From the
The voltage-dependent anion-selective channel (VDAC) in
mitochondrial outer membranes is formed by a polypeptide (M
The mitochondrial outer membrane contains numerous copies of a
polypeptide (M
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.
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).
Immunoglobulin (Ig) fractions
were prepared from sera by precipitation with 50% ammonium sulfate,
resuspended in PBS
Western blots of fungal
mitochondrial proteins probed with the antisera used in subsequent
experiments contained a single strong band at M
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
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
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
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.
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
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
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
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
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
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.
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).
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.
, 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).
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).
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.
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.
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.''
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.
-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.
-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).
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
) 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
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.