JOURNAL

OF STRUCTURAL

Structure

BIOLOGY

108, 227-237 (1992)

of Paracrystalline Arrays on Outer Membranes Rat-Liver and Rat-Heart Mitochondria C.A. MANNELLA,"+

Wadsworth Center York 12201-0509;

for Laboratories *Department

A. RIBEIRO,~ B. COGNON, AND

D. D'ARCANGELIS

and Research, New York State Department of Health, Empire State of Biomedical Sciences. School of Public Health, and iDepartment 1Jniversit.v of New York at Albany, Albany, New York 12203 Received

November

20. 1991.

and

m revised

form

of

February

Plaza,

Box

of Biologwal

509, Albany, New Sctences. State

19, 1992

liver and rat-heart mitochondria. A model for the basic organization of the arrays is presented, as inferred from correlation averaging of side views of tubular arrays and top views of planar arrays. The fundamental motif is one of parallel rows of projecting bilobed subunits. The phosphotungstate-induced arrays are compared with those observed on surfaces of other mitochondria and with those formed by iontransport ATPases in other kinds of membranes. Also described in this report are first experiments aimed at determining possible functional components of the arrays. There is no evidence of specific decoration of the phosphotungstate-treated mitochondria with hexokinase-labeled colloidal gold, suggesting the absence of the channel protein, VDAC, which is the mitochondrial binding site for hexokinase.

Crystalline arrays are induced in outer membranes of rat-liver and rat-heart mitochondria by phosphotungstate and silicotungstate. The basic structure of the arrays has been determined by correlation averaging of electron microscopic images of side views of tubular arrays and en face views of planar arrays. The arrays consist of rows of bilobed projecting subunits and are similar (in lattice parameters and projected subunit dimensions) to periodic arrays of ion transport ATPases, e.g., arrays of Ca” ATPase induced by vanadate in sarcoplasmic reticulum. Hexokinase-labeled colloidal gold particles do not specifically decorate the arrays, suggesting that the hexokinase receptor (VDAC channel) is not a component of the arrays. t” 1992 Academic Press. Inc. ___..

INTRODUCTION Parsons (1963) was the first to report that the negative stain phosphotungstate induces the formation of paracrystalline arrays of projecting subunits in outer membranes of rat-liver mitochondria. The nature of these paracrystals has remained a mystery, although they resemble structures subsequently reported to occur on the surfaces of mitochondria from other tissues in the absence of phosphotungstate (see Discussion). Apparently, binding of this metalcontaining complex anion to the mitochondrial outer membrane induces one or more of the components of this membrane to laterally segregate and cryst,allize, a process that has been observed in other membrane systems, e.g., the vanadate-induced crystallization of Ca”’ -ATPase in sarcoplasmic reticulum (Taylor et al., 19841. Because of increasing interest in the structural and functional components of the mitochondrial outer membrane, we have set out to characterize the phosphotungstate-induced paracrystalline arrays. In the present report, we describe the formation of arrays observed using phosphotungstate and the related complex anion, silicotungstate, with both rat-

MATERIALS ANDMETHODS Inductton of paracrystalline arrays on mitochondrial outer Mitochondria were isolated from livers and hearts membranes of Wistar rats by procedures described in detail elsewhere (Mannella and Parsons, 1977; Tyler and Gonze, 1967). Final pellets of both types of mitochondria were resuspended in MSE buffer 10.225 M mannitol, 0.075 M sucrose, 1.0 m&f EDTA, adjusted to pH 7.0 with NaOH) unless otherwise indicated. The procedure used to generate paracrystalline arrays on the outer membranes of rat-liver and rat-heart mitochondria was based on that of Parsons 119631. An aliquot of a concentrated rnitochondrial suspension (30-60 mg protein/m! of MSE buffer) is ddded to 90 vol of potassium or sodium phosphotungstate ~0.5 2%. pH 6.5-7.2) at room temperature. After a short time (lo-30 min, see below), 10 p.1 of the suspension is deposited and air-dried on a freshly glow-discharged, carboniformvar-coated specimen grid, which is subsequently examined by electron microscopy. It was found that the paracrystalline arrays could be induced in mitochondria up to 2 days after isolation, although the data presented in this report were generated using mitochondria within a few hours of their isolation. Hexokinase binding to mitochondria. Purified rat-brain hexokinase (Polakis and Wilson, 19821 was generously provided as a lyophilized powder by John E. Wilson (Michigan State University). For experiments involving direct binding of the enzyme to mitochondria, the enzyme was reconstituted in medium containing 0.1 M glucose, 10 m&f thioglycerol, 0.5 n&f sodium phosphate 227 104723477’92 $5 00 ( opyright L 1992 by Academic Press, Inc. Ali righti: of reproduction in any form resenzed

226

MANNELLA

(pH 7.0). For use as an electron microscopic probe, hexokinase was adsorbed to colloidal gold particles of diameter 15-30 nm, made by reduction of a 0.01% solution of chloroauric acid with citrate (Geoghegan and Ackerman, 1977). One milliliter of the gold suspension (prebuffered to pH 7.0 with 5 m&f Tris-HCl) was mixed with 20-30 pg hexokinase (approx. 1.5 units, see below), using a stock solution of 0.125 mg protein/ml of 5 mM TrisHCl, pH 7.0. The amount of hexokinase was determined for each colloidal gold fraction as twice the amount required to stabilize the gold against salt-induced aggregation, at pH 7.0, which is just above the p1 of the rat-brain protein, 6.35-6.45 (Polakis and Wilson, 1985). Prebuffering of the gold suspension was found to be necessary for formation of a stable hexokinase-gold complex. The protein-gold suspension was centrifuged (15 6OOg, 30 min in an Eppendorf Model 54141 and the deep red pellet was resuspended in 0.25 ml of a solution containing 5 m&f Tris-HCl (pH 7.0) and 0.1% polyethylene glycol (M, 20 000). (Final gold concentration in these colloidal gold suspensions was 0.04%.) Colloidal gold suspensions also were prepared that were stabilized with only polyethylene glycol (M, 20 000, 0.2%, pH 7.0) for use in control experiments. For experiments involving mitochondrial binding of hexokinase (native or bound to colloidal gold), the final mitochondrial wash and resuspension was done with hexokinase binding medium (HB medium, 0.25 M sucrose, 10 n-&f glucose, 5 m&f KCl, and 20 mM Hepes, pH 7.51, generally containing 1 m&f phenylmethylsulfonyl fluoride. To quantitate the extent of binding of rat-brain hexokinase to mitochondria, aliquots of the enzyme were incubated for 30 min at room temperature with mitochondria (0.5 mg protein) in 0.5 ml of HB medium containing 4 n-&f MgClz. The suspension was centrifuged (15 6OOg, 10 min, 4”C), and the mitochondrial pellet was washed and resuspended with 1.0 ml HB medium. The mitochondria were then repelleted and resuspended at room temperature in a final volume of 2.7 ml of hexokinase assay buffer (2.75 nu’l4 glucose, 16.5 m&f MgCl,, 1.0 mhf dithiothreitol, 0.5 mM sodium cyanide, 13 kg/ml oligomycin, 27.5 mM Hepes, pH 8.5). Hexokinase activity was measured according to Rose and Warms (1967) in terms of the oxidation of product, glucose-g-phosphate, upon addition of 1.0 mM NADP, 2 units glucose-g-phosphate dehydrogenase, and 5.0 n&f ATP to the mitochondrial suspension. Formation of NADPH in the suspension was measured with a DW-2 UV-Vis spectrophotometer (American Instrument Corp., Urbana, IL), using an extinction coefficient of 6.22 n-&-i cm-i. (One unit of hexokinase is defined as that which produces 1 (*mole glucose-6-phosphate per min at 25°C.) Binding of hexokinase to the mitochondria was found to be reversed (generally 60-70%) by addition to the incubation medium of either 3 n&f ATP or 5 m&f glucose-6-phosphate, as expected (Rose and Warms, 1967). For electron microscopic localization of hexokinase binding, mitochondria were mixed with hexokinase-labeled (or polyethylene glycol-labeled) colloidal gold during treatment with phosphotungstate to induce formation of outer-membrane arrays. In the experiment illustrated by Fig. 8A, rat-liver mitochondria (final concentration l-l.5 mg protein/ml) were first incubated in 1% potassium phosphotungstate (10 min, room temperature) containing 3 m&f MgCl,, after which 0.1 vol of 20x HB-S medium (HB medium without sucrose and containing 3 mM MgCl,) was added, followed by 1 vol of hexokinase-gold (0.02% final gold concentration). After an additional 10 min, lo-p1 aliquots of this suspension were deposited on specimen grids, partially blotted, washed with a buffer (10 mM Hepes, pH 7.5, 10 mM MgCl,) containing 1% glutaraldehyde (EM grade, Polysciences, Inc., Warrington, PA), blotted, air-dried, and directly examined in the electron microscope. In the experiment illustrated by Figs. 8B and 8C, similar sequential incubations of rat-liver mitochondria in potassium phosphotungstate and hexokinase-gold were employed. After 20 min, an equal volume of HB-S medium containing 3% glutaraldehyde was added to the suspension and, after 20

ET AL. min at room temperature, the mitochondrial suspension was centrifuged (15 6OOg, 20 min, 4°C). The mitochondrial pellets were subsequently postfixed with 1.3% osmium tetroxide in Millonig’s phosphate buffer (Dawes, 1979) for 1 hr at room temperature dehydrated with a graded ethanol series, and embedded in Epon (Luft, 1961). Pellets were washed extensively with 0.06 M sodium phosphate buffer (pH 7.0) after fixation and osmification to minimize formation of an electron-dense precipitate that occurred on membranes pretreated with phosphotungstate. Thin sections were cut using an LKB 8800 ultramicrotome (LKB Instruments, Inc., Washington, DC) and stained with uranyl’lead (Dawes, 1979). Electron microscopy and image processing, Electron micrographs were recorded from both negatively stained and thinsectioned mitochondrial specimens on Kodak 4489 and SO163 films (Eastman-Kodak, Rochester, NY) using Philips EM300 and EM420-T electron microscopes (Philips Electronics, Mahwah, NJ) operated at 100 kV. Membrane images were screened for crystallinity by optical diffraction from the negatives, using coherent radiation from a He-Ne laser (Jodon Engineering Associates, Ann Arbor, MI). Selected images of crystalline membrane arrays were digitized with a PDS 1OlOA flatbed scanning microdensitometer (Perkin-Elmer, Garden Grove, CA). Averages of images of negativey stained mitochondrial arrays were computed using the SPIDER image processing system (Frank et al., 1981) implemented on a VAX 11/780 computer or MicroVAX 3100 and 3500 workstations (Digital Equipment Corp., Maynard, MA). Side views of array subunits were averaged using a correlation alignment procedure (Frank, 1981). Projecting particles were selected from straight edges of flattened tubular arrays, translationally aligned by cross-correlation with a well-defined reference particle, and summed. Averages of planar crystal fields were obtained by a procedure (Mannella et al., 1986) involving quasi-optical Fourier filtration (Goldfarb et al., 1979) to obtain a preliminary average, which is subsequently used as a reference for correlation analysis (Saxton, 1980; Frank, 1982; Kessel et al., 1984). RESULTS

Formation of Paracrystalline Arrays Membranes of Rat-Liver and Rat-Heart Mitochondria

in the Outer

Following procedures based on those described by Parsons (1963), it was found that distinctive paracrystalline arrays of projecting subunits form on the outer membranes of mitochondria isolated from rat liver after incubation of mitochondria in potassium or sodium phosphotungstate (see Materials and Methods.) The same type of arrays also were observed under these conditions using rat-heart mitochondria. The general appearance of rat-liver mitochondria after incubation in phosphotungstate is illustrated in Fig. 1. The inner-membrane compartments may display varying degrees of contraction or “tubularThe outer membrane is usually pulled ization.” away from the inner membrane, and it is in these separated, distended regions that the distinctive outer-membrane arrays are found. Typically, the arrays appear as corset-like constrictions, 90-140 nm wide and up to 700 nm in length, connecting two regions of protruding membrane. The paracrystalline substructure of the arrays on these distended membranes is visually apparent in such images, and

MITOCHONDRIAL

1. pot,assium

Electron micrograph phosphotungstate.

of linear

paracrystalline

OUTER-MEMBRANE

arrays

can be readily confirmed by optical diffraction from the negatives. (A computed diffraction pattern from a typical array is included in Fig. 2.) Images of pelleted phosphotungstate-treated mitochondria that have been embedded in plastic indicate that the outer-membrane arrays are actually cylinders or tubes with circular cross section and diameters of 60-80 nm (see Figs. 8B and 8C below). The greater width of the arrays in negative stain (90-140 nm) is consistent with flattening of the tubes during drying on the grids. Formation of paracrystalline arrays on the mitochondrial outer membrane was found to be a function of phosphotungstate concentration and time of incubation. Arrays were consistently observed in mitochondrial suspensions incubated for 10-15 min in 2% phosphotungstate, at an average frequency of about l-3 arrays per mitochondrion. Array formation was less consistent and generally required increased incubation times (20-30 min) with 1% phosphotungstate. Arrays could usually be found at low frequency after 30-60 min incubation of mitochondrial preparations in phosphotungstate at concentrations as low as 0.5%, but not with 0.2% solutions.

(arrows)

induced

229

ARRAYS

in outer

membranes

of rat-liver

mitochondria

by 2%

Paracrystalline outer-membrane arrays were not observed when rat-liver mitochondria were incubated with the negative stains uranyl acetate (1 or 2%, pH 4.31, uranyl oxalate (l%, pH 7.0), or ammonium molybdate (1 or 2%, pH 7.0). Similarly, incubation of mitochondria with either sodium phosphate (50 mM, pH 7.0, counterstained with molybdate), ATP (5 m&f, pH 7.0, also counterstained with molybdate), or sodium tungstate (l%, pH 7.0) did not result in detectable array formation. However, the outer-membrane arrays were induced by 2% sodium silicotungstate (pH 7.01, a negative stain closely related to phosphotungstate (Mergner et al., 1972). An important characteristic of the arrays formed in silicotungstate was that they were sometimes planar instead of cylindrical or tube-shaped. This proved to be useful for subsequent computer-averaging procedures (as described below). The 2% phospho- and silicotungstate solution that were most effective at inducing formation of mitochondrial outer-membrane arrays are considerably hypotonic, 50-58 mOsm (Mergner et al., 1972). It was found that inclusion of 0.25 M sucrose in incubation medium containing 2% phosphotungstate

230

MANNELLA

ET

AL.

l/6 nm

l/4.3 FIG. 2. Paracrystalline nature of phosphotungstate-induced mitochondrial array in a distended region of an outer membrane of a rat-liver mitochondrion. the Fourier transform) from the region of the membrane indicated by arrows in text) are indicated.

greatly inhibited formation of the tubular arrays on rat-liver mitochondria. This suggests that rupture of the outer membrane may be a prerequisite for formation of this type of array. This might simply be due to the fact that formation of tubular arrays involves “pinching off’ of significant areas of the outer mitochondrial membrane, which is not possible if the membrane is intact and enclosing the inner membrane. Formation of the mitochondrial outer-membrane arrays appears to be reversible. For example, arrays were not observed if, following deposition on a carbon-coated grid of a mitochondrial suspension in l-2% phosphotungstate, the grid was washed with water prior to counterstaining with uranyl acetate (l%, pH 4.3) or uranyl oxalate (l%, pH 6.8). However, it was found that the arrays could be stabilized after their formation in phosphotungstate by glutaraldehyde fixation. Figure 3 shows an outermembrane array in a mitochondrial suspension that was incubated for 30 min in 1% phosphotungstate, fixed for 2 min (on the grid) with an equal volume of 0.2% glutaraldehyde in 1% phosphotungstate, washed 1 min with water, and counterstained with 1% uranyl acetate. In general, mitochondrial outermembrane arrays observed after fixation and removal of phosphotungstate appeared to be more disordered (partly “unwound”) than those observed in the presence of phosphotungstate. In other experiments, it was found that the arrays could be fixed in suspension (i.e., prior to deposition on carbon-coated specimen grids), indicating that spreading of the membranes on a carbon substrate is not part of the mechanism of array formation. Conversely, prefixation of intact or water-lysed mitochondria in sus-

nm

outer-membrane arrays. (Left) Electron micrograph of an (Right) Computed diffraction pattern (power spectrum of in the image. Spacings of the major reflections (explained

pension before incubation with phosphotungstate was found to inhibit induction of the outer membrane arrays. This is consistent with the arrays forming by lateral segregation of existing components in the membrane plane, which would be inhibited by cross-linking of membrane proteins. Correlation Averaging of the Mitochondrial

of Edge and en Face Views Outer-Membrane Arrays

As noted above, the outer-membrane arrays observed upon incubation of rat-liver and rat-heart mitochondria with phosphotungstate are tubes or cylinders, which undergo varying degrees of flattening in the course of drying in the negative stain. Each array is composed of bands, 8.5 nm wide, aligned nearly normal to the long axis of the tubes (Fig. 2, left). The transverse bands are evident in the par-

FIG. 3. Phosphotungstate-induced outer-membrane array that has been uranyl acetate following glutaraldehyde

rat-liver negatively fixation.

mitochondrial stained with

1%

MITOCHONDRIAL

OUTER-MEMBRANE

tially unraveled tubular array of Fig. 3, suggesting that they are the basic structural unit from which the arrays are assembled. The bands are actually rows of white (stain-excluding) subunits, which can be seen to project from edges of the tubes (Figs. 2 and 4). Sometimes the tubes have a striated appearance (Fig. 41, with alternating white and black stripes running parallel to the long axis of the tubes, suggesting that the subunits on adjacent bands of these arrays are in register. Average side views of the array subunit were formed by aligning and summing particle projections along straight edges in linear array regions. Selection of individual projecting particles was difficult because of their close packing; in fact, discrete particles could not be unambiguously identified in most array edges. (No attempt was made to merge averages from different array edges, which would require classification of large numbers of “edge averages” to sort out variations in particle staining and “rocking.“) One subunit side average is presented in the inset of Fig. 4, showing a particle with a bilobed structure 6 nm wide and extending 5-6 nm from the membrane surface. Typically, there is a slight asymmetry in the size and shape of the two

FIG. 4. Side view of the subunit of phosphotungstate-induced mitochondrial outer-membrane arrays. An electron microscopic image of two paracrystalline arrays (black arrowheads) on a distended piece of the outer membrane of a rat-liver mitochondrion. Unlike the array in Fig. 2, the array near the center of this field has a striated appearance (evident when the array is viewed obliquely down its cylinder axis) suggesting that the subunits of adjacent rows are nearly in register, (Inset) An average (9.5 x 8.5 nm) of six projecting subunits selected from the array edge indicated by the white arrow.

ARRAYS

231

lobes in these averages, with an indication that the ends of the lobes are in contact, or nearly so. The tubular arrays do not display long-range crystalline order. The tubes are often curved or bent, with gaps between groups of close-packed subunit bands (see Fig. 4). Even along reasonably straight array segments, subunits of adjacent bands are not normally in register over significant distances. This paracrystallinity is evident in the diffraction pattern from the array in Fig. 2. The subunit rows or bands in this array are oriented parallel to the vertical direction (J in the image). While the (h,O) reflections, corresponding to the “interband” spacing of 8.5 nm, are sharp, the reflections along the k = (t)l layer lines are horizontally blurred arcs, consistent with stacking disorder between the bands. This situation is further complicated by the fact that the bands in the layers on either side of the collapsed tubes tend to be parallel. Thus, the density contributions from the two sides cannot usually be separated. All of these factors make obtaining average projections of individual layers of the arrays in the collapsed tubes by computer processing techniques a formidable task. Ordered arrays are occasionally observed on flat, nontubular regions of mitochondrial outer membranes, in particular, when the mitochondria have been incubated with silicotungstate (e.g., Fig. 5A). That these planar arrays are related to the tubular arrays is indicated by comparison of diffraction patterns from images of the two types of arrays (Figs. 2 and 5B). The lattice parameters of the planar arrays (a = 8.5 nm, b = 6.0 nm, y = 84 +- 2”) correspond closely to the fundamental spacings in the tubular arrays, i.e., an 8.5-nm interband repeat and a 6-nm repeat along the bands. In addition, profiles of projecting particles on the folded edges of the planar arrays (e.g., Fig. 5A) are indistinguishable from those of the tubular arrays. The appearance of some of these planar arrays (not shown) suggests that they may represent early stages in the formation of the tubular arrays. In any event, diffraction from these mitochondrial outer-membrane arrays indicates that they are frequently better ordered than the tubular arrays. and the two sides in overlapped vesicles are often separable in Fourier space (Fig. 5B). We have been able to obtain en face averages of these two-dimensional crystals in both single- and double-layer regions using Fourier and correlationaveraging procedures. Averages of both sides of the double-layered membrane array of Fig. 5A are presented in Figs. 5C-5F. The array is seen to be composed of rows of close-packed, white (stainexcluding), biconcave or dumbbell-shaped subunits, with a long dimension (normal to the rows) of 5.5-6 nm and a short dimension which varies from 3 nm at

232

MITOCHONDRIAL

OUTER-MEMBRANE

the widest part to 2 nm at the “waist.” There are two regions of stain accumulation in the array. The most heavily stained loci are roughly circular in shape (2-2.5 nm in diameter), situated between adjacent subunits in the rows. There are also “grooves” of stain that run between the rows of the arrays. These grooves are generally less heavily stained than the intersubunit locus, although the staining of the two features is roughly equivalent in the example of Fig. 5. [As noted above, it was not possible to obtain reliable en face averages from the tubular arrays. However, in a few cases where we could almost (but not quite fully) separate the two sides of a tube in Fourier averaging, the results were generally consistent with those obtained with the planar arrays.] Figure 6 is a schematic representation of the three-dimensional organization of the mitochondrial outer-membrane tubular arrays inferred from the side and en face projection averages. Binding of Hexokinase to the Mitochondrial Membrane; Effects of Phosphotungstate

233

I&+/

----_/ --___/ -_-_ /I______

8.5 nm FIG. 6. Schematic model of the organization of the cylindrical arrays induced in rat mitochondrial outer membranes by phosphotungstate. The arrays are represented as stacks of rings composed of bilobed subunits. When flattened, the rings form bands or rows such as those observed in negatively stained specimens. The dumbbell-shaped outline of the stain-excluding subunit in the planar array is interpreted as the axial projection of the two protruding lobes visualized in side views.

Outer

As noted in the Introduction, it is likely that the arrays induced in the mitochondrial outer membrane by phosphotungstate represent a lateral segregation of one or more components of this membrane. Some inferences regarding the functional identity of these components may be drawn from structure alone (see Discussion). However, positive identification of the subunits requires localization of known mitochondrial activities to the specialized outer-membrane regions represented by the arrays. In the present study, we investigated whether the phosphotungstate-induced arrays might be the site of binding of hexokinase to the mitochondrial outer membrane (Rose and Warms, 1967). To this end, experiments were undertaken to determine whether rat-brain hexokinase, previously shown to bind to the outer membranes of rat-liver mitochondria (Polakis and Wilson, 19851, binds specifically to the arrays. The experiments involved incubation of mitochondria with hexokinase (free or adsorbed to colloidal gold) under conditions which favor array formation, followed by electron microscopic examination of negatively stained or thin-sectioned specimens. -

ARRAYS

---.-__

Before undertaking these electron microscopic experiments, the effects of phosphotungstate on hexokinase binding to mitochondria were determined. Typically, rat-brain hexokinase bound to the ratliver mitochondrial fractions used in these experiments at levels of 30-65 mU/mg mitochondrial protein. This degree of binding is slightly lower than that previously reported for binding of tumor hexokinase to normal rat-liver mitochondria, 85 mU/mg protein (Rose and Warms, 1967), and is considerably lower than that of tumor hexokinase binding to tumor mitochondria, 273 mU/mg protein (Bustamante et al., 1981). It was found that phosphotungstate partially inhibits mitochondrial hexokinase binding in the concentration range that induces outermembrane array formation (O&2%). However, as indicated in Fig. 7, the inhibition of hexokinase binding by phosphotungstate could be overcome by increasing the amount of hexokinase added to the mitochondria. In the binding experiments described below, hexokinase concentrations were above those needed to overcome the inhibition of binding by phosphotungstate. In general, there was no indication of specific binding of hexokinase to the phosphotungstate-

___--

FIG. 5. Computer-averaged en fuce projections of planar mitochondrial outer-membrane arrays induced by 2% sodium silicotungstate. 1A) Electron micrograph of part of a rat-liver mitochondrial outer membrane containing a flat crystalline area demarcated by arrows. (B) Diffraction pattern computed for a 75 x 75 nm region near the center of the crystalline patch in A. The Fourier maxima on the reciprocal lattices of the two membrane layers present in the collapsed vesicle in A do not overlap anywhere in the pattern, allowing independent averaging of the two crystalline layers. The (LO) and (O,l) reflections of one of the two lattices are indicated. Note that the register of the two layers differ somewhat; lattice angles of the arrays in C and D are 82” and 86”, respectively. (CD) Quasi-optical Fourier averages and iE,F) correlation averages (with contours added) of the two superimposed arrays in the crystalline patch of A. The rows of white ‘stain-excluding) bilobed subunits are apparent in these averages. The arrowheads in E and F point to sites of heavy stain accumulation situated between adjacent subunits.

MANNELLA

ET AL. DISCUSSION

0

70

HK

140

ADDED

210

(mu)

FIG. 7. Effect of phosphotungstate on binding of rat-brain hexokinase to rat-liver mitochondria. Plots of hexokinase activity recovered in mitochondrial pellets after incubation (20 min, room temperature) of 0.5 ml mitochondrial suspensions with indicated amounts of hexokinase in the presence (circles) and absence (squares) of 1% potassium phosphotungstate,(see Materials and Methods for experimental details).

induced arrays on the outer membranes of rat-liver mitochondria. In experiments in which mitochondria were incubated with free hexokinase prior to negative staining with phosphotungstate, images of outer-membrane arrays were indistinguishable from those of control (no hexokinase added) mitochondria, i.e., there were no new features in the images that might correspond to bound hexokinase (results not shown). In other experiments, mitochondria were incubated with hexokinase adsorbed to colloidal gold particles; incubations were performed either before or after incubation of the mitochondria in phosphotungstate to induce array formation. In both negative-stained (Fig. 8A) and thin-sectioned (Figs. 8B and 8C) specimens, numerous hexokinase-labeled colloidal gold particles could be found on various regions of mitochondrial outer membranes but were rarely found on the tubular arrays. (For comparison, almost no gold particles were found on mitochondrial membranes in control experiments using polyethylene glycol-labeled colloidal gold.) Table 1 summarizes particle counts from two different hexokinase-binding experiments, in which less that 5% of the hexokinase-gold particles fell on or within one particle diameter of the tubular arrays in the fields recorded. While the total area occupied by arrays in these fields was also small (corresponding to a few percent of the total membrane area), particle densities on the arrays never exceeded (in fact, rarely approached) that on other regions of the mitochondrial outer membranes. Thus, there is no indication from these experiments of specific attachment of hexokinase to the phosphotungstate-induced outer mitochondrial membrane arrays.

The ordered arrays induced by phosphotungstate and silicotungstate in the outer membranes of ratliver and rat-heart mitochondria are composed of parallel rows of bilobed projecting subunits. The periodic arrays with best long-range order are the infrequently observed planar crystals induced by silicotungstate. The more common paracrystalline tubular arrays are apparently composed of rings of subunits aligned roughly normal to the long axis of the tubes. The register between adjacent rings is generally poor, although it is possible that this disorder may result from irregular flattening of the tubes during drying in the negative stains. Therefore, imaging experiments with phosphotungstateinduced tubular arrays embedded in vitreous ice will be undertaken to determine whether the native arrays have better inherent order. If so, such cylindrical arrays would be preferred specimens for attempting three-dimensional reconstruction from multiple tilted views (Amos et al., 1982), or from individual projections if the nonflattened arrays should display helical symmetry (DeRosier and Klug, 1968). Numerous examples of linear particle arrays have been observed on surfaces of mitochondria from various tissues and organisms. However, for the most part, the constituent particles are larger and spaced farther apart than the subunits in the phosphotungstate-induced outer-membrane arrays of rat mitochondria. For example, linear arrays of 7-8 nm particles have been observed in freeze-fracture replicas of mitochondrial outer membranes in the central axons of an insect (Smith et al., 1977). The particles are spaced E-20 nm apart along the rows, with 20-25 nm spacings between adjacent parallel rows. Aligned bands of twinned particles have been observed in thin-section electron micrographs on mitochondrial surfaces in the fungus Pythium ultimturn (Bracker and Grove, 1970). The particles are 5-8 nm wide and project 12-18 nm from the membrane surface. The center-to-center spacing between particles in each pair is about 10 nm. Spacings of 15 nm occur between subunit pairs along the rows and 29 nm between adjacent rows. Also, “ladder”-like arrays of particles have been observed on freezeetched surfaces of guinea-pig sperm mitochondria (Friend and Heuser, 1981). The individual particles in the sperm arrays are 4-5 nm in diameter, arranged 3 per “rung” with center-to-center spacings of 6-7 nm between particles in the rungs, 7-8 nm between rungs, and 25 nm between ladders. In contrast, the subunits in the induced rat-liver mitochondrial outer-membrane arrays have lobes that are only 3-4 nm in diameter and project 5-6 nm from the membrane surface, with a center-to-center separation of 4 nm. The bilobed rat-liver mitochondrial subunits are positioned every 6 nm along the

MITOCHONDRIAL

OUTER-MEMBRANE

ARRAYS

235

FIG. 8. Localization of binding of hexokinase-labeled colloidal gold to rat-liver mitochondria pretreated with potassium phosphotungstate. Experimental details are under Materials and Methods. (A) Electron micrograph of a distended outer membrane in a suspension of mitochondria that was deposited on a specimen grid and glutaraldehyde fixed. Note the large number of gold particles on the membrane regions on either side of the tubular array (arrowheads), which itself contains no gold particles. (B) Electron microg-raph of a field in a thin-sectioned mitochondrial pellet from Experiment 1 in Table 1. Tubular arrays are indicated by arrowheads. There are numerous hexokinase-gold particles on mitochondrial peripheries and detached outer membranes in fields such as these. (C) Detail in a micrograph of a field from the same specimen as B containing a tubular array cut normal to its long axis, revealing its circular cross section. ‘Magnification same as B.)

rows and adjacent rows have an 8.5-nm spacing. There is no clear indication of the functional nature of the components of any of the linear arrays previously observed in mitochondrial outer membranes. The phosphotungstate-induced mitochondrial outer-membrane arrays are strikingly similar in overall appearance and geometry to another class of membrane crystals: the “dimer ribbons” of iontransport ATPases observed in sarcoplasmic reticulum (Taylor et al., 1984,1986; Castellani et al., 19851 and plasma membranes (Mohraz et al., 1983; Hebert :,*tal., 1982). For example, vanadate induces the for-

TABLE1 Binding

Experiment” 1 2

of Hexokinase-Labeled to Mitochondrial

Colloidal Outer-Membrane

Total No. of arrays in fields 94 49

a Mitochondria were incubated tungstate and hexokinase-labeled hedded and sectioned as described

Total No. of particles on membranes __~ 1159 339

Gold Particles Arrays

% Particles on arrays 1.5 4.7

sequentially with 1% phosphocolloidal gold, then fixed, emunder Materials and Methods.

mation of cylindrical periodic arrays of Ca2+ATPase on sarcoplasmic reticulum which, although better ordered, are very similar in overall morphology to the arrays induced by phosphotungstate in mitochondrial outer membranes. Arrays of Ca2+ATPase are composed of parallel rows of subunit dimers, each half of the dimer having a maximum projected diameter of 3.5 nm and protruding 6 nm from the membrane surface. The rows are organized into a two-dimensional lattice with one dimer per unit cell of parameters a = 11.4 nm, b = 6.6 nm, y = 78”. (The bilobed particles of the mitochondrial arrays have similar projected dimensions in a somewhat smaller unit cell: a = 8.5 nm, b = 6.0 nm, y = 84”.) Interestingly, the side projections of the subunits of both types of arrays are also similar, with indications in each case that the tops of the two halves of the dimers are in contact. (Compare Fig. 4 above with Fig. 7 in Taylor et al., 1986). In the case of Ca2 + -ATPase, three-dimensional reconstruction of the membrane arrays has shown that the two subunits within the protein dimer are in fact connected by a linear bridge (Taylor et al., 1986; Castellani et al., 1985). Computation of the approximate molecular volume of the surface (protruding) domain of the

236

MANNELLA

subunits in the mitochondrial arrays, based on analogy with projections of the Ca’+-ATPase array for which a three-dimensional volume has been obtained (Taylor et al., 1986) suggests a subunit molecular weight of 80-100 kDa (40-50 kDa per lobe). The similarities between the induced rat-liver mitochondrial outer-membrane arrays and those of cation-transport ATPases raise the possibility that the mitochondrial array subunit may be a transport ATPase of some kind. The existence of an active cation transporter in the mitochondrial outer membrane seems unlikely in light of this membrane’s large passive permeability to small molecules and ions, generally attributed to the high density of VDAC channels present in this membrane (Colombini, 1979). Nonetheless, the possibility that this subunit may be composed of a protein structurally related to ion transport ATPases should be explored. An interesting feature of the en face projection images of the rat-liver mitochondrial outermembrane arrays is the 2-25nm-diameter negative-stain accumulatiion centered between adjacent pairs of bilobed projecting subunits. These densely stained loci are similar to those observed in periodic arrays induced in fungal mitochondrial outer membranes by phospholipase A, (Mannella, 1982; Mannella et al., 1986), which have been shown by threedimensional reconstruction to represent stain-filled transmembrane VDAC channels (Mannella et al., 1984). Three-dimensional reconstruction will be needed to determine whether the dense circular features in the rat-liver arrays represent surface accumulations of negative stain trapped between protruding subunits or stain-filled pores. The failure of hexokinase-labeled colloidal gold to bind to the arrays indicates that the VDAC channel, identified as the mitochondrial receptor for hexokinase (Linden et al., 1982; Fiek et al., 1982), is probably not a component of the arrays. However, the present experiments do not rule out the possibility that the VDAC channel might be present in the arrays in an inaccessible form, i.e., as part of a complex with another protein containing a large protruding domain. One such peripheral protein might be hexokinase itself, which is a 96 OOO-kDa protein with two structurally related domains (Schwab and Wilson, 1989). However, it was found that the ratliver mitochondria used in these experiments have no detectable endogenous hexokinase activity, consistent with the results of others (Rose and Warms, 1967; Polakis and Wilson, 1985; Bustamante et al., 1981). Also, in preliminary immunoelectron microscopic experiments, antibodies to rat-brain hexokinase (gift of J. Wilson, Michigan State University) did not bind to rat-liver mitochondrial paracrystalline arrays. Thus, it is unlikely that the projecting domains of the array subunits correspond to hexokinase.

ET AL. The authors are indebted to Dr. John Wilson (Michigan State University) for generously providing samples of rat-brain hexokinase and antibodies against this protein, and to Drs. Wilson and Joachim Frank (Wadsworth Center for Laboratories and Research) for helpful discussions in the course of this work. This work is supported by Grant DMB-8916315 from the National Science Foundation. REFERENCES Amos, L. A., Henderson, R., and Unwin, N. (1982) Threedimensional structure determination by electron microscopy of two-dimensional crystals, Prog. Biophys. Molec. Biol. 39, 183231. Bracker, C. E., and Grove, S. N. (1970 Surface structure on outer mitochondrial membranes of Pythium ultimum, Cytobiologie 3, 229-239.

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Polakis, P. G., and Wilson, J. E. (1982) Purification of highly bindable rat brain hexokinase by high performance liquid chromatography (HPLC), Biochem. Biophys. Res. Commun. 107, 937-943. Polakis, P. G., and Wilson, J. R. (19851 An intact hydrophobic N-terminal sequence is critical for binding of rat brain hexokinase to mitochondria, Arch. Biochem. Biophys. 236, 328-337. Rose, I. A., and Warms, J. V. B. (1967) Mitochondrial hexokinase: Release, rebinding, and location, J. Bial. Chem. 242, 1635-1645. Saxton, W. 0. (1980) Matching and averaging over fragmented lattices, in Baumeister, W., and Vogell, W. (Eds.), Electron Microscopy at Molecular Dimensions, pp. 244-255, SpringerVerlag, Berlin. Schwab, D. A., and Wilson, J. E. (19891 Complete amino acid sequence of rat brain hexokinase, deduced from the cloned cDNA, and proposed structure of a mammalian hexokinase, PFOC. Natl. Acad. Sci. USA 86, 2563-2567. Smith, D. S., Jarlfors,U., and Cayer, M. L. (1977) Structural cross-bridges between microtubules and mitochondria in central axons of insect (Pariplaneta americana), J. Cell Sci. 27, 255-272. Taylor, K., Dux, L., and Martinosi, A. (1984) Structure of the vanadate-induced crystals of sarcoplasmic reticulum CaATPase, J. Mol. Biol. 174, 193-204. Taylor, K. A., Dux, L., and Martinosi, A. (1986) Threedimensional reconstruction of negatively stained crystals of the Ca-ATPase from muscle sarcoplasmic reticulum, J. Mol. Biol. 187, 417427. Tyler, D. D., and Gonze, J. (1967) The preparation chondria from laboratory animals, in Estabrook, Pullman, M. E. (Eds.), Methods in Enzymology, 75-77, Academic Press, New York.

of heart mitoR. W., and Vol. 10, pp.