Anti-peptide antibodies and proteases as structural probes for the lactose/H+ transporter of Escherichia coli: a loop around amino acid residue 130 faces the cytoplasmic side of the membrane

From the amino acid sequence of the Escherichia coli lactose/H+ transporter, 7 hydrophilic segments were selected, 8-13 amino acids in length, and chemically synthesized, and anti-peptide antibodies were raised in rabbits. Apart from the antiserum to the synthetic COOH terminus (P408-417), which reacted strongly with the lactose/H+ transporter and has previously been used to localize the COOH terminus on the cytoplasmic face of the membrane, only those antibodies directed against the peptide corresponding to amino acid residues 125-135 (P125-135) exhibited a marked reaction with the transporter, while antibodies to the five other peptides reacted very weakly or not at all, suggesting that most of the hydrophilic segments are conformationally restricted or buried in the interior of the protein. Thermolysin treatment destroys the epitope on the transporter which is recognized by anti-P125-135 antibodies. Comparison of the kinetics and the extent of proteolysis of the transporter in right-side-out or inside-out cytoplasmic membrane vesicles or in reconstituted proteoliposomes suggests that the hydrophilic sequence from amino acid 125 to amino acid 135 is accessible to thermolysin only from one side, corresponding to the cytoplasmic face of the membrane. Furthermore, the experiments demonstrate that the transporter is inserted bimodally in a nonpreferential fashion into the proteoliposomes, confirming earlier results using antibodies to the synthetic COOH terminus of the transporter in conjunction with carboxypeptidase A treatment.     

Adenine nucleotide and phosphate transport systems of mitochondria. Relative location of sulfhydryl groups based on the use of the novel fluorescent probe eosin-5-maleimide

Eosin-5-maleimide is impermeable to the inner mitochondrial membrane, exhibiting essentially no reactivity with matrix glutathione or with beta-hydroxybutyrate dehydrogenase located on the matrix surface of the inner membrane. In intact mitochondria, eosin-5-maleimide is unreactive with the ADP/ATP antiporter even under conditions which promote maximal labeling by N-[3H]ethylmaleimide (i.e., ADP present). However, eosin-5-maleimide readily labels the ADP/ATP antiporter in "inverted" inner membrane vesicles even in the presence of N-ethylmaleimide. Labeling is prevented if the vesicles are prepared from mitochondria pretreated with carboxyatractyloside. In contrast to the ADP/ATP antiporter, essential sulfhydryl groups of the Pi/H+ symporter are accessible to eosin-5-maleimide in intact mitochondria with optimal inhibition of phosphate transport being observed at 25 degrees C. Eosin-5-maleimide also prevents labeling of the Pi/H+ symporter by N-[3H]ethylmaleimide. These results show that essential sulfhydryl groups of the ADP/ATP antiporter and the Pi/H+ symporter have differing reactivities and locations in functionally intact mitochondria. With respect to eosin-5-maleimide, sulfhydryl groups of the ADP/ATP carrier occur in two distinct classes, both of which are inaccessible in intact mitochondria. Only one class, depending on conditions, can be exposed in submitochondrial particles. In contrast, sulfhydryl group(s) of the Pi/H+ symporter behave as a single reactive class which is readily accessible in mitochondria at 25 degrees C.     

Mitochondrial phosphate transport. Import of the H+/Pi symporter and role of the presequence

The rat liver mitochondrial phosphate transporter contains a 44-amino acid presequence. The role of this presequence is not clear since the ADP/ATP carrier and the brown fat uncoupling protein, related members of a family of inner membrane anion transporters, lack a presequence and contain targeting information within the mature protein. Here, we present evidence that the rat liver mitochondrial phosphate transporter can be synthesized in vitro, imported into mitochondria, and processed to a protein of Mr 33,000. Import requires the membrane potential and external nucleotide triphosphate. The presequence inserts into the outer mitochondrial membrane, and import proceeds via a process similar to other proteins destined for the inner membrane or matrix. A mutant phosphate transporter lacking 35 amino acids at the NH2 terminus of the presequence has little capacity for mitochondrial import. The rat liver phosphate transporter is also imported and processed by rat kidney mitochondria and by mitochondria from the yeast Saccharomyces cerevisiae. A site-directed mutation of the N-ethyl-maleimide reactive cysteine 41 does not affect import or processing. The results presented show that optimal import of the mitochondrial phosphate transporter, unlike the ADP/ATP carrier and the brown fat uncoupling protein, is dependent on a presequence. As these carriers are believed to have evolved from a single gene, it seems likely that the H+/Pi carrier, known to be present in prokaryotes, appeared first and that subsequent evolutionary events leading to the other anion carriers eliminated the presequence.     

Energy-linked anion transport. Cloning, sequencing, and characterization of a full length cDNA encoding the rat liver mitochondrial proton/phosphate symporter

A full length cDNA clone encoding the precursor of the rat liver mitochondrial phosphate transporter (H+/Pi symporter) has been isolated from a cDNA library using a bovine heart partial length phosphate transporter clone as a hybridization probe. The entire clone is 1263 base pairs in length with 5'- and 3'-untranslated regions of 16 and 168 base pairs, respectively. The open reading frame encodes for the mature protein (312 amino acids) preceded by a presequence of 44 amino acids enriched in basic residues. The polypeptide sequence predicted from the DNA sequence was confirmed by analyzing the first 17 amino-terminal amino acids of the pure phosphate transporter protein. The rat liver phosphate transporter differs from the bovine heart transporter in 32 amino acids (i.e. approximately 10%). It contains a region from amino acid 139 to 159 which is 37% identical with the beta-subunit of the liver mitochondrial ATP synthase. Amino acid sequence comparisons of the Pi transporter with Pi binding proteins, other H+-linked symporters, and the human glucose transporter did not reveal significant sequence homology. Analysis of genomic DNA from both rat and S. cerevisiae by Southern blots using the rat liver mitochondrial Pi carrier cDNA as a probe revealed remarkably similar restriction patterns, a finding consistent with the presence in lower and higher eukaryotes of homologous Pi carrier proteins. This is the first report of the isolation, sequencing, and characterization of a full length cDNA coding for a protein involved in energy-coupled Pi transport.     

Binding of malonate to the inner membrane of rat liver mitochondria

The binding of malonate to the external face of the mitochondrial inner membrane has been investigated by using mitoplasts and on the opposite face, by using inside-out oriented vesicles prepared from sonicated mitoplasts. The external face of the inner membrane displays a single class of binding sites whereas two classes are observed in vesicles. In trypsin treated vesicles, only high-affinity sites are evidenced. The disappearance of the low affinity sites is correlatively related with the loss of succinate dehydrogenase activity. Both high-affinity sites of mitoplasts and vesicles are sensitive to the presence of malate; they are also masked by 2-butylmalonate, phosphate, citrate and mercurials. Our results suggest that the internal and external high-affinity sites of the inner membrane are involved in the dicarboxylate transport system.     

C-terminal-specific monoclonal antibodies against the human red cell glucose transporter. Epitope localization with synthetic peptides

Mice were immunized with human red cell glucose transporter for production of monoclonal antibodies. Four peptides were synthesized that correspond to relatively hydrophilic segments of the human HepG2 glucose transporter (Mueckler, M., Caruso, C., Baldwin, S.A., Panico, M., Blench, I., Morris, H.R., Allard, W. J., Lienhard, G.E., and Lodish, H.F. (1985) Science 229, 941-945), including a C-terminal segment. After identification of hybridomas that were positive for the red cell glucose transporter, enzyme-linked immunosorbent assays were done with the synthetic peptides in solution to detect peptide-binding monoclonals. The very hydrophilic C-terminal peptide 478-492 (P2), but no other peptide, gave strong and selective inhibition of antibody binding to the glucose transporter. Two C-terminal-specific monoclonal antibodies were selected. The binding of these two antibodies to immobilized inside-out vesicles of human red cell membranes could be inhibited with the peptide P2. The antibodies did not react with right-side-out vesicles. The binding of these C-terminal-specific antibodies to the glucose transporter, to immobilized vesicles, and to the peptide P2 was enhanced by the presence of the peptide 218-232 (P1), although the peptide P1 alone showed no reaction with these antibodies. This suggests that the C terminus and the segment 218-232 of the red cell glucose transporter are exposed at the cytoplasmic face of the membrane and interact in the transporter. The C-terminal-specific monoclonal antibodies reacted strongly in Western blotting with the human red cell glucose transporter.     

The orientation of halorhodopsin in the cell membrane of halobacteria

The orientation of the light-driven chloride pump, halorhodopsin, in the membrane was determined using antibodies directed against a synthetic peptide which represents the C-terminal segment of the protein. Antibodies against this decapeptide did not bind to right-side-out cell vesicles. Partial inversion by sonication or lysis under low salt conditions exposed this COOH-terminal antigenic site. Antibody binding was removed by preincubation with the decapeptide. The COOH terminus of the molecule is therefore located on the cytoplasmic surface of the membrane.     

Immunochemical study of cholesterol-hydroxylating cytochrome P-450. Topology of the peptide chain of the heme protein in the phospholipid membrane

The topology of adrenocortical cytochrome P-450scc in inner mitochondrial membrane was studied. To determine the polypeptide chain parts exposed to matrix or cytosol, two approaches were used, i.e. i) limited proteolysis of membrane-bound cytochrome P-450scc followed by the detection of the peptides formed by immunoblotting; ii) binding of monospecific antibodies against cytochrome P-450scc as well as fragments F1 and F2 representing N- and C-terminal sequences of the hemeprotein, to membrane structures (mitoplasts and submitochondrial particles). The data obtained confirm the transmembrane orientation of cytochrome P-450scc molecule, since antibodies against the hemeprotein as well as fragments F1 and F2 were found to be bound both on the matrix and cytosol surfaces of the inner mitochondrial membrane. It was shown that region 250-257 in cytochrome P-450scc connecting domains F1 and F2 is exposed to the matrix. A model of molecular organization of cytochrome P-450scc in inner mitochondrial membranes is proposed.     

Structural characterization of the avian retrovirus reverse transcriptase and endonuclease domains

The enzymatic domains of the avian retrovirus polymerase (pol) gene have been mapped by the use of peptide antibodies and COOH-terminal amino acid analysis. The processed pol beta polypeptide is cleaved in vivo to yield alpha and pp32. Rabbit antibodies were directed against synthetic peptides whose sequence was deduced from the known pol sequence of Rous sarcoma virus, Prague C (Schwartz, D.E., Tizard, R., and Gilbert, W. (1983) Cell 32, 853-869). The RNase H active site of pol was located in the NH2-terminal region of the alpha DNA polymerase subunit. The COOH terminus of the alpha subunit was found to be immediately adjacent to the NH2 terminus of the pp32 pol protein. COOH-terminal amino acid analysis of pp32 revealed that this protein is also processed. From the deduced amino acid sequence of pol, it appears likely that pol encodes an additional 4100-dalton polypeptide located at its extreme COOH terminus. The enzymatic domains on beta appear to map in the following order: RNase H-DNA polymerase-DNA endonuclease. Hydrophilicity analysis and secondary structure predictions of wild type Rous sarcoma virus pol products and mutated pp32 possessing single amino acid changes permit further structural evaluation of the multifunctional pol protein.     

Mitochondrial energy-linked nicotinamide nucleotide transhydrogenase. Membrane topography of the bovine enzyme

The mitochondrial energy-linked nicotinamide nucleotide transhydrogenase is a homodimer of monomer Mr = 109,228. Hydropathy analysis of its cDNA-deduced amino acid sequence (1043 residues) has indicated that the molecule is composed of 3 domains: a 430-residue-long hydrophilic N-terminal domain which binds NAD(H), a 200-residue-long hydrophilic C-terminal domain which binds NADP(H), and a 400-residue-long hydrophobic central domain which appears to be made up mainly of about 14 hydrophobic clusters of approximately 20 residues each. In this study, antibodies were raised to the hydrophilic N- and C-terminal domains cleaved from the isolated transhydrogenase by proteolytic digestion, and to a synthetic, hydrophilic pentadecapeptide, which corresponded to position 540-554 within the central hydrophobic domain. Immunochemical experiments with mitoplasts (mitochondria denuded of outer membrane) and submitochondrial particles (inside-out inner membrane vesicles) as sources of antigens showed that essentially the entire N- and C-terminal hydrophilic domains of the transhydrogenase, as well as epitopes from the central pentadecapeptide, protrude from the inner membrane into the mitochondrial matrix, where the N- and C-terminal domains would be expected to come together to form the enzyme's catalytic site. Treatment of mitoplasts with several proteolytic enzymes indicated that large protease-sensitive masses of the transhydrogenase are not exposed on the cytosolic side of the inner membrane, which agreed with the exception that the central highly hydrophobic domain of the molecule should be largely membrane-intercalated. Trypsin, alpha-chymotrypsin, and papain had little or no effect on the mitoplast-embedded transhydrogenase. Proteinase K, subtilisin (Nagarse), thermolysin, and pronase E each split the mitoplast-embedded enzyme into two fragments only, a fragment of approximately 70 kDa containing the N-terminal hydrophilic domain, and one of approximately 40 kDa bearing the C-terminal hydrophilic domain. The cleavage site of proteinase K was determined to be A690 -A691, which is located in a small hydrophilic segment within the central hydrophobic domain. This protease-sensitive loop appears to be exposed on the cytosolic side of the inner membrane. The proteinase K-nicked enzyme containing two peptides of 71 and 39 kDa was isolated from mitoplasts and shown to have high transhydrogenase activity.     

Intramitochondrial localization of alanine aminotransferase in rat-liver mitochondria: comparison with glutaminase and aspartate aminotransferase

Summary The removal of the outer mitochondrial membrane and hence of constituents of the intermembrane space in rat-liver mitochondria using digitonin showed that phosphate-dependent glutaminase, alanine and aspartate aminotransferase were localized in the mitoplasts. Further fractionation of mitoplasts following their sonication resulted in 90% of glutaminase, 98% of alanine aminotransferase and 48% of aspartate aminotransferase being recovered in the soluble fraction while the remainder of each enzyme was recovered in the sonicated vesicles fraction. These results indicated that glutaminase and alanine aminotransferase were soluble matrix enzymes, the little of each enzyme recovered in the sonicated vesicles fraction being probably due to entrapment in the vesicles. Aspartate aminotransferase had dual localization, in the inner membrane and matrix with the high specific activity in sonicated vesicles confirming its association with the membrane. Activation experiments suggested that the membrane-bound enzyme was localized on the inner side of the inner mitochondrial membrane.     

Determinants of substrate affinity for the oligopeptide/H+ symporter in the renal brush border membrane.

We and others have shown previously the existence of high and low affinity systems for oligopeptide transport in kidney brush border membrane vesicles (BBMV). In the present study we investigated the relationship between the structure of substrates and their affinity for interaction with the high-affinity oligopeptide/H+ transporter in kidney BBMV. Based on competition experiments using [3H]Gly-Gln as a probe we determined the Ki values for more than 60 selected peptides. For a high-affinity interaction with the carrier site the following structural features of substrates are required: (a) both a free amino and carboxyl terminus; (b) the amino group and peptide bond nitrogen located in the alpha-position; (c) a trans peptide bond rather than the cis configuration; (d) L-alpha-amino acid isomers in both COOH and NH2 termini, although D-isomers of hydrophobic amino acids are acceptable in the NH2 terminus; and (e) a backbone of less than 3 amino acid residues. A striking finding of the present study is that, for peptides satisfying these minimal structural requirements, the primary determinant of affinity is hydrophobicity. The fact that there is a highly significant (p less than 0.001) correlation between Ki and hydrophobicity allows the prediction of the affinity for any di- or tripeptide composed of alpha-amino acids in the L-form.     

Transmembrane topography of the mitochondrial phosphate carrier explored by peptide-specific antibodies and enzymatic digestion

Two peptides corresponding to the amino acid sequences 1-10 (N-terminal peptide) and 303-313 (C-terminal peptide) of the bovine heart mitochondrial phosphate carrier have been synthesized. After being coupled to ovalbumin, they were injected into rabbits to raise polyclonal antibodies. The specificity of the generated antibodies was tested by enzyme-linked immunosorbent assay (ELISA) and/or Western blot. Anti-N-terminal antibodies and anti-C-terminal antibodies exclusively reacted with the corresponding terminal peptide, they also reacted with the isolated phosphate carrier as well as with the phosphate carrier protein in mitochondrial lysates. Both anti-N-terminal and anti-C-terminal antibodies bound to freeze-thawed mitochondria, indicating that both termini of the membrane-bound phosphate carrier are exposed to the cytoplasmic side of the inner mitochondrial membrane. These immunological data were complemented with results concerning enzymatic cleavage of the membrane-bound phosphate carrier by carboxypeptidase A and by an arginine-specific endoprotease. Carboxypeptidase A markedly decreased the binding of anti-C-terminal antibodies to phosphate carrier in freeze-thawed mitochondria. Arg-endoprotease cleaved the phosphate carrier in inside-out submitochondrial particles, but not in right-side-out particles, yielding two fragments of similar apparent molecular weight (Mr approximately equal to 14.5K), which were immunodetected only by the anti-N-terminal antiserum, and a fragment of Mr approximately equal to 17K which was detected only by the anti-C-terminal antiserum. It appears, therefore, that Arg-endoprotease cleavage sites of the phosphate carrier are present only at the matrix side of the inner mitochondrial membrane, at Arg-140 and/or Arg-152.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and reconstitution of an N-ethylmaleimide-sensitive phosphate transport protein from rat liver mitochondria

An N-ethylmaleimide-sensitive phosphate transport protein has been isolated from rat liver mitochondria, substantially purified, and reconstituted into phospholipid vesicles. Purified inner mitochondrial membrane vesicles depleted of F1-ATPase by urea treatment proved to be the most satisfactory starting material. Treatment of these membrane vesicles with Triton X-100 resulted in solubilization of the phosphate transport protein. Further purification was achieved using hydroxylapatite powder. Polyacrylamide gel electrophoresis of the purified fraction in sodium dodecyl sulfate indicated the presence of two Coomassie blue-staining bands with apparent Mr's of 30,000 and 35,000. Labeling of the 35,000 Mr band by the Pi transport inhibitor diazobenzene sulfonate was reduced markedly by prior treatment of the mitochondria with the inhibitor N-ethylmaleimide. The purified fraction containing both proteins could be reconstituted into liposomes prepared from purified asolectin. Phosphate efflux from these vesicles was inhibited by N-ethylmaleimide, by the impermeant mercurial agent, p-chloromercuribenzoate, and by diazobenzene sulfonate. Treatment of the purified fraction with N-ethylmaleimide prior to incorporation into liposomes resulted in a reconstituted system incapable of catalyzing Pi efflux. These studies summarize the first detailed attempt to purify the Pi/H+ transport system from rat liver mitochondria and emphasize the need to commence the purification with purified inner membrane vesicles depleted of F1-ATPase. In addition, these studies show that the final fraction contains a reconstitutively active transport system which when incorporated into phospholipid vesicles has its essential sulfhydryl groups oriented outward. Finally, it is shown that the purified fraction also contains a 30,000 Mr component.     

Reconstitution of the mitochondrial non-selective Na+/H+ (K+/H+) antiporter into proteoliposomes

Mitochondria contain two Na+/H+ antiporters, one of which transports K+ as well as Na+. The physiological role of this non-selective Na+/H+ (K+/H+) antiporter is to provide mitochondrial volume homeostasis. The properties of this carrier have been well documented in intact mitochondria, and it has been identified as an 82,000-dalton inner membrane protein. The present studies were designed to solubilize and reconstitute this antiporter in order to permit its isolation and molecular characterization. Proteins from mitoplasts made from rat liver mitochondria were extracted with Triton X-100 in the presence of cardiolipin and reconstituted into phospholipid vesicles. The reconstituted proteoliposomes exhibited electroneutral 86Rb+ transport which was reversibly inhibited by Mg2+ and quinine with K0.5 values of approximately 150 and 300 microM, respectively. Incubation of reconstituted vesicles with dicyclohexylcarbodiimide resulted in irreversible inhibition of 86Rb+ uptake into proteoliposomes. Incubation of vesicles with [14C]dicyclohexylcarbodiimide resulted in labeling of an 82,000-dalton protein. These properties, which are also characteristic of the native Na+/H+ (K+/H+) antiporter, lead us to conclude that this mitochondrial carrier has been reconstituted into proteoliposomes with its known native properties intact.     

COOH-terminal requirements for the correct processing of a phosphatidylinositol-glycan anchored membrane protein

Placental alkaline phosphatase (PLAP) is anchored to the plasma membrane by a phosphatidylinositol-glycan (PI-G) moiety. During processing of nascent PLAP, a 29-residue COOH-terminal peptide is cleaved out and the PI-G moiety is attached to the newly created COOH terminus of the mature protein. To investigate the structural requirements of the COOH terminus of the nascent protein for PI-G tailing and anchoring to the plasma membrane, we have transfected COS cells with wild type and mutant forms of cDNA encoding human prepro-PLAP. Utilizing a series of COOH-terminal deletion mutants of prepro-PLAP, it was found that to be PI-G-tailed the newly synthesized protein must possess an uncharged, predominantly hydrophobic amino acid sequence of a minimal length in the COOH-terminal peptide. While forms of prepro-PLAP with 17 consecutive hydrophobic residues in the terminal sequence yielded PI-G-tailed and membrane-bound products, prepro-PLAP mutants with 13 or fewer of such residues yielded hydrophilic proteins that were no longer PI-G-tailed but efficiently secreted into the medium. Studies using cassette mutants demonstrated that the precise amino sequence of the COOH-terminal region could be altered as long as minimal hydrophobicity and length was maintained.     

Perspectives on the mitochondrial multiple conductance channel

A multiple conductance channel (MCC) with a peak conductance of over 1 nS is recorded from mitoplasts (mitochondria with the inner membrane exposed) using patch-clamp techniques. MCC shares many general characteristics with other intracellular megachannels, many of which are weakly selective, voltage-dependent, and calcium sensitive. A role in protein import is suggested by the transient blockade of MCC by peptides responsible for targeting mitochondrial precursor proteins. MCC is compared with the peptide-sensitive channel of the outer membrane because of similarities in targeting peptide blockade. The pharmacology and regulation of MCC by physiological effectors are reviewed and compared with the properties of the pore hypothesized to be responsible for the mitochondrial inner membrane permeability transition.     

Stoichiometry of subunit e in rat liver mitochondrial H(+)-ATP synthase and membrane topology of its putative Ca(2+)-dependent regulatory region

Previous studies have revealed that residues 34-65 of subunit e of mitochondrial H(+)-ATP synthase are homologous with the Ca(2+)-dependent tropomysin-binding region for troponin T and have suggested that subunit e could be involved in the Ca(2+)-dependent regulation of H(+)-ATP synthase activity. In this study, we determined the content of subunit e in H(+)-ATP synthase purified from rat liver mitochondria, and we also investigated the membrane topology of a putative Ca(2+)-dependent regulatory region of subunit e using an antibody against peptide corresponding to residues 34-65 of subunit e. Quantitative immunoblot analysis of subunit e in the purified H(+)-ATP synthase revealed that 1 mol of H(+)-ATP synthase contained 2 mol of subunit e. The ATPase activity of mitoplasts, in which the C-side of F(0) is present on the outer surface of the inner membrane, was significantly stimulated by the addition of the antibody, while the ATPase activity of submitochondrial particles and purified H(+)-ATP synthase was not stimulated. The antibody bound to mitoplasts but not to submitochondrial particles. These results suggest that the putative Ca(2+)-dependent regulatory region of subunit e is exposed on the surface of the C-side of F(0) and that subunit e is involved in the regulation of mitochondrial H(+)-ATP synthase activity probably via its putative Ca(2+)-dependent regulatory region.     

Peptide-specific antibodies as probes of the orientation of the glucose transporter in the human erythrocyte membrane

Antibodies were raised in rabbits against synthetic peptides corresponding to the N-terminal (residues 1-15) and the C-terminal (residues 477-492) regions of the human erythrocyte glucose transporter. The antisera recognized the intact transporter in enzyme-linked immunosorbent assays (ELISA) and Western blots. In addition, the anti-C-terminal peptide antibodies were demonstrated, by competitive ELISA and by immunoadsorption experiments, to bind to the native transporter. Competitive ELISA, using intact erythrocytes, unsealed erythrocyte membranes, or membrane vesicles of known sidedness as competing antigen, showed that these antibodies bound only to the cytoplasmic surface of the membrane, indicating that the C terminus of the protein is exposed to the cytoplasm. On Western blots, the anti-N-terminal peptide antiserum labeled the glycosylated tryptic fragment of the transporter, of apparent Mr = 23,000-42,000, showing that this originates from the N-terminal half of the protein. The anti-C-terminal peptide antiserum labeled higher Mr precursors of the Mr = 18,000 tryptic fragment, although not the fragment itself, indicating that the latter, with its associated cytochalasin B binding site, is derived from the C-terminal half of the protein. Antiserum against the intact transporter recognized the C-terminal peptide on ELISA, and the Mr = 18,000 fragment but not the glycosylated tryptic fragment on Western blots.