pedersen, P. L. (1974) J. Biol. Chem. 249, 2593) yields an approximate minimum turnover for this process of 3500 min-* at 0". This turnover number is at least 20-fold greater than similarly calculated values for adenine nucleotide transport and succinate oxidation in rat liver mitochondria at this temperature. Taken together these results suggest that the NEM-sensitive phosphate transport system in rat liver mitochondria has an unusually high catalytic activity compared to other mitochondrial processes, and that at least one of the five NEM-binding proteins is likely to be an essential component of this transport system.
Phosphate transport in rat liver mitochondria is catalyzed by two transport systems; one is dependent on the membrane pH gradient (l-4), whereas the second exchanges Pi for dicarboxylate ions (2,3,(5)(6)(7). Both transport systems are sensitive to mercurial reagents, such as p-MB' and mersalyl (5,8,9), * This work was supported in part by grants from the United States Public Health Service (CA 10951) and the National Science Foundation (BMS 74-14071 Hepes, N-2-hydroxyethylpiperazine-N'-2-ethane-whereas only the pH gradient-dependent phase of transport is sensitive to NEM (10).
In addition, the NEM-sensitive phosphate transport system has an extremely high activity, with a V,,,,, at 0" of 205 nmol x min -1 x mg-1 (11). This rapid rate is consistent with proposals that pH gradient-dependent transport of phosphate, which can be thought of as a phosphate-hydroxyl antiport activity (5), plays an essential role in such mitochondrial processes as oxidative phosphorylation (12,13), cation transport (14,15), and net uptake or efflux of numerous anionic metabolites (2 dodecyl sulfate was present, a precipitation with ice-cold acetone was employed, as previously described (21). Crystalline bovine albumin was used as standard.  (25), or tricarboxylates (25), and transport of adenine nucleotides (13) are not inhibited by NEM. Results presented in Fig. 1 are consistent with this view and show that NEM also fails to inhibit permeation of substances such as CO, and acetate, which penetrate the mitochondrial inner membrane by simple diffusion (5,26). This is demonstrated by measurements of passive swelling of mitochondria in ammonium salts of phosphate, bicarbonate, and acetate. Swelling is observed in solutions containing these three anions, but not in the presence of Cl-, which does not penetrate the membrane (Fig. 1A) (5). NEM specifically inhibits swelling in ammonium phosphate, whereas swelling in ammonium acetate or bicarbonate is not affected (Fig. 1B).

Specificity
Labeling  (27), phosphate was found to have no effect on the inhibition of transport by NEM. For this reason, an alternative method of protection using the reversible sulfhydryl group inhibitor p-MB was investigated.
The phosphate swelling experiments summarized in Fig. 4 indicate that p-MB and NEM react with the same sulfhydryl group of the phosphate transport system. Fig. 4, A and B show that whereas inhibition of swelling by p-MB is reversed by thiol reagents such as dithiothreitol, NEM irreversibly inhibits swelling.
As noted in Fig. 4C, p-MB added in amounts sufficient to inhibit swelling in ammonium phosphate protects  Table I    Sensitivity of Phosphate Transport to NEM after p-MB Protection and Blocking of -SH Groups-Mitochondria were treated first with sufficient p-MB to inhibit phosphate transport and then with an excess of NEM. After restoration of transport with dithiothreitol and removal of excess sulfhydryl reagent by centrifugation and resuspension, the concentration dependence of reinhibition of transport by NEM was determined. As shown in Fig. 6, the concentration needed to produce half-maximal inhibition is reduced approximately 7-fold, from 10 nmol/mg in untreated mitochondrial to 1.5 nmol/mg. Thus, this treatment has sensitized phosphate transport to inhibition by NEM, because it has reduced the levels of accessible, reactive membrane -SH groups. Mitochondria treated in this manner are referred to below as "inhibitor-sensitized." Labeling of Inhibitor-sensitized Mitochondria with [*H]NEM-Mitochondria were treated with [aH]NEM after inhibitor sensitization, as described above, and the inner membrane fraction then was isolated. As noted in Table II,  This treatment was carried out as follows: mitochondria (5 mg/ml) in isolation medium were treated successively at 0" with p-MB, 12.5 nmol/mg for 2 min; then NEM, 125 nmol/mg for 5 min; and finally an excess of dithiothreitol(O.5 mM) for 5 min. The mitochondria then were diluted to about 2 mg/ml with isolation medium and centrifuged. The pellet fraction was suspended in isolation medium, recentrifuged to remove any residual dithiothreitol, and then suspended to a concentration of 100 mg/ml for transport assays. Mitochondria were inhibitor-sensitized with p-MB as described in Fig. 6 and then were treated with [*H]NEM (2.4 nmol/mg of protein), under the same conditions as in Table I. After dithiothreitol addition and centrifugation, these labeled mitochondria were subfractionated as described under "Methods."   Fig. 7 resolved by gel electrophoresis in sodium dodecyl sulfate. Apparent molecular weights were determined by comparison with standard curves of mobility uersus log molecular weight using ["Clacetyl standard proteins, as described in Fig. 7

70
[3H]NEM/mg of protein, a 6-fold decrease over the direct labeling procedure (See Table I). An analysis of the labeled inner membrane proteins by polyacrylamide gel electrophoresis in sodium dodecyl sulfate is shown in Fig. 7 Fig. 3, membranes isolated from unsensitized mitochondria contain at least 10 NEM-labeled components.
In contrast, membranes isolated from inhibitor-sensitized mitochondria contain one major peak of apparent molecular weight 32,000 which contains approximately 40% of the total label. The remaining label is distributed among four minor peaks ranging in apparent molecular weight from 47,000 to 95,000 (Fig. 7B).
The 32,000 molecular weight component is present in the amount of 160 pmol/mg of inner membrane protein, and the four minor components in amounts ranging from 40 to 80 pmol/mg of inner membrane (Table III). These values range from 5 to 17% of the concentration of cytochrome oxidase in the inner membrane. where phosphate transport is inhibited by NEM at least 10 inner membrane proteins are labeled (Fig. 3).

Results
In order to enhance the specificity of NEM for reaction with protein components of the phosphate transport system, p-MB, a reversible inhibitor of both protein sulfhydryl groups and the phosphate transport system was employed. When this agent was added initially to mitochondria to inhibit phosphate transport, unlabeled NEM could be added to block irreversibly most of the membrane sulfhydryl groups unrelated to transport. Addition of dithiothreitol to remove p-MB and restore transport (Fig. 4), followed by the addition of labeled NEM, resulted in a 6-fold decrease in the membrane-bound NEM observed under unprotected conditions (Tables I and II) (Table III) by 0.25, the fraction of inner membrane protein comprising the mitochondrial fraction, and by 1.5 to correct for the fact that inhibitor-sensitized mitochondria retained approximately 67% of their original P, transport activity.
Sixty picomoles per milligram of mitochondria is about 25% of the concentration of respiratory chain components such as cytochrome a (17) and approximately the same level as the estimated concentration of atractyloside binding sites of the adenine nucleotide transport system (28). If it is assumed that there is-a 1:l correspondence between the NEM-reactive protein and the active site for phosphate transport, then for a V,,,., of 205 nmol x min-1 x mg-' at 0" (ll), a turnover number of approximately 3500 min-1 can be calculated.
Similar calculations of the turnover number for succinate oxidation and adenine nucleotide transport give values of 110 and 175 min-' at O", respectively (17,28,29). The unusually high catalytic activity of the phosphate transport system at this low temperature may relate to the mechanism of transport. A "gated pore" mechanism (30) involving relatively little protein movement would require a much lower activation energy than a "mobile carrier" mechanism (31,32). Measurements of the temperature dependence of phosphate transport are being made to test this possibility. The five polypeptides labeled with NEM are possible components of the transport system which catalyzes P,/OHm exchange in rat liver mitochondria.
Although it seems unlikely that all of these components are parts of the PJOH-carrier, it is not unreasonable to suggest that as many as two of these polypeptides may be associated with this process. Pertinent here are the recent results of Fonyo (33) which suggest that at least two sulfhydryl groups may be associated with the transport system catalyzing Pi/OH-exchange. These results, therefore, provide a basis for future progress toward isolation of the phosphate transport system. The [sH]NEM-labeled proteins can be used as markers for the isolation of the same proteins from untreated mitochondrial membranes, which then could be tested for activity in a reconstitution assay for phosphate transport. Experiments currently are being carried out toward this objective, with special attention being given to the 32,000 molecular weight component which contained 40% of the total NEM bound to the inner membrane.