©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Peptide-specific Antibodies as Probes of the Topography of the Voltage-gated Channel in the Mitochondrial Outer Membrane of Neurospora crassa(*)

Scott Stanley (1) (3), James A. Dias (3) (2), Dora D'Arcangelis (1), Carmen A. Mannella (1) (3)(§)

From the (1)Division of Molecular Medicine and the (2)Division of Genetics, Wadsworth Center, New York State Department of Health, Albany, New York 12201-0509 and the (3)Department of Biomedical Sciences, School of Public Health, The University at Albany, State University of New York, Albany, New York 12201-0509

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The voltage-dependent anion-selective channel (VDAC) in mitochondrial outer membranes is formed by a polypeptide (M 31,000) coded by a nuclear gene whose cDNA sequence is known for several organisms. Antibodies have been raised against synthetic peptides corresponding to four different regions in the predicted sequence of the VDAC polypeptide of the fungus Neurospora crassa (residues 1-20, amino terminus; 195-210, 251-268, and 272-283, carboxyl terminus). Specificity of the antibodies has been characterized in terms of binding to peptides or fungal mitochondria on microtiter plates and binding to mitochondrial proteins of several species in Western blots. Reactivity of three of the four antibodies with fungal mitochondria in suspension increases with lysis of outer membranes, indicating that the respective epitopes (including those near the amino and carboxyl termini) are exposed on the surface of the outer membrane that faces inside the mitochondrion. Preincubation of mitochondria with a polyanion that modulates VDAC voltage dependence strongly inhibits binding of the antibody against residues 251-268, whose epitopes are on the outer mitochondrial surface.


INTRODUCTION

The mitochondrial outer membrane contains numerous copies of a polypeptide (M 31,000) that forms large conductance, voltage-gated ion channels when incorporated into phospholipid bilayers or liposomes(1, 2, 3, 4) . This voltage-dependent, anion-selective channel (VDAC)()(5) (also known as mitochondrial porin) is thought to represent the main pathway for diffusion of polar solutes through the mitochondrial outer membrane. Circular dichroism spectra of fungal VDAC in detergents and liposomes are very similar to those of bacterial porins(41, 42) , indicating that the two classes of large passive diffusion pores have similar secondary structure. Bacterial porins have been shown by electron (6) and x-ray crystallography (7, 8, 9) to be ``-barrels,'' i.e. 16- or 18-strand antiparallel -sheets twisted to form a cylinder with most nonpolar residues facing out and most polar groups lining the interior of the lumen. Electron microscopic images of fungal VDAC in frozen-hydrated, two-dimensional crystals are consistent with a -barrel having a diameter of 3.8 nm at the C backbone(10) , somewhat wider than bacterial porin and in agreement with the known permeability properties of mitochondrial porin(4, 11) . Several structural models have been proposed for VDAC based on results from electron microscopy, computer analyses of available sequences, accessibility of regions of the protein to either proteases or an antibody against the amino terminus, and electrical characteristics of site-directed mutants(12, 13, 14, 15, 16) . While all models for the VDAC open state posit a -barrel, there is considerable disagreement on the number of -strands (from 12 to 19) and on the disposition of the amino-terminal segment, which may fold as an amphiphilic -helix(17, 18) . One model places the amino-terminal helix in the lumen wall(13) , while other models have the helix outside the lumen(14, 15, 18) . The current state of affairs, summarized by Mannella et al. (11), is that no structural model for VDAC successfully accounts for all the available data.

To address this situation, a battery of polyclonal antibodies is being raised against peptides corresponding to various regions of the fungal VDAC polypeptide. These will be used to determine the accessibility of epitopes in these regions of the VDAC protein and to functionally map the protein. This report characterizes antibodies directed against four regions of fungal VDAC: residues 1-20 (the amino terminus), 195-210, 251-268, and 272-283 (the carboxyl terminus). Binding of the antibodies to fungal mitochondria is correlated with outer membrane intactness (to determine the side of the outer membrane on which epitopes are exposed) and with preincubation of lysed mitochondria with VDAC effectors.


EXPERIMENTAL PROCEDURES

Preparation of Fungal Mitochondria

Mitochondria were isolated from a wall-less strain of the fungus Neurospora crassa (FGSC 326, fz, sg, arg-1 (B369), cr-1 (B123), aur(34508), os-1 (B135) A) as described by Mannella(19) . Final mitochondrial suspensions contained approximately 15 mg of protein/ml of isolation medium (IM), consisting of 0.3 M sucrose, 1 mM sodium EDTA, 0.3% bovine serum albumin (BSA) (fraction V, Sigma), pH 7.0. All solutions were made with water deionized by the Milli-Q system (Millipore), and all chemicals were obtained from Sigma unless otherwise indicated.

Outer Membrane Integrity

Intactness of mitochondrial outer membranes was determined by measuring the accessibility of the respiratory chain to exogenously added holocytochrome c, which cannot penetrate the intact outer membrane(19, 20, 21) . Mitochondria were preincubated (5 min, room temperature, 30 µg of protein/ml) in IM or water (which fully lyses the outer membranes), and the succinate:cytochrome c oxidoreductase activities, v(IM) and v(water), were monitored as rates of increase of A using an Ultraspec III spectrophotometer (Pharmacia Biotech Inc.) and reaction conditions described by Mannella(19) . The fraction of mitochondria with lysed outer membranes in the original suspension is given by the ratio v(IM)/v(water).

In general, outer membranes of fungal mitochondria maintain integrity for several hours in buffers like IM that contain isoosmotic sucrose but lyse over the same time period in buffers in which 0.14-0.15 M KCl or NaCl are the osmoticants (due to swelling of the inner membrane compartment). Outer membrane lysis was prevented by inclusion of hyperosmotic (0.4 M) sucrose in salt-containing buffers used for antibody incubations (see below).

Peptide Synthesis

Four peptides corresponding to segments of the VDAC polypeptide of N. crassa inferred from its cDNA sequence (17) were prepared by the Wadsworth Center's Peptide Synthesis core facility: MAVPAFSDIAKSANDLLNKD, HKVNSQVEAGSKATWN, LREGVTLGVGASFDTQKL, and THKVGTSFTFES. The peptides were made on an automated synthesizer (model 431, Applied Biosystems) using 9-fluorenylmethoxycarbonyl chemistry with standard cycles on 4-hydroxymethyl-phenoxymethylcopolystyrene, 1% divinylbenzene resin. Composition and purity of the peptides were confirmed by amino acid analysis on a Systems Gold model 126 (Beckman Instruments) and by electrospray mass spectroscopy on an MAT TSQ 700 (Finnigan, San Jose, CA).

Preparation of Immunogen

Synthetic peptides were coupled to hemocyanin (from Limulus polyphemus or keyhole limpets) with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl (EDAC) (22). Each peptide (4 mg/ml final concentration) was combined with carrier and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl in molar ratio 100:1:200 in 0.1 M sodium phosphate (pH 7), vortexed, and incubated for 16-24 h at room temperature with continuous rotary mixing. Peptides containing Tyr were incubated for an additional 4 h at room temperature with an equal volume of 1 M hydroxylamine to reverse modifications in the aromatic ring. Peptide 1-20 was also coupled to hemocyanin using glutaraldehyde. Peptide (4 mg/ml final concentration) and carrier protein were combined at a molar ratio of 100:1 in 0.1 M sodium phosphate (pH 7.0). Glutaraldehyde was added at a final concentration of 0.25%, and the mixture was agitated for 30 min at room temperature. In all cases, the conjugate was dialyzed overnight at 4 °C against several hundred volumes of PBS (0.14 M NaCl, 0.01 M sodium phosphate buffer, pH 7.4).

Production of Polyclonal Antipeptide Antibodies

Two Nys:(FG) female rabbits (2.5-3.0 kg) were used per peptide conjugate. 1 month after collection of control sera, rabbits were injected subcutaneously in multiple sites on the lower back using 2 mg of the peptide:hemocyanin conjugate emulsified in Freund's complete adjuvant (Pierce) at a 1:1 volume ratio of conjugate to adjuvant. The rabbits were generally put on monthly cycles of blood collection (from inner marginal ear veins) and reimmunization, with 1 mg of peptide:hemocyanin conjugate emulsified in an equal volume of Freund's incomplete adjuvant (Pierce). Near maximum immunogenic response (based on criteria described below) was generally elicited in the animals after the third immunization. Failure to immunize a rabbit in the month prior to serum collection resulted in a marked decrease in antibody production that was restored with subsequent immunization. At least one animal within each pair immunized with a given peptide conjugate showed a sufficient immunogenic response to be used in subsequent experiments.

Immunoglobulin (Ig) fractions were prepared from sera by precipitation with 50% ammonium sulfate, resuspended in PBS, and dialyzed overnight against 10 mM Tris-HCl (pH 7.0) (final volume of the Ig fraction was equal to that of the starting serum fraction). Ig fractions were used for all experiments except as indicated. Sera and Ig fractions were stored at -90 °C.

Binding of Antibodies to Immobilized Peptides

Antibodies were initially characterized in terms of binding to their peptide antigens using enzyme-linked immunosorbent assays (ELISAs). Procedures employed are described in detail by Weiner et al.(23) . All incubations were done with gentle agitation on a Titer Plate Shaker (Lab-Line Instruments, Melrose Park, IL). Briefly, Immulon I microtiter plates (96 well, Dynatech Laboratories, Chantilly, VA) were coated with 10 µg of peptide per well, incubated with antipeptide antibody (diluted 1:100), followed by goat-anti-rabbit IgG conjugated with alkaline phosphatase (Fisher BioTech, Pittsburgh, PA). To determine bound antibody in each well, p-nitrophenyl-phosphate substrate in diethanolamine buffer (Bio-Rad) was added, and A was measured with an EL340 microtiter plate reader (Bio-Tek Instruments, Winooski, VT). In these and other ELISAs described below, determinations were routinely made in triplicate for each experimental condition. The relative titer of the different antisera in terms of reactivity toward their respective peptides in solid phase ELISAs is summarized in .

Western Blot Analysis of Antibodies

Protein specificity of the antipeptide antibodies was determined by Western blotting. Polyacrylamide gel electrophoresis of mitochondrial proteins solubilized in SDS was done with Laemmli buffers (24) on a Mini-Protean II apparatus (Bio-Rad). Approximately 0.2 mg of fungal mitochondrial protein was loaded per slab gel (12.5% polyacrylamide) using a single-slot comb. Proteins were transferred from gels to nitrocellulose membranes (Bio-Rad) by electroblotting (25) with the Mini-Trans-Blot system (Bio-Rad) at 100 V constant voltage for 1.25 h. Prestained protein markers (Bio-Rad) were used to monitor the electrotransfer process. Nitrocellulose membranes were incubated with antipeptide antibody (diluted 1:400 to 1:100), followed by goat-anti-rabbit IgG adsorbed to colloidal gold (5-10-nm diameter), and silver enhancing was employed, using reagents and protocols in the AuroProbe BL Plus and IntenSE BL kits (Amersham).

Western blots of fungal mitochondrial proteins probed with the antisera used in subsequent experiments contained a single strong band at M 31,000, corresponding to the molecular mass of VDAC (see Fig. 1). The relative intensities of labeling of this band by different bleeds from the same rabbit correlated well with the relative binding of the same sera in peptide ELISAs. The relative reactivities of the different antibodies in Western blots were anti(272-283) > anti(1-20) > anti(195-210) anti(251-268). This agreed with the relative titers for peptide binding () except for anti(272-283), which reacted least strongly with its immobilized immunizing peptide. Weak diffuse labeling was variably observed above the VDAC band in Western blots, similar to that observed with electrophoresis of bacterial porins(26, 27) , and attributed to incomplete extraction of endogenous lipids from the proteins(27) .


Figure 1: Western blots of N. crassa mitochondrial proteins probed with antipeptide antibodies (right lane in each pair) or with the corresponding pre-immune sera (left lane) used at the following dilutions: anti(1-20), 1:200; anti(195-210) and anti(251-268), 1:100; anti(272-283), 1:400. For anti(1-20), a and b refer to peptides conjugated to carrier by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl and glutaraldehyde, respectively. The M of VDAC (31,000) is indicated by the marker at left.



Western blotting experiments were also done with rat heart mitochondria (prepared by the procedure of Bowman and Tedeschi (28) and loaded at 5-10 µg of protein per lane) and with yeast strains expressing VDAC from different species (gift of Michael Forte, Oregon Health Sciences Univ.(29) ). For the latter experiments, total cellular protein from 1.0 ml of 60-h cultures (grown at 32 °C) was precipitated with trichloroacetic acid as described by Yaffe and Schatz (30) and loaded in one lane.

Mitochondrial Solid Phase ELISAs and Peptide Competition Experiments

Mitochondria with lysed outer membranes were immobilized on Immulon I microtiter plates by a modification of a procedure originally developed for bovine-heart mitochondria by De Pinto et al.(15) . All incubations were done with continuous gentle shaking as described above. Following addition to each well of 0.1 ml of mitochondrial suspension containing approximately 40 µg of protein in STE buffer (0.3 M sucrose, 0.01 M Tris, 1 mM sodium EDTA, pH 7.2), the plates were incubated (2 h, room temperature), washed with 1% BSA in STE, blocked (1.5 h, room temperature) with 5% BSA (Amersham) in STE, and washed with STE. Primary and secondary antibody incubations and alkaline phosphatase reaction development and recording were done as described above for peptide ELISAs, except all dilutions and washes were done in SKPEB buffer (0.4 M sucrose, 0.12 M KCl, 0.01 M sodium phosphate buffer, 1 mM sodium EDTA, and 1% BSA, pH 7.4). It was found that antibodies against all four VDAC peptides react with lysed mitochondria immobilized on microtiter plates, indicating that epitopes within the corresponding segments of membrane-bound VDAC are accessible to the antibodies. In general, relative levels of binding of the different antibodies to immobilized lysed mitochondria corresponded approximately to their labeling intensities in Western blots, i.e. anti(272-283) anti(1-20) > anti(195-210) > anti(251-268).

Peptide-competition experiments were used to determine whether the antipeptide antibodies were recognizing the expected regions of the VDAC protein in mitochondrial membranes. Prior to reacting with immobilized mitochondria by the above protocol, each antipeptide antibody was diluted 1:350 in SKPEB and incubated (30 min, room temperature) with each of the four peptides in serial dilutions ranging from 0.05 to 500 µg of peptide/ml. It was found (Fig. 2) that binding of each antibody to mitochondria was specifically inhibited by low concentrations of its immunizing peptide. In each case, immunizing peptide in the concentration range 0.1-10 µg of peptide/ml reduced the extent of antibody binding to that of preimmune sera (i.e.A in the range 0.1-0.35). Inhibition of antibody binding to mitochondria was also observed with peptides other than those against which the antibodies were raised, but this nonspecific inhibition occurred only at much greater peptide concentrations (0.1-1 mg/ml) and was generally incomplete.


Figure 2: Specificity of binding of antipeptide antibodies to mitochondrially bound VDAC. Antibodies (1:350 dilution) were preincubated with indicated concentrations of peptides prior to incubation with lysed fungal mitochondria immobilized on microtiter plates. Absorbance is proportional to the concentration of mitochondrially bound antibody.



Mitochondrial Back-titration ELISAs

Fungal mitochondria (unpretreated or lysed by osmotic swelling followed by two cycles of freezing and thawing in liquid nitrogen) were serially diluted (11-173 µg/ml of SKPEB) and incubated (1 h, room temperature) with antipeptide antibodies (variously diluted 1:2000 to 1:10,000 in SKPEB) in polypropylene microtubes (Laboratory Product Sales, Rochester, NY) that had first been blocked with 5% BSA (Amersham) in SKPEB. Mitochondria were pelleted (12,000 rpm, 1 h, 4 °C, model 5415C microfuge, Brinkmann Instruments, Westbury, NY), and the supernatants (containing unbound antibody) were added to wells in microtiter plates coated with fungal mitochondria. The plates were incubated, washed, and developed with alkaline phosphatase-linked secondary antibody as described above.

Indirect Immunoelectron Microscopy

Mitochondria (untreated or hypoosmotically lysed) were diluted to approximately 1.5 mg/ml in 1% BSA-PBS, i.e. 0.15 M NaCl, 10 mM sodium phosphate buffer (pH 7.2) containing 10 µg/ml fatty acid- and globulin-free BSA (incubation of fungal mitochondria in this buffer for 30-40 min did not cause significant lysis of outer membranes). 5-µl aliquots of the mitochondrial suspensions were immediately deposited on freshly glow-discharged, carbon/formvar-coated, 300-mesh electron microscopy specimen grids for 2 min, and excess buffer was blotted with filter paper (this and subsequent steps were done at room temperature in a humidity chamber to minimize evaporation from grids during incubations). Specimen grids were incubated for 15 min with 10 µl of antipeptide antibody diluted 1:100 to 1:50 with 1% BSA-PBS. After blotting excess buffer, the grids were washed for 2 min with 1% BSA-PBS and incubated for 20 min with protein A adsorbed to colloidal gold (20-nm diameter) (Polyscience, Inc., Warrington, PA) diluted 1:1 with 1% BSA-PBS. The grids were partially blotted, washed for 1 min (with 10 mM Tris-HCl, 1 mM sodium EDTA, pH 7), negatively stained for 1.5 min with 2% ammonium molybdate(31) , thoroughly blotted, and air dried. Electron micrographs were recorded from the specimens on SO163 or 4489 film (Eastman Kodak Co.) using an EM420-T or EM301 electron microscope (Philips Electron Instruments, West Nyack, NY) operated at 80 or 100 kV. To quantify binding of colloidal gold to mitochondria in these fields, numerous micrographs were taken at random and printed at equivalent enlargement. Counts were made visually of gold particles on (or within one particle diameter of) mitochondria and of those not on mitochondria. A digitizing pad (model GP6-040, Science Accessories Corp., Stratford, CT) was used to determine the total area occupied by mitochondria in the fields, allowing calculation of the mean particle densities on and off the membrane crystals for each micrograph.


RESULTS

Binding of the Antipeptide Antibodies to Peptides and to Fungal and Non-fungal VDAC-Peptides corresponding to four different regions in the fungal VDAC polypeptide (residues 1-20, 195-210, 251-268, 272-283) were conjugated to hemocyanin and used to immunize rabbits. For each peptide, antisera were obtained that reacted with the immunizing peptide, labeled a single strong band in Western blots of fungal mitochondria at the M expected for VDAC, and exhibited binding to immobilized lysed mitochondria that was specifically inhibited by the immunizing peptide (see ``Experimental Procedures''). By these criteria, it was concluded that each antiserum is specific for the segment in the VDAC polypeptide corresponding to the respective immunizing peptide.

Western blot experiments were used to check the reactivity of the antipeptide antibodies with VDAC from other species. The results of these experiments are summarized in . None of the antibodies reacted with proteins from rat-heart mitochondria or from yeast cells expressing human VDAC. In contrast, three of the four antipeptide antibodies labeled a protein at M 31,000 (presumably VDAC) in wild-type yeast. There appeared to be a general correlation between the intensity of labeling of the yeast VDAC band by an antipeptide antibody and the percent amino acid identity between the immunizing fungal VDAC peptide and the corresponding region in the nonfungal polypeptide. Cross-reactivity of the antibodies with nonfungal VDAC was detectable only when the amino acid identity between the fungal and nonfungal subsequences was above about 40%.

Accessibility of Epitopes on the Mitochondrial Outer Membrane

To determine whether epitopes are exposed on the outer or inner face of the outer mitochondrial membrane, reactivities of antipeptide antibodies toward mitochondria with predominantly intact and lysed outer membranes were compared by both a back-titration ELISA and immunoelectron microscopy. For both kinds of experiments, relative intactness of outer membranes was determined by the cytochrome c accessibility assay described under ``Experimental Procedures.''

Varying concentrations of mitochondria (predominantly intact or lysed) were incubated with a constant dilution of antibody. Mitochondria were pelleted, and the supernatants were assayed for unbound antibody by an ELISA using plates coated with lysed mitochondria. The depletion of antibody from each antiserum as a function of mitochondrial protein concentration is shown in Fig. 3, typical of results obtained several times for each antibody. Three of the antibodies, anti(1-20), anti(195-210), and anti(272-283), are bound much more effectively by mitochondria after lysis of the outer membranes, i.e. depletion of antibody from the supernatant occurs at considerably lower mitochondrial protein concentrations. Furthermore, when the data for binding of these antibodies by non-lysed mitochondria are replotted after multiplying the mitochondrial protein concentration by the fraction of mitochondria with broken outer membranes, the data points fall on the curve for fully lysed mitochondria (Fig. 3, A, B, and D). This result strongly argues that binding of these three antibodies directly correlates with outer membrane lysis and not with some other possible effect of hypoosmotic treatment. The fourth antibody, anti(251-268), behaves differently; its depletion curves for lysed and non-lysed mitochondria (Fig. 3C) are approximately parallel and level off at low mitochondrial concentration. The simplest interpretation of these results is that the three antibodies whose binding at low mitochondrial concentrations increases markedly with outer membrane lysis are recognizing VDAC epitopes located predominantly on the inner membrane-facing surface of the outer membrane. The absence of a similar effect with anti(251-268) suggests that the majority of its epitopes are located on the external surface of the outer membrane.


Figure 3: Effect of mitochondrial outer membrane lysis on binding of antipeptide antibodies. Mitochondria with 75% intact (, solidline) and fully lysed (, brokenline) outer membranes were incubated in suspension at the indicated protein concentrations with antipeptide antibodies at the following dilutions: A, anti(1-20), 1:6000; B, anti(195-210), 1:3000; C, anti(251-268), 1:2000; D, anti(272-283), 1:10,000. Absorbance is proportional to the antibody remaining in the supernatants after pelleting the mitochondria. The data for the 75% intact mitochondria are also plotted () after multiplying the mitochondrial protein concentration by the fraction of broken outer membranes (0.25).



Indirect immunoelectron microscopy also was used to assess the disposition of the epitope(s) for one of the antibodies, anti(1-20). The results, summarized in I, agree well with the results described above for the back-titration ELISAs. When lysed and non-lysed fungal mitochondria are incubated sequentially with antibody and protein A gold, the lysed mitochondria are labeled with a significantly greater density of gold particles, suggesting increased accessibility of the epitope(s) of anti(1-20).

Effect of Channel Modulators on Binding of Antipeptide Antibodies

Several agents have been identified that alter the voltage-gating characteristics of VDAC. Experiments were undertaken to determine whether incubation of fungal mitochondria (immobilized on microtiter plates after outer membrane lysis) with these effectors alters the binding of antipeptide antibodies. Two of the effectors used were aluminum (added as AlCl), which inhibits voltage-dependent closure of VDAC(32) , and NADH, which increases the channel's voltage sensitivity(33) . When incubated with mitochondria at concentrations that affect VDAC gating, neither chemical was found to have a statistically significant effect on antibody binding (results not shown). Several large polyanions have been found to decrease VDAC gating potential(34) , one of which is a 10-kDa copolymer of maleate, styrene, and methacrylate that also inhibits mitochondrial uptake of adenine nucleotides(35) . Micromolar levels of this modulator polyanion were found to inhibit binding of three of the four antipeptide antibodies to lysed fungal mitochondria in a concentration-dependent fashion, with greatest inhibition observed for anti(251-268) (Fig. 4).


Figure 4: Effect of the modulator polyanion at concentrations of 1 µM (open bars) and 5 µM (solid bars) on binding of antibodies (diluted 1:1000) to lysed mitochondria. Values are expressed as the mean after subtraction of background (i.e. binding of preimmune serum) and normalization of controls (no polyanion) to 1.0, with the standard error of each determination (made in triplicate) indicated.




DISCUSSION

The results of this study (summarized in ) indicate that peptide-specific antibodies have considerable utility as probes of the topography of the VDAC channel in the mitochondrial outer membrane. Three of the four antibodies display a latency of binding to fungal mitochondria that correlates directly with outer membrane lysis, indicating that their epitopes are exposed at the interior surface of the outer membrane. One of these antibodies is anti(1-20), in apparent contradiction with a previous report (15) that lysis of beef-heart mitochondria does not increase binding of an antibody against the amino-terminal of VDAC. There are at least two possible explanations for the discrepancy between the current and earlier studies. One is that the amino-terminal segment of VDAC might cross the outer membrane and that the antibodies used in the two studies recognize epitopes at opposite ends of the peptide. Another possibility is suggested by our observation that salt-containing buffers like those used in the previous study cause mitochondrial outer membranes to rupture over long periods of time (greater than 1 h). This loss of mitochondrial integrity was prevented in the back-titration experiments of Fig. 3by including hyperosmotic concentrations of sucrose in the buffer. Since mitochondrial outer membrane integrity was not monitored in the earlier beef-heart study, it is possible that outer membranes of what were considered to be intact mitochondria lysed during the experiments, exposing internal epitopes to the antibodies.

It is unknown which subregions of the VDAC peptides serve as epitopes for the respective antibodies. However, in the case of anti(1-20), clues about a likely recognition site are provided by cross-reactivity of the antibody with yeast and human VDAC (see ). This antibody reacts well with yeast VDAC, yet the immunizing peptide has significant homology with the yeast sequence only in the segment 14-20, making this 7-residue-long region a likely linear epitope. The sequence of human VDAC, which is not recognized by anti(1-20), agrees with fungal VDAC at only two residues in the same region. That segment 14-20 may be an epitope for anti(1-20) is consistent with the finding (43) that this antibody reacts with a yeast VDAC construct missing residues 1-8.

As noted in the Introduction, existing structural models for VDAC are based on a -barrel motif, although the postulated numbers and identities of the transmembrane -strands vary considerably(12, 13, 15, 16, 44) . The ability of anti(272-283) to bind to mitochondria is unexpected, since its immunizing peptide almost coincides with a segment that is a consensus candidate for a transmembrane -strand, 271-281 (), and so should be inaccessible. A similar situation exists with anti(251-268) since segment 253-263 is another consensus -strand candidate, although residues 264-268 might comprise an accessible linear epitope at one membrane surface.

The simplest interpretation of the reactivities of anti(272-283) and, to a lesser extent, of anti(251-268) with membrane-bound VDAC is that the models are wrong and these are not transmembrane -strands. However, there is another possible explanation, namely that some -strands may not always be in a transmembrane configuration. For example, electrophysiological studies on mutant yeast VDACs (36) suggest that several transmembrane -strands in the carboxyl-terminal region may leave the lumen during partial closure of the channel. Thus, the ability of anti(272-283) and anti(251-268) to bind to isolated mitochondria could indicate that some or all of the VDAC channels are in the partially closed state. The free energy difference between the open and partially closed states of VDAC is so small (about one-third that of a hydrogen bond) that the states have been referred to as ``quasi-degenerate''(37) . Therefore, it is possible that VDAC in the mitochondrial outer membrane in vitro (and perhaps in vivo) is in dynamic equilibrium between two (or more) different functional states and that antibodies may react with epitopes that are transiently exposed in any of the states. Of course, the existence of a dynamic population of mixed channel conformers complicates the interpretation of experiments like those presented in this paper. For example, the two hypotheses for the location of the amino-terminal -helix (i.e. forming part of the lumen or extending away from it) cannot be distinguished by the binding of anti(1-20) to mitochondria, since this part of VDAC (like the carboxyl-terminal region) also may leave the lumen wall upon closure(36) . This situation may be simplified experimentally by working with systems in which VDAC is ``locked'' in a particular conformation (e.g. two-dimensional crystals and voltage-clamped bilayers).

The fact that aluminum and NADH do not change the extent of binding of any antibody to lysed mitochondria indicates that, under the conditions of these experiments, these channel effectors do not lock the channels into states with altered epitope accessibility. In contrast, the modulator polyanion selectively inhibits binding of several antibodies to lysed mitochondria. Since the polyanion favors the partially closed state of VDAC, this inhibition may reflect reduced epitope accessibility in this state. Alternatively, since the polyanion is large (M 10,000), its inhibition of antibody binding may be steric, reflecting proximity of its binding site(s) to the corresponding epitopes. A likely example of steric blocking of antibody binding to VDAC is observed in experiments with mitochondria preincubated with cytochrome c (results not shown)(45) . Electron microscopic results indicate that this protein binds to two-dimensional crystals of VDAC in a region occupied by the amino-terminal -helix(18, 38) . Binding of anti(1-20) to mitochondria is inhibited about 50% by preincubation of mitochondria with 0.17 mM cytochrome c, while binding of anti(272-283) is unaffected. In the case of the modulator polyanion, the strongest inhibition occurs with anti(251-268), whose epitopes are exposed on the external surface of the outer membrane. Binding of the polyanion to the outer surface of mitochondria is consistent with this macromolecule's inhibition of adenine nucleotide uptake by intact mitochondria in vitro(35) .

  
Table: Relative titer of antisera for the respective immunized peptide


  
Table: Cross-reactivity of antipeptide antibodies with VDAC of human and yeast mitochondria


  
Table: Binding of antibodies against VDAC peptide (1-20) to Neurospora mitochondria as a function of outer membrane integrity in immuno-gold electron microscopy experiments

Experiment 1 was done with antibody against glutaraldehyde-coupled peptide-hemocyanin conjugate, and experiments 2 and 3 were done with antibody against EDAC-coupled conjugate. R, ratio of the density (number per unit area) of protein-A-labeled colloidal gold particles on mitochondria (D) to the particle density in the background (D). , standard deviation. P, probability of the result (R/R > 1) occurring by chance according to the t test. SE, standard error of the difference.


  
Table: Summary of binding of antipeptide antibodies to mitochondrially bound VDAC



FOOTNOTES

*
This research was supported by Grant MCB-9219353 from the National Science Foundation. 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: Wadsworth Center, Empire State Plaza, Box 509, Albany, NY 12201-0509. Tel.: 518-474-2462; Fax: 518-486-4901; E-mail: carmen@tethys.ph.albany.edu.

The abbreviations used are: VDAC, voltage-dependent anion-selective channel; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay.


ACKNOWLEDGEMENTS

We thank Dr. Michael Forte (Oregon Health Sciences University) for the yeast strains used in Western blotting experiments, Dr. Svetlana Konstantinova (currently at Moscow State University) for samples of rat-heart mitochondria used for Western blotting, and Drs. Marco Colombini (University of Maryland, College Park) and Tamas König (Semmelweis University, Budapest) for a sample of the modulator polyanion.


REFERENCES
  1. Colombini, M. (1979) Nature279, 643-645 [Medline] [Order article via Infotrieve]
  2. Benz, R. (1985) CRC Crit. Rev. Biochem.19, 145-190 [Medline] [Order article via Infotrieve]
  3. Wunder, U. R., and Colombini, M. (1991) J. Membr. Biol.123, 83-91 [Medline] [Order article via Infotrieve]
  4. Colombini, M. (1994) in Current Topics in Membranes, pp. 73-101, Academic Press, New York
  5. Schein, S. J., Colombini, M., and Finkelstein, A. (1976) J. Membr. Biol.30, 99-120 [Medline] [Order article via Infotrieve]
  6. Jap, B. K., Downing, K., and Walian, P. J. (1990) J. Struct. Biol.103, 57-63 [Medline] [Order article via Infotrieve]
  7. Weiss, M. S., Wacker, T., Weckesser, J., Welte, W., and Schulz, G. E. (1990) FEBS Lett.267, 268-272 [CrossRef][Medline] [Order article via Infotrieve]
  8. Paupitt, R. A., Schirmer, T., Jansonius, J. N., Rosenbusch, J. P., Parker, M. W., Tucker, A. D., Tsernoglou, D., Weiss, M. S., and Schulz, G. E. (1991) J. Struct. Biol.107, 136-145 [Medline] [Order article via Infotrieve]
  9. Schirmer, T., Keller, T. A., Wang, Y.-F., and Rosenbusch, J. P. (1995) Science267, 512-514 [Medline] [Order article via Infotrieve]
  10. Mannella, C. A., Guo, X. W., and Cognon, B. (1989) FEBS Lett.253, 231-234 [CrossRef]
  11. Mannella, C. A., Forte, M., and Colombini, M. (1992) J. Bioenerg. Biomembr.24, 7-19 [Medline] [Order article via Infotrieve]
  12. Forte, M., Guy, H. R., and Mannella, C. A. (1987) J. Bioenerg. Biomembr.19, 341-350 [Medline] [Order article via Infotrieve]
  13. Blachly-Dyson, E., Peng, S. Z., Colombini, M., and Forte, M. (1990) Science247, 1233-1236 [Medline] [Order article via Infotrieve]
  14. Mannella, C. A. (1990) Experientia46, 137-145 [Medline] [Order article via Infotrieve]
  15. De Pinto, V., Prezioso, G., Thinnes, F., Link, T. A., and Palmieri, F. (1991) Biochemistry30, 10191-10200 [Medline] [Order article via Infotrieve]
  16. Rauch, G., and Moran, O. (1994) Biochem. Biophys. Res. Commun.200, 908-915 [CrossRef][Medline] [Order article via Infotrieve]
  17. Kleene, R., Pfanner, N., Pfaller, R., Link, T., Sebald, W., Neupert, W., and Tropschug, M. (1987) EMBO J.6, 2627-2633 [Abstract]
  18. Guo, X. W., Smith, P. R., Cognon, B., D'Arcangelis, D., Dolginova, E., and Mannella, C. A. (1995) J. Struct. Biol.114, 41-59 [CrossRef][Medline] [Order article via Infotrieve]
  19. Mannella, C. A. (1982) J. Cell Biol.94, 680-687 [Abstract/Free Full Text]
  20. Douce, R., Mannella, C. A., and Bonner, W. D., Jr. (1973) Biochim. Biophys. Acta292, 105-116 [Medline] [Order article via Infotrieve]
  21. Mannella, C. A., and Bonner, W. D. (1975) Biochim. Biophys. Acta413, 213-225 [Medline] [Order article via Infotrieve]
  22. Wood, J. N. (1984) in Methods in Molecular Biology: Proteins (Walker, J. M., ed) Vol. 1, pp. 264-266, Humana Press, Clifton, NJ
  23. Weiner, R. S., Andersen, T. T., and Dias, J. A. (1990) Endocrinology127, 573-579 [Abstract]
  24. Laemmli, U. K. (1970) Nature227, 680-685 [Medline] [Order article via Infotrieve]
  25. Towbin, H., and Gordon, J. (1984) J. Immunol. Methods72, 313-340 [CrossRef][Medline] [Order article via Infotrieve]
  26. Reid, J., Fung, H., Gehring, K., Klebba, P. E., and Nikaido, H. (1988) J. Biol. Chem.263, 7753-7759 [Abstract/Free Full Text]
  27. Holzenburg, A., Engel, A., Kessler, R., Manz, H. J., Lustig, A., and Aebi, U. (1989) Biochemistry28, 4187-4193 [Medline] [Order article via Infotrieve]
  28. Bowman, C. L., and Tedeschi, H. (1984) Biochim. Biophys. Acta731, 261-266
  29. Blachly-Dyson, E., Zambronicz, E. B., Yu, W. H., Adams, V., McCabe, E. R. B., Adelman, J., Colombini, M., and Forte, M. (1993) J. Biol. Chem.268, 1835-1841 [Abstract/Free Full Text]
  30. Yaffe, M. P., and Schatz, G. (1984) Proc. Natl. Acad. Sci. U. S. A.81, 4819-4823 [Abstract]
  31. Munn, E. A. (1968) J. Ultrastruct. Res.25, 362-380 [Medline] [Order article via Infotrieve]
  32. Dill, E. T., Holden, M. J., and Colombini, M. (1987) J. Membr. Biol.99, 187-196 [Medline] [Order article via Infotrieve]
  33. Zizi, M., Forte, M., Blachly-Dyson, E., and Colombini, M. (1994) J. Biol. Chem.269, 1614-1616 [Abstract/Free Full Text]
  34. Mangan, P. S., and Colombini, M. (1987) Proc. Natl. Acad. Sci. U. S. A.84, 4896-4900 [Abstract]
  35. Benz, R., Wojtczak, L., Bosch, W., and Brdiczka, D. (1988) FEBS Lett.231, 75-80 [CrossRef][Medline] [Order article via Infotrieve]
  36. Peng, S., Blachly-Dyson, E., Forte, M., and Colombini, M. (1992) Biophys. J.62, 123-135 [Abstract]
  37. Colombini, M. (1989) J. Membr. Biol.111, 103-111 [Medline] [Order article via Infotrieve]
  38. Mannella, C. A., Ribeiro, A. J., and Frank, J. (1987) Biophys. J.51, 221-226 [Abstract]
  39. Mihara, K., and Sato, R. (1985) EMBO J.4, 769-774 [Abstract]
  40. Kayser, H., Kratzin, H. D., Thinnes, F. P., Gotz, H., Schmidt, W. E., Eckart, K., and Hilschmann, N. (1989) Biol. Chem. Hoppe-Seyler370, 1265-1278 [Medline] [Order article via Infotrieve]
  41. Shao, L., Van Roey, P., Kinnally, K. W., and Mannella, C. A. (1994) Biophys. J.66, 21 (abstr.)
  42. Shao, L. (1994) Biophysical Studies of the Conformational and Functional States of the Mitochondrial Voltage-dependent Anion-selective Channel, Ph.D. thesis, The University at Albany, SUNY
  43. Koppel, D., Masters, P., Shao, L., and Mannella, C. A. (1995) Biophys. J.68, 145 (abstr.)
  44. Mannella, C. A., Dolginova, E., Stanley, S., D'Arcangelis, D., Lawrence, C. E., and Neuwald, A. F. (1995) Biophys. J.68, 145 (abstr.)
  45. Stanley, S. (1994) Immunological-Topographical Analysis of the Mitochondrial Voltage-dependent Anion-selective Channel, M.S. thesis, The University at Albany, SUNY

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.