The arrangement of mitochondrial membranes and a new structural feature of the inner mitochondrial membranes

J O U R N A L OF U L T R A S T I ~ U C T U R1~ R E S E A R C H

59, 292-319 (1977)

The Arrangement of Mitochondrial Membranes and a New Structural Feature of the Inner Mitochondrial Membranes 1 FRITIOF S. SJ6STRAND

Department of Biology and Molecular Biology Institute, University of California, Los Angeles, California 90024 Received December 20, 1976 Rat heart muscle mitechondria were analyzed by means of high-resolution electron microscopy of ultrathin sections of tissue prepared according to the techniques of SjSstrand and Barajas [J. UItrastruct. Res. 25, 121 (1968)]. These techniques aim at reducing the extent of changes in protein conformation as compared to conventional preparatory procedures. It was possible to discriminate between three different types of membranes in mitochondria. Two of these types of membranes formed membranes bounding the mitochondrion, while the third type constituted the inner mitechondrial membranes. The first two types of membranes could be distinguished by the difference in the thickness, 40-50/~ for the outermost and 60-70/k for the innermost component. The inner mitochondrial membranes which form the cristae differed by 100% in thickness as compared to the innermost, thicker component of the bounding membranes and furthermore showed uniformly distributed, relatively intensely stained regions located at the surface of the membrane facing away from the matrix space. The thickness of these membranes was measured to be 125/k, half the total thickness of the cristae, 250 A. Intramembranous spaces separating the two membrane components at the surface of the mitochondrion and the two membranes of the cristae (intracristal spaces) were considered to be artifactual because they appeared when the mitochondria had been exposed to adverse experimental conditions, while they were absent in material where the treatment intended to involve minimal modification of the structure by reducing the extent of the changes in protein conformation. The two inner mitechondrial membranes composing the cristae were proposed to form compact, predominantly nonpolar regions. Some aspects of the functional significance of such regions are discussed.

It is generally accepted t h a t there are only two types of membranes in mitochondria, the inner and the outer membranes. It is also accepted t h a t the inner membranes constitute a closed system consisting of two parts, the cristae mitochondrales and a peripheral part extending aloIig the inner surface of the outer membrane. According to this concept, the cristae mitochondrales are mere folds of this peripheral membrane, and the inner membranes can be thought of as parts of a sac with a number of folds extending toward the center of the sac. It is generally accepted t h a t the two parts of the inner 1 This work was supported by USPHS Grants NB02889 and EY-00097 and by NSF Grants GB-7859 and BMS 74-20390. Additional support from the UCLA Molecular Biology Institute is gratefully acknowledged.

membranes are chemically and therefore functionally identical. This concept of a folded sac was presented by Palade in 1952 and was considered by him at t h a t time to represent the entire membrane structure of mitochondria (Fig. 1A). The folds would be open and the space in the folds, the intracristal space, would therefore communicate with the surrounding cytoplasm. Furthermore, the cristae did n o t reach all the way across the diameter of the mitochondrion, but left a central matrix space free. When Palade (1953) later confirmed the observations made by Sj6strand in 1952 (1953) t h a t the mitochondrion was bounded by an additional membrane, he placed this membrane as a sac enclosing the m e m b r a n e system he had observed. This additional membrane was then re-

292 Copyright © 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISSN 0022-5320

293

MITOCHONDRIAL MEMBRANE STRUCTURE

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PALADE 1952 A

SJOSTRAND 1952 B

FIG. 1. The two concepts regarding the structure of mitochondria proposed in 1952 by Palade and SjSstrand, respectively. Two versions are presented of Palade's concept of 1952 because it is unclear from his description w h a t he considered to be the relationship between the cristae and the surface membrane. Palade emphasized t h e cristae as being ridges extending from the surface m e m b r a n e (redrawn from AnderssonCedergren, 1959).

fel:red to as the outer membrane. Palade also assumed that, in addition to the matrix space, there was a space extending between the inner and the outer membranes and t h a t this space communicated freely with the intracristal space. According to the independent discovery in 19522 of an elaborate structure of mitochondria by SjSstrand (1953)3, the mitochondria consisted of an outer double membrane enclosing a space in which discshaped inner double membranes extended across almost the entire diameter of the mitochondrion (Fig. 1B). This structure resembled t h a t of the outer segments of photoreceptor cells (SjSstrand, 1948, 1949, 1953). The inner ~nembrane discs had, ">"These observations were reported a t the EMSA meeting in Cleveland, August 1952. This paper was submitted for publication on August 22, 1952.

however, the shape of pieces of a jig-saw puzzle. The matrix spaces that extended between the inner membrane discs communicated along the edges of these inner membranes. Contact points between inner and outer membranes were observed and continuity between the inner membrane discs and the innermost component of the double outer membrane was described (SjSstrand and Hanzon, 1954). However, the frequency at which such a continuity could be observed was low and could only allow for stalk-like connections between the inner membrane discs and the innermost component of the double outer membrane. The question of whether these connections were real or due to artifactual fusion of the membranes was left open. According to the concept t h a t was proposed by SjSstrand, the double membranes formed solid structures and were not

294

FRITIOF S. SJOSTRAND

bounding any intramembranous space. An intracristal space was therefore not considered to exist according to this concept. Furthermore, the two membranes at the surface were considered to form a compact composite surface structure. Both of these membranes were therefore included as parts of a composite outer membrane. The two components of this double membrane were later (SjSstrand, 1960) referred to as two membrane elements of the outer membrane, one being the innermost and the other the outermost components of the double membrane. The two membrane components of the cristae were also considered as two membrane elements of a compact, composite membrane disc structure. The outer and the inner membranes were assumed to be structurally and functionally different. Three-dimensional reconstructions by Andersson-Cedergren (1959) and by Daems and Wisse (1966) have shown that the cristae are connected to the innermost component of the surface double membrane by about 100-~ thick stalks, showing that the cristae are not wide folds of this component, but also revealing that a certain continuity is likely to exist between the innermost component of the outer double membrane and the cristae. These connections were, however, confined to a few spots at the edge of the inner membrane discs. The differences in these two concepts are of a basic nature, and it will eventually be important that a concept which reflects the actual situation in the native state of mitochondria is considered. This will become more and more important as we realize that many functions of the mitochondrion, like that of the respiratory chain, depend on a particular structural organization, and that biochemical data alone are insufficient to develop an understanding of how such complex systems function. To settle the question of which concept is closest to the native situation, it was required that new and improved preparatory

procedures be developed. The analysis of mitochondria in thin sections of tissues a n d of mitochondrial fractions has shown that the structure of mitochondria is very labile and that, for instance, swelling of mitochondria is a common fixation defect. The central matrix space described by Palade (1952), for instance, appears as a consequence of extensive swelling of the mitochondria. Since conventional procedures used for preparing tissues for thin sectioning involve complete denaturation of the membrane proteins, it seemed reasonable to question whether preparing specimens under such conditions really could reveal the arrangement of the mitochondrial membranes in the intact in vivo state. With denaturation of the membrane proteins, secondary changes in membrane structure and membrane arrangement are likely to take place since the balance between nonpolar interaction and charge interaction will be completely changed. This, in turn, will lead to different conditions for protein-water interaction, which could result in changes in the distribution of water. It therefore appeared appropriate to analyze this problem when applying the methods of SjSstrand and Barajas (1968) which aim at limiting the extent of protein denaturation in connection with the preparation of biological material for thin sectioning. The application of these methods gave results which support the concept originally presented by SjSstrand (1953). In this paper, observations will be discussed which indicate the existence of three different types of mitochondrial membranes: two different membranes forming two membrane elements of the outer membrane, and a third type of membrane constituting the membrane elements of the cristae. In addition, a new structural feature of the inner mitochondrial membranes was 'observed. MATERIALS AND METHODS Mitochondria were analyzed when in situ in rat heart muscle tissue as well as after isolation. Heart

MITOCHONDRIAL MEMBRANE STRUCTURE muscle tissue was prepared according to the method of SjSstrand and Barajas (1968). The first step in this method aims a t reducing the risks for protein denat u r a t i o n w h e n w a t e r is replaced by an embedding medium by stabilizing the protein conformation through intra- a n d intermolecular cross-linking with glutaraldehyde as a cross-linking agent. The tissue was cross-linked' for 3-7 m i n through injection directly into the left ventricle of a 1% glutaraldehyde solution in 0.3 M phosphate buffer adjusted to pH 7.4. D e n a t u r a t i o n of the proteins through prolonged exposure to glutaraldehyde (Lenard a n d Singer, 1968) was thus avoided. The second step involved dehydration in ethylene glycol, which is the weakest known protein denaturing organic solvent (Tanford et al., 1955, 1962). The material was exposed to this solvent for 1-7 min. The dehydration was brief enough to prevent complete dehydration. The proteins thus retained some of the hydration water, which reduced the risks for extensive denaturation. When small pieces of papillary muscle a n d of t h e wall of the r i g h t ventricle were prepared, these pieces were first washed in buffer and t h e n dehydrated for 7 min in 100% ethylene glycol. Dehydration in 70-80% ethylene glycol is possible when using Vestopal as t h e embedding medium. With small specimens such as single-cell preparations, the m a t e r i a l can be transferred to Vestopal without previous dehydration. The latter method is presently being explored in our laboratory. In the t h i r d step, the m a t e r i a l was infiltrated with a n embedding medium with excellent sectioning properties. Th~s embedding medium should be as polar as possible. Vestopal was found to be the best embedding medium presently available. The dehydrated pieces of tissue were transferred to Vestopal without activator and initiator after careful blotting of the pieces of tissue with filter paper. After 3-4 days, the tissue was transferred to Vestopal with activator and initiator. The fourth step was t h e solidification of Vestopal, which is due to a cross-linking reaction. To avoid h e a t denaturation, this was pursued a t temperatures of 40°C or lower. Mitochondrial fractions were prepared according to a method worked out as a modification of t h a t of Cleland a n d Slater (1953). Two to four rats were used for most experiments. They were killed by decapitation. The h e a r t s were removed as quickly as possible and transferred to ice-cold suspending m e d i u m within 60 sec after dec a p i t a t i o n . T h e h e a r t s were cut open a n d washed to remove blood by changing the medium twice. After reducing the volume of t h e medium to a few milliliters, each h e a r t was cut up separately in small pieces using a pair of scissors. The chopped h e a r t tissue was washed in several changes of medium until no traces of blood contaminated t h e medium.

295

After decanting the medium, the h e a r t muscle tissue was transferred to a n ice-cold glass or porcelain m o r t a r containing 1 g of washed sand per heart. The homogenization is the critical step in the preparatory procedure. It was considered i m p o r t a n t t h a t a m i n i m u m of t r a u m a be applied in order to release the mitochondria. The grinding was therefore pursued with light pressure, this pressure being confined almost to t h a t due to the weight of the pestle. ~ It was also considered i m p o r t a n t t h a t the mitochondria, after being released, not be exposed to any additional t r a u m a . To make this possible, the tissue was ground repeatedly for only short periods with a~ r a t h e r large volume of medium which allowed the released mitochondria to diffuse away from the grinding surfaces. Furthermore, the medium with the released mitochondria was removed and new medium was added after each period of grinding. The following schedule was used for the grinding. Twenty milliliters of medium was added to the chopped h e a r t muscle tissue before grinding. After 30 sec of gentle grinding and after allowing the larger particles to sediment, some of the supernat a n t liquid was removed and 4 ml of medium was added. This was repeated five times, b u t now with only 15 sec of grinding each time. The material collected after the first grinding, which contained some red blood cells, was discarded. The fluid was t h e n decanted a n d the residue was washed with 10 ml of medium. This method of grinding the h e a r t muscle tissue improved the quality of t h e fractions considerably as compared to grinding continuously for 3 rain with the tissue dispersed in a small volume of medium, which was the procedure of Cleland and Slater (1953). The collected homogenate containing mitochondria was centrifuged for 3 min a t 755g (2500 rpm) in a Sorvall SS34 rotor in a Sorvall Model SS33 centrifuge in a cold room at +3 ° to +4°C. The s u p e r n a t a n t was transferred to clean centrifuge tubes and centrifuged a t 7700g (8000 rpm) for 7.5 min. The pellet was resuspended in 30 ml of medium. After a second centrifugation, this time a t 9750g (9000 rpm), the pellet was dispersed in 3 ml of medium not containing any albumin. The medium used for homogenization contained 100 mM tris(hydroxymethyl)aminomethane.HC1 buffer, 300 mM sucrose, 10 mM Versene, and 1% albumin (fatty acid free). The pH of the medium was adjusted to 7.4 with 12 N HC1. Elimination of the Tris buffer from this medium gave identical results. The respiratory control ratio was used as one indicator for the quality of the mitechendrial fractions. This ratio was determined using a polarographic Clark oxygen electrode. The medium used for the determination had the following composition: 200 mM mannitol, 50 mM sucrose, 20 mM Tris buffer

296

FRITIOF S. SJOSTRAND

and 10 mM KH2PO4. The pH was t i t r a t e d to 7.4 by adding 12 N HC1. Succinate was used as substrate and ADP as phosphate accepter. After the ADP h a d been consumed, respiration was uncoupled by adding dinitrophenol and the rate of respiration was compared to t h a t during phosphorylation of ADP. These rates were identical. The respiratory control ratio varied between 3 and 5.5 for different preparations. A n o t h e r indicator t h a t was used to evaluate the degree to which the mitochondria h a d been damaged was the c h a n g e in respiratory control ratio with time w h e n the fractions were aged a t 0°C. The m o s t stable preparations m a i n t a i n e d the respiratory control to a considerable extent during aging, with no appreciable change after 24 h r of aging and less t h a n 50% change after a 5- to 7-day period of aging. For electron microscopy, the isolated mitochondria were suspended in the medium without album i n and were cross-linked for 4-7 rain in 1% glutaraldehyde. A freshly opened ampoule of glutaraldehyde was diluted by adding the medium without albumin to m a k e a 2% solution of glutaraldehyde. A n equal volume & t h i s glutaraldehyde solution and of the dispersion of mitochondria were mixed. The cross-linking was stepped by dilution of the suspension with medium. The diluted mitochondria dispersion was t h e n centrifuged for 8 rain a t 4340g (6000 rpm). The pellet was dispersed in 100% ethylene glycol for 3-4 m i n for dehydration. The mitochondria were pelleted by I rain of centrifugation in a Microfuge centrifuge, and t h e pellet was transferred to Vestepal after cutting offthe bottom of the centrifuge tube. Samples of the residue after grinding were prepared in a n identical way. The 3- to 4m i n dehydration time was m e a s u r e d from the mom e n t the m a t e r i a l was dispersed in ethylene glycol to the dispersion of the material in Vestopal. Samples of t h e residue after grinding were prepared in a n identical way. The exchange of Vestopal for ethylene glycol is slow because of the limited miscibility of ethylene glycol in Vestopal. The infiltration of Vestopal is speeded up considerably if the m a t e r i a l is centrifuged repeatedly in fresh Vestopal. This way, the infiltration time can be reduced to between 5 and 10 min. After repeated centrifugation, the m a t e r i a l was t h e n transferred directly to gelatin capsules one-fourth filled with Vestopal. The mitochondria were dispersed in this volume of Vestopal, and the capsule was filled a n d allowed to cross-link for 24-48 h r a t 40°C or for i week a t r o o m temperature. When infiltration was speeded up this way, Vestepal with initiator and activator was used all the way. Crosslinking of Vestopal can also be achieved a t cold room t e m p e r a t u r e by m e a n s of a weak uv light source such as a sterilization lamp. The m a t e r i a l was sectioned on an LKB Ultrotome I using glass knives. The sections appeared gray to

dark gray in reflected light when floating off the knife edge onto water. Only the t h i n n e s t dark gray sections could be used for high-resolution electron microscopy. The sections were stained with a n 8% uranyl acetate solution in w a t e r at 60°C, 40°C, or room temperature. No difference was observed in the structure of the mitochondria when comparing, different s t a i n i n g temperatures. The sections were collected either on filmed single slot (1 × 2 ram) grids or were mounted on holy films on 100-mesh grids. The holy films were prepared according to the method described by SjSst r a n d (11967). The hole size was adjusted to 1-2 ~m. When ~he t h i n sections extended over holes of larger diameter, they were unstable and became distorted. In the ease of sections mounted on holy films, it was important t o stain the sections a t a t e m p e r a t u r e not exceeding 40°C because the sections broke when exposed to the electron b e a m after being stained a t a higher temperature, even when they extended over holes with a d i a m e t e r less t h a n 1 ~m. A Hitachi HU-11 electron microscope equipped with short focal length objective lens pole pieces and 30- or 50-tLm objective apertures was used. Contamir/ation was practically eliminated by m e a n s of cold traps placed above the specimen stage and below the objective-intermediate lens system. The electron micrographs were t a k e n at magnifications of 40,000-60,000×. To achieve ~'minimum exposure" of the photographed area to the electron beam, a n area close to t h a t to be photographed was used for focusing. The latter area was photographed as soon as possible after it h a d been moved into the field of view by moving the stage. The m e a s u r e m e n t s were carried out on prints at a magnification of 180,000× using a m e a s u r i n g ocular. The dimensions of the m e m b r a n e s were compared u n d e r conditions where the m e m b r a n e s showed identical orientation. This was necessary in order to eliminate errors through variations in dimensions caused by the varying degrees of compression introduced during sectioning. Measurements of the thickness of t h e i n n e r and outer m e m b r a n e s as well as of individual m e m b r a n e elements were compared on the same mitochondrion, a n d the differences were expressed as percentage differences. OBSERVATIONS

The Surface Membranes Rat heart muscle mitochondria, whether cross-linked in situ (Figs. 2 and 3) or after fractionation (Fig. 4), appear with densely arranged inner membranes which were lightly stained in comparison to the staining of the matrix. In isolated mitochon-

FIG. 2. Section through rat h e a r t muscle tissue showing a large number ofmitochondria. The difference in dimensions of the cristae and of the surface membranes is obvious. The variation in the thicknesses of the former membranes in different mitochondria is due to compression caused by the sectioning, x 93 000. 297

FIG. 3. Mitechondria in a fragment of rat h e a r t muscle tissue collected from the residue after third grinding to prepare mitochondrial fraction electron micrograph by F. Kretzer. x 44 000. 298

FIG. 4, Isolated rat heart muscle mitochondrion. The cristae stand out with high contrast due to the high opacity of the matrix. Opaque regions are present in the middle of the cristae. The section is in this case relativety thick. As a consequence of extensive superposition, the opaque regions in the middle of the cristae are r a t h e r large, x 180 000. 299

300

FRITIOF S. SJOSTRAND

dria, the individual mitochondria were thin with two faintly stained layers sepabounded by a surface membrane which rated by some opaque material located in consisted of two lightly stained compo- the middle of the boundary. This situation nents (Figs. 5 and 6). The innermost com- was also observed sometimes in isolated ponent appeared as a continuous lightly mitochondria (Fig. 8). Such boundaries in stained layer. This layer was clearly de- in situ mitochondria were of about the limited peripherally by a layer of opaque same thickness as a single crista. This is material located along its peripheral sur- remarkable since such a boundary consists face and centrally by the opaque matrix. of four membranes arranged in parallel in This made the surfaces of this layer stand addition to some material interposed beout with good contrast in spite of its low tween the two mitochondrial surfaces. opacity. A second lightly stained, thinner In whole isolated mitochondria that layer was observed outside this layer. It were negatively stained without previous was delimited by tlie opaque layer located fixation, a single bounding membrane was on the outside of the first component and observed which measured about 100/k in frequently by opaque material along its thickness (Fig. 9). peripheral surface (Figs. 5 and 6). When The surface membranes in isolated mithis latter opaque coat was absent, the low tochondria offer a favorabIe condition for opacity of this layer made it difficult to observing a substructure because when observe it clearly. Rather high magnifica- they are oriented obliquely to the direction tion electron micrographs of good resolu- of the electron beam, it is possible to obtion were then required to observe this tain face-on views without interference of second faintly stained layer. This layer other structural components. The surface frequently appeared to be discontinuous. membranes then form a wedge-like edge The opaque layer interposed between extending into clear plastic. Only electron the two lightly stained layers at the sur- scattering in the plastic will then interfere face was often interrupted by lightly with the imaging and no other material stained material. When two mitochondria will be superimposed in the image of the were in close apposition, the most periph- surface membranes. Figure 10 shows such eral lightly stained layers of the two mito- a situation, and it seems clear that there is chondria could either appear separated by a globular substructure in the surface opaque material (Fig. 7) or fused to form structure of the mitochondria. Opaque an irregularly arranged and faintly areas that are observed can be interpreted to belong to the opaque layer interposed stained particulate material (Fig. 5). This description applies to isolated mito- between the two lightly stained surface chondria and not to mitochondria in situ. layers. The fact that the opaque material In the latter case (Figs. 2 and 3), the struc- appears as opaque areas of limited extenture bounding the mitochondria appeared sion in the obliquely oriented surface • as a single lightly stained layer. This layer structure indicates that superposition acwas, however, split into two components counts for the fact that a more or less in some small regions in the in situ mito- continuous opaque layer is observed in chondria of the residue collected after cross sections of the outer membrane. Of the two lightly stained layers at the grinding the heart muscle tissue. Such splitting up of the outer membrane was surface of the mitochondria, the outermost not observed in in situ mitochondria in one was measured to be 40-50/~ and the heart muscle tissue cross-linked by perfu- innermost one 60-70/k thick (Table I). The sion with glutaraldehyde. Where two mi- difference in thickness between these two tochondria were closely apposed in situ, components was easily observed directly in the bounding structure appeared rather the electron micrographs (Figs. 5-7).

]FIG. 5. I s o l a t e d r a t h e a r t m u s c l e m i t o c h o n d r i a . T h e s u r f a c e m e m b r a n e s a r e s h o w n w i t h t h e i n n e r m o s t c o m p o n e n t , ic, s e p a r a t e d from t h e o u t e r m o s t c o m p o n e n t , oc, by a n o p a q u e layer. T h e c r i s t a e a r e o r i e n t e d m o r e or less o b l i q u e l y in r e l a t i o n to t h e d i r e c t i o n of t h e e l e c t r o n b e a m . O p a q u e r e g i o n s a r e t h e n s e e n d i s t r i b u t e d r a n d o m l y over t h e s e o b l i q u e l y o r i e n t e d cristae, x 200 000. 301

FIG. 6. Isolated mitechondria. The two outer membrane elements are shown with the innermost membrane element, ic, considerably thicker t h a n the outermost, oc. Inner membranes seen partially in profile and partially in face-on views. Opaque re~ions give a speckled appearance to the inner membranes in the latter case. x 180 000. 302

FIG. 7. Isolated mi'mchondria. At the boundary indicated by the arrows, where two rnitochondria are in close apposition, the outermost m e m b r a n e elements, oc, of the apposed outer membranes are separated by opaque material. The t.wo m e m b r a n e elements of the outer membrane, oc and ic, are furthermore separated by opaque material. This boundary structure should be compared with t h a t ofmitochondria in situ in Figs. 2 and 3, where no such layer of opaque material between the two m e m b r a n e elements is present, x 120 000. 303

304

FRITIOF S. SJOSTRAND

FIG. 8. Two isolated mitochondria t h a t are in close apposition. The boundary between t h e two mitochondria is indicated by arrows. Notice how t h i n this boundary formed by four m e m b r a n e elements is. In this case, t h e boundary appears similar to t h a t observed in closely apposed mitochondria in situ. × 110 000.

FIG. 9. Whole r a t h e a r t muscle mitechondrion negatively stained, unfixed. The stain has entered the m a t r i x space. The i n n e r m e m b r a n e s are observed in profile view as is the outer m e m b r a n e . The l a t t e r appears as a single, about 100-A thick m e m b r a n e , while the cristae measure about 300 A in thickness. × 100 000.

MITOCHONDRIAL MEMBRANE STRUCTURE

305

FIG. 10. Isolated mitochondrion with outer m e m b r a n e sectioned a t an oblique angle. A particulate substructure (arrows A) is seen in the m e m b r a n e elements of t h e outer m e m b r a n e when viewed under these conditions. A more opaque zone (arrows B) is located a distance inside the edge of the outer membrane. The opacity is due to the presence of opaque particles. When the outer m e m b r a n e is observed in a profile view, these particles are imaged superimposed. Due to this superpositioning, the two m e m b r a n e elements appear to be separated by a continuous opaque layer in the profile view. × 190 000.

T)ie Cristae Membranes

Mitochondria that were cross-linked in situ showed no intracristal space, while such a space was observed in some inner membranes of freshly prepared isolated mitochondria (Fig. 11). The frequency of occurrence of intracristal spaces increased with aging of the mitochondrial fractions. When present, the intracristal space made it possible to reveal clearly that the inner membranes were positively stained (Fig. 11) by comparing the opacity of the membrane elements to that of the unstained space which separated the two membrane elements. A particulate substructure was observed in closely apposed inner membranes when

they were oriented obliquely. This usually required extremely thin sections mounted on holy films (Fig. 12). The individual particles in the particulate substructure appeared as lightly stained areas delimited by somewhat more intensely stained boundaries. The dimensions of these particles ranged between 30 and 50/~. The mean thickness of the inner membranes when not compressed by the sectioning was measured to be 250/k (Table I). When measured in the direction of compression, the mean thickness was 190 /~. The thickness of the individual membrane elements in the cases where an intracristal space separated them varied between 90 and 170/~. On .an average, the thickness of the single membrane ele-

306

FRITIOF S. SJOSTRAND

ments was about 50% of that of the cristae (Table I).

A New Structural Component of the Cristae Membranes It was characteristic that positively stained areas were distributed evenly in the center of the cristae membranes. When the membranes were observed in profile views (Figs. 4 and 11), these stained areas measured 60-80/~ in diameter and sometimes showed indications of an angular or square shape. These areas could exceed this dimension in a direction parallel to the plane of the membranes (Fig. 4), particularly in thicker sections. In obliquely oriented inner membranes, opaque areas were observed uniformly and randomly distributed within the membranes, giving a speckled appearance to the membranes (Figs. 5, 6, 10, 13, and 14). These opaque areas were of a fairly uniform size and sometimes showed an indication of an angular, square, or rectangular shape. The smallest areas measured about 40/~ in diameter. Since the opaque areas observed in obliquely oriented membranes were the only components that showed an opacity that well exceeded the average opacity of the membranes, it seems obvious that the opaque areas observed in profile views of the membranes are identical to those observed in face-on views. The difference in size and in opacity can be accounted for by superpositon in the case of the profile views. Such superposition must occur since the thickness of the sectidns was considerably greater than the average distance between the opaque areas observed in face-on views of the membranes. DISCUSSION

The application of preparatory methods that involve reduced risks for extensive denaturation of proteins makes the mitochondrial membranes in ultrathin sections appear vastly different from their appearance in material prepared according to

conventional methods. The membranes appear less opaque than the matrix but are still positively stained, they show no indications of the presence of any continuous, uninterrupted layers, and they are 2.5 to 3 times thicker than in conventional preparations. The interpretation of this appearance of the membranes has been discussed in earlier papers by Sj6strand and Barajas (1968, 1970), Sj6strand and Kretzer (1975), and SjSstrand (1976). It is consistent with what can be expected to be the appearance of a membrane that consists of a close aggregation of globular protein molecules with an addition of lipids, because such an aggregation would exclude the stain from being bound to the predominantly nonpolar interior of globular proteins and to nonpolar regions occupied by the hydrocarbon tails of lipid molecules.

The Differentiation between Three Types of Membranes in Mitochondria In conventionally prepared material, the mitochondria appear to be bounded by two membranes (Sj6strand, 1953) which are both of the same thickness (about 50 /~). A difference in total thickness of the outer double membrane as compared to the cristae double membranes was reported by SjSstrand and Hanzon (1954) for mitochondria in exocrine pancreas cells. However, the thicknesses of the individual membrane elements in the outer double membrane and the crista double membranes were insignificant. In sectionstained material, all these membrane elements appear triple layered and of the same thickness and appearance. In the present study, the two membranes at the surface of the mitochondria appeared to be structurally different from the cristae membranes. The most obvious indication of a structural difference was the difference in thickness of the membranes. The thickness of the pair of apposed membranes of the cristae was measured to

MITOCHONDRIAL

MEMBRANE TABLE

307

STRUCTURE

I

THE THICKNESS OF MITOCHONDRIAL MEMBRANES a Mitochondrion

Cristae

Outer membrane

Apposed membrane elements

Single membrane elements

Innermost component

Outermost component

A

B

C

D

(A)

(£)

(h)

B as % of A

C as % o f 1 / 2 A

•

242 268 250 252

217 219 170 169

66 75 60 57

52 68 60 62

55 56 48 45

48 62 71 73

M

253£

194£

65A

61£

51%

63%

1 2 3 4 8 9 10 11 12 13 14 28 29 31 32 33 34

244 243 234 234 243 241 240 241 236 251 244 251 252 271 253 241 233

M

110

47

56 51 46 6O 53 46 54 56 59 54 58 5O 46 55 66 7O 67

CA)

D as % of C

II

IF 5 6 17 21

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The m e a s u r e m e n t s of t h e t h i c k n e s s e s of t h e m i t o c h o n d r i a l m e m b r a n e s h a v e been divided u p into t h r e e groups. A t t h e top of t h e t a b l e a r e collected t h e m e a s u r e m e n t s m a d e on cristae m e m b r a n e s w h e n these m e m b r a n e s in t h e s a m e m i t o c h o n d r i o n w e r e o r i e n t e d in two p e r p e n d i c u l a r directions, one of w h i c h w a s close to t h e direction of t h e sectioning. The two values for c r i s t a e t h i c k n e s s t h u s correspond to t h e t h i c k n e s s of t h e l e a s t compressed a n d of t h e m o s t compressed merobranes. In t h e second g r o u p of m e a s u r e m e n t s , t h e values for cristae t h i c k n e s s above 230 £ h a v e b e e n collected, while t h e t h i r d g r o u p of v a l u e s a t t h e bottom of t h e t a b l e s h o w s m e a s u r e m e n t s w h e r e t h e cristae m e a s u r e d less t h a n 230 A in thickness. It is obvious t h a t compression slightly reduces t h e difference in t h i c k n e s s b e t w e e n t h e i n n e r m o s t c o m p o n e n t of t h e o u t e r m e r o b r a n e a n d t h e single cristae m e m b r a n e elements. ~L, p e r p e n d i c u l a r to knife edge; I], p a r a l l e l to knife edge.

be 160 to 270/~. This considerable variation in the thickness can be wholly accounted for by differences in the degree of compression of the membranes caused by

nonreversible plastic flow of the embedding material and the membrane material during sectioning. That this variation in the thickness of

FIG. 11. Isolated mitochondria with some cristae split by intracristal spaces (arrow). The m e m b r a n e elements of the cristae are positively stained, while the intercristal space is unstained. Notice the distribution of opaque regions in the cristae and in the separated m e m b r a n e elements of the cristae. × 180 000. 308

MITOCHONDRIAL MEMBRANE STRUCTURE

the cristae membranes really could be accounted for by different degrees of compression was shown by measuring the thickness of membranes in the same mitochondrion when the profiles of the membranes were bent in such a w a y that measurements could be made on different parts of the same membranes with mutually perpendicular orientations (symbols at top of Table I). It was also verified from measurements made on pictures where the direction of the sectioning was indicated by knife marks. We are therefore justified in selecting the mean value 250/~ for cristae oriented parallel to the direction of sectioning (the first group of values in Table I) as the most representative value for the thickness of the cristae. Since the two membrane elements of the cristae membranes are closely apposed, the thicknesS of the individual membrane elements will then be 125 •. The thickness of the individual membrane elements when separated by an intracristal space was found to be on an average about 50% of that of the cristae with apposed membrane elements. If we now compare the thickness of the membrane elements of the cristae with that of the innermost membrane at the mitochondrial surface, which was 60-70 A, it becomes obvious that there is about 100% difference in these dimensions. The measurements accounted for in Table I show that the thickness of this outer membrane component was only 50% of the thickness of half the total thickness of the cristae in situations where the deformation due to compression was limited. This difference was slightly less in cases where the membranes had been compressed by the sectioning. Table I also shows the same differences when the thicknesses of the cristae membranes were measured on individual crista membranes separated by an intracristal space. These differences in the thickness of the membranes make it justifiable to conclude

309

that the membrane elements of the cristae are structurally different from the innermost component of the two membranes at the surface of the mitochondrion. If we now consider the two membranous components at the surface of the mitochondrion, it becomes obvious that they differ structurally. This is shown both by the difference in thickness of these components, 40-50/~ for the most peripheral and 60-70/~ for the other component, and by the greater lability of the outermost component. The lability of the latter component is shown by the high frequency of isolated mitochondria in which this component is missing over part of the surface. The preparatory procedure therefore is likely to damage it to the extent that it is partially disrupted. This difference in stability of the two membranous components at the surface can be explained by a difference in lipid content. The fact that no differences in the structure of these membranes can be observed in specimens prepared according to conventional methods can be explained by the extensive changes that must occur in the membrane structure in connection with denaturation of membrane proteins. It is pertinent to point to the fact that the triple-layered ~unit membrane" pattern which is generally observed in such preparations occurs irrespective of the proportions of lipids and proteins. It is thus observed in practically pure protein membranes such as artificial cytochrome oxidase membranes and in lipid-depleted mitochondrial membranes (Fleischer et al., 1965), as well as in artificial pure lipid membranes (Stoeckenius, 1959) and predominantly lipid membranes such as the layers of the myelin sheath (Sj6strand, 1953, 1954). The thing these membranes have in common is the presence of polar and nonpolar groups. Therefore, it is likely that the triple-layered appearance results from an artifactual sequestering of nonpolar groups of lipids and of denatured proteins in the center of the modified mem-

310

MITOCHONDRIAL MEMBRANE STRUCTURE brahe with polar and Charged groups accumulated at the two surfaces. This interpretation is reasonable from a thermodynamic point of view. When the protein molecules denature, the unfolding of the peptide chains will expose nonpolar amino acid side chains to the aqueous medium. The entropy change associated with such exposure will favor aggregation of nonpolar groups and their shielding from contact with water by polar groups. These are, in principle, the same conditions that determine the native conformation of globular proteins and the aggregation of lipid molecules in bilayers. Both of these cases are characterized by an accumulation of nonpolar side chains in the interior with polar and charged groups located at the surface of the globular protein molecules and at the surfaces of the bilayer, respectively. Due to the entanglement and secondary cross-linking of the unfolding peptide chains during denaturation, the sequestering of nonpolar amino acid side chains will involve aggregates of peptide chains and not individual chains. These aggregates will remain located close to where the protein molecules originally were positioned in case the denaturation proceeds slowly. Therefore, if the protein molecules were components of a membrane, they will aggregate in the form of a membrane structure, but the structure of this membrane will be very different from t h a t of the native membrane. The staining pattern of the triple-layered structure agrees with this interpretation since the staining is likely to involve charged sites which would be. located at the two surfaces of the medifled membrane.

311

The Arrangement of Mitochondrial Membranes A striking feature of the present material was the absence of any intracristal space in mitochondria preserved in situ, and the .rare occurrence of such a space in isolated mitochondria provided t h a t fresh mitochondria fractions had been prepared for electron microscopy. With aging, intracristal spaces appeared more frequently t h a n in fresh mitochondria fractions. The absence of an intracristal space could be explained by shrinkage due to the dehydration method used. However, there were no intracristal spaces observed in freeze-dried, cold-embedded material where the conditions for shrinkage are less drastic (SjSstrand and Kretzer, 1975). That the preparatory technique preserves such spaces when present is shown by the fact t h a t intracristal spaces could be observed in isolated mitochondria and t h a t the frequency of appearance of such spaces increased with aging. In a recent study by SjSstrand and Bernhard (1976) applying cryo-ultramicrotomy, it was further shown t h a t when this method was applied under conditions where the risks for denaturation of the membrane proteins were reduced, no intracristal space could be observed. Any change in the procedure that increased the risks for protein denaturation, on the other hand, lead to the appearance of an intracristal space. It is also characteristic t h a t no intracristal space is observed after conventional aldehyde fixation or after potassium permanganate fixation. The cristae then appear as a five-layered structure. If the tis-

FIG. 12. Isolated mitochondria showing inner and outer membranes with a particulate substructure (arrows). The inner membranes are more or less obliquelyoriented, partially making them appear in a faceon view. The diameters of the particles vary between 30 and 50 A. The innermost element, ic, of the outer membrane is seen in profile view and also shows a particulate substructure. Picture of very thin section mounted on holy film and therefore viewedwithout interferenceby electron scattering in any support film. At the lower edgeofthe picture a hole in the sectionwas includedto showthat the picture was taken closeto focus and that the particulate structure is not due to phase contrast granularity. Some distortion of the membrane arrangement is due to compressioncaused by sectioning. × 300 000.

312

MITOCHONDRIAL MEMBRANE STRUCTURE

sue is postfixed with osmium tetroxide after aldehyde fixation, however, intracristal spaces appear, as they do after osmium fixation of fresh tissues. It is therefore obvious that osmium tetroxide treatment can produce intracristal spaces where no such spaces were present. It is also characteristic that no indications of the presence of an intracristal space can be seen in published pictures of freeze-fractured mitochondria when fractured in situ. It therefore is reasonable to propose that the presence of an intracristal space is artifactual. We then arrive at a concept of the structure of the inner mitochondrial membranes (or the cristae) originally proposed by Sjhstrand (1953) that the two membrane elements of the cristae form a composite compact membrane. The dimensions of the membrane elements that were disclosed by Sj6strand and Barajas (1968) for rat kidney mitochondria are confirmed here for rat heart muscle mitochondria and were also confirmed by Sjhstrand and Bernhard (1976) on freeze-sectioned rat heart muscle mitochondria. These dimensions make it obvious that this composite membrane is of a thickness, 250 to 270/~, which is large in comparison to the average dimensions of globular protein molecules. Proteins and lipids therefore must form a three-dimensional aggregate (Sjhstrand and Barajas, 1968, 1970). That this aggregate consists of closely packed molecules is shown by the observations on freeze-sectioned material (Sjhstrand and Bernhard, 1976). These sections were neg-

313

atively stained and the stain was excluded from entering the cristae membranes. The opaque layer interposed between the two membranous components at the surface of the mitochondrion indicates that there is a considerable mass of charged sites present between these two components. It is now characteristic that such a layer is not observed in mitochondria when preserved in situ in heart muscle tissue using the same method of cross-linking and embedding as was used for the isolated mitochondriao Nor was any such layer observed in mitochondria in the heart muscle fragments collected as residue after grinding the heart muscle tissue where the total treatment had been identical to that to which the isolated mitochondria had been subjected, with the only exception being that the latter mitechondria had been released from the muscle cells. Furthermore, when analyzing whole, unfixed, negatively stained isolated mitechondria in drop preparations, only the profile of a single surface membrane was observed. The thickness of the profile of this membrane, about 100/~, agrees with that determined by Muscatello et al. (1972) for isolated liver mitochondria. The measurements of the dimension of the outer membrane appear reasonably reliable under these conditions since they were made on membrane profiles observed with maxim u m contrast, which requires that the membrane be oriented parallel to the beam. A thickness of 100/~ corresponds to the

FIG. 13. Isolated mitochondria showing inner membranes in different orientations. Light printing aims at enhancing the contrast of the opaque regions which give a speckled appearance to the inner membranes, where they have been sectioned more or less tangentially. P a r t of the inner membranes are oriented perpendicularly to the plane of the sections, while in the areas between these sharply outlined membrane profiles of inner membranes are oriented more or less parallel to the plane of the section and therefore are viewed face-on. At arrows A, an inner membrane changes its orientation from perpendicular to close to parallel to the plane of the section. It is then possible to see how the distribution of opaque areas in the membrane changes from t h a t of being confined to the center of the membrane profile to a random distribution over the entire membrane. At the same time, the size of the opaque areas decreases. Arrows marked by the letter B point to fenestrations in the inner membranes viewed face-on. A few opaque areas have been indicated by arrows marked by the letter C. × 180 000.

FIG. 14. H i g h m a g n i f i c a t i o n v i e w o f o p a q u e r e g i o n s i n i n n e r m e m b r a n e s o f i s o l a t e d m i t e c h o n d r i o nSome . such regions are indicated by arrows. Light print of electron micrograph to make the opaque areas appear in good contrast, x 420 000. 314

MITOCHONDRIAL MEMBRANE STRUCTURE sum of the thicknesses of the two surface membranes as determined in the present study. This dimension therefore would correspond to the total thickness of these two membranes when closely apposed. These observations together make it justifiable to propose that the two light layers that were observed at the surface of the mitochondrion in isolated mitochondria are closely apposed to form one composite m e m b r a n e in the native state of the mitochondrion. This composite membrane consists of two structurally different membrane elements. The justification of this conclusion could be questioned on the basis that the methods used for preparing the specimens in the cases where no intramembranous space was observed involved dehydration that could make such a space disappear through water removal. However, the isolated mitochondria had been exposed to treatment identical to that of the in situ mitochondria. Furthermore, when a layer was observed between the two surface membranes, it contained intensely positively stained material. It is difficult to imagine a closing of a space containing such material and located between the two membranes without any remnants of this material with high affinity for electron stains being left interposed between the two membranes. No such material could be observed in the in situ, cross-linked mitechondria. A similar separation of the two membrane elements of the cristae with an intensely stained layer of material interposed between the two membrane elements is a common modification of the cristae structure that occurs during aging of mitochondrial fractions. The Intramembranous Spaces Under certain experimental conditions, the two membrane components of the crista and of the surface membrane are separated by a space that is referred to here as the intramembranous space. In the case of the cristae, the intramembranous space is the intracristal space.

315

Intramembranous spaces can develop easily in connection with injury to mitochondria caused by the isolation procedure, by incubation in artificial media, and by aging of mitochondrial fractions. After such spaces have been developed, they can be made subject to extensive studies. The fact that they can be subjected to analysis does not prove that these spaces are present in intact in vivo mitochondria. The results that have been obtained in such studies are difficult to interpret, particularly when they are based on electron microscopy, since in that case, in addition to the experimental conditions mentioned above, the preparatory procedures used for electron microscopic analysis themselves can produce such spaces. In this connection, it seems justified to point to the studies of "membrane conformation" in mitochondria during different metabolic states. Of such studies, that of Muscatello et al. (1972) appears meaningful. A comparison was made of isolated whole rat liver mitochondria when prepared in a state of phosphorylation of ADP in the presence of different substrates, state 3, and mitochondria prepared in state 4 either before or after a period of phosphorylation of ADP. The differences in the appearance of the mitochondria were most obvious. In addition to a reduction of the volume of the mitochondria during phosphorylation, the distribution of the negative stain that had entered the mitochondria was strikingly different. It is rather obvious that in the nonphosphorylating mitechondria (state 4), the negative stain entered the intracristal spaces while little or no stain appeared in the matrix space. The stain in that case was dispersed in spaces which, in the image, project as thin lines extending from the surface of the mitechondria. This corresponds to what would be expected if the stain were located in narrow intracristal spaces, but not if it were located in the matrix space, which is rather wide in isolated liver mitochondria which have assumed a spherical shape due to damage

316

FRITIOF S. SJOSTRAND

caused by the preparatory procedure. In the phosphorylating (state 3) mitechondria, the stain was located in wide open spaces that communicate centrally. This is what would be expected if the stain were located in the matrix. This interpretation becomes obvious from the following consideration. The fact that in sections through isolated spherical liver mitochondria the membrane profiles are predominantly located around the periphery and few extend through the center shows that the inner membranes have a larger part of their mass located at the periphery. Consequently, there must then be a considerably less dense arrangement of inner membranes in the center of these isolated spherical mitochondria. The pictures of negatively stained whole liver mitochondria can be interpreted to reflect this distribution of membrane mass within the mitochondrion. In state 3, the mitochondria thus show an accumulation of unstained material peripherally, while the stain is preferentially located centrally in rather large spaces. If the stain were located in widened intracristal spaces, on the other hand, the stain would be most densely arranged peripherally, where the inner membranes are most densely arranged, and not in the center. If the inner membranes had become extended, "increased folding," there still would be a considerable amount of stain located peripherally. The stain furthermore delimits septa that extend from the periphery toward the center or across the mitochondria. The thicknesses of these septa correspond to the thicknesses of the inner membranes according to the measurements made on material prepared with techniques aimed at limiting the conformational changes of membrane proteins or on whole negatively stained heart muscle mitochondria. They therefore are likely to constitute the cristae. There were no indications of an intracristal space in these septa. With this interpretation of the pictures of Muscatello et al., their study indicates

that the permeability properties of the outer mitochondrial membrane are determined by the metabolic state of the mitochondrion to the extent that this membrane practically excludes stain from entering the matrix space when the respiratory chains are not phosphorylating, while the stain enters freely during phosphorylation. To account for the different location of the stain, it is necessary to assume that it is the innermost membrane element of the outer membrane that determines the permeability of this membrane. This conclusion is based on the fact that the innermost membrane element is the only membrane component that could exclude the stain from the matrix without preventing it from entering the intracristal spaces during state 4 conditions. The latter conclusion is based on the fact that the cristae are continuous with the innermost membrane element of the surface membrane through stalk-like connections~ T h e stain can therefore enter the cristae when the two membrane elements of the cristae become separated to form an intracristal space. The outer membrane element, on the other hand, must be permeable for the stain in both state 3 and state 4. Furthermore, during phosphorylation, no stain could be observed in intracristal spaces, indicating that the two membrane elements of the cristae which were separated during state 4 become closely apposed during state 3. This study therefore supports the conclusion that intracristal spaces appear as a consequence of abnormal conditions, in this case represented by isolation of mitechondria and incubation at 25°C when the mitochondrial metabolism is stopped due to lack of substrate for phosphorylation. It also shows that this change is reversible.

Substructures in Inner Membranes The most obvious substructures that were observed in the inner mitochondrial membranes were the positively stained regions. These regions were stained by either uranyl acetate or phosphotungstic

MITOCHONDRIAL MEMBRANE STRUCTURE acid. The staining indicates that both negatively and positively charged groups are accumulated in these regions within the membranes. The fact that the size of these opaque regions was considerably larger in membranes oriented parallel to the electron beam than in membranes oriented obliquely is explained by superposition, the conditions for which are maximal in membranes oriented parallel to the beam. Since opaque regions were confined to the middle of the cristae when observed in profile view, and since superposition was involved, these regions must be preferentially located at a certain level within the membrane. When the membranes were even slightly tilted in relation to the direction of the electron beam, the opaque regions were found distributed over the entire crisae. The preferred level must furthermore be at that surface of the membrane elements which is located away from the matrix. This location is also obvious when observing membrane elements that have been separated by an intracristal space. Opaque areas were then observed located at the surface of the membrane elements which faced this space. Since the staining must involve charged groups, this location of the opaque regions means that charged groups are accumulated at this surface and that they might be shielded off more or less completely from the matrix by predominantly nonpolar regions in the membranes. I t also shows a polarity of the membrane elements of the cristae. The presence of a high concentration of charged groups at this level can also explain the tendency of the membrane elements of the cristae to separate to form an intracristal space because of the affinity for water of the charged groups. The presence of the opaque regions with their characteristically uniform distribution in the membranes can be interpreted to show that there is a certain orderly arrangement of the components within the membranes and that membrane molecules are not randomly distributed. It can fur-

317

thermore be assumed that the regions within which the stained groups are accumulated are sufficiently large to involve stained groups belonging to several molecules, since a massive staining to this extent is unlikely to occur in a single molecule like a globular protein molecule. Such an association of molecules would be in agreement with the concept that the enzyme molecules in the membrane are present in the form of multimolecular complexes. A denaturation of the membrane proteins with secondary accumulation of charged groups in regions of these dimensions is a possibility which is considered less likely to explain the staining of the inner membranes because it seems unlikely that denaturation would give rise to order of this kind. Since the opaque regions are rather intensely stained, they are likely to contain a comparatively high density of charged sites. Assuming that the respiratory chain components were associated in complexes of a particular organization like that proposed by Sj6strand and Barajas (197{}), there would be a considerable accumulation of charged groups in these complexes. The large number of opaque regions and their fairly uniform size and distribution in the membranes shows that they reflect the sites of a major component of these membranes. It then seems justifiable to tentatively propose that these sites are the sites of respiratory chain complexes, although there are at present no ways to exclude that other multimolecular complexes could be involved. The opaque regions would correspond to a limited region within the respiratory chain complex and/ or some other multimolecular complexes where charged groups are particularly densely arranged and would not reflect the actual dimensions of any such complex. In addition to the opaque areas, it was possible to observe indications of a particulate substructure in both inner and outer membrane elements. The particles had the dimensions of globular protein molecules. In addition to the size of these particles, 30

318

FRITIOF S. sJOSTRAND

to 50 A, matching that of globular protein molecules, they were stained the way it can be predicted that globular proteins would stain when in a state close to their native conformation; i.e., the staining was confined to the periphery of the particles, while the interior of the particles showed a low opacity. It was also obvious that there were no continuous, uninterrupted layers present in these membrane elements. The analysis therefore did not give support to the concept that these membranes consist of a backbone structure formed by a continuous, uninterrupted lipid bilayer. Neither were any interrupted layered regions observed. This would indicate that the lipids are dispersed in aggregates of rather small dimensions comparable in size to those of the particulate substructure or are present in more specific lipid-protein complexes. It was obvious that the cutting of the thinnest possible sections caused a certain perturbation of the membrane structure due to compression. It is important to improve the sectioning technique in such a way that sectioning damage is reduced when cutting at the limit of section thickness. It is also important to consider the effects due to beam damage. However, it seems unlikely that the particulate substructure would be caused by beam damage, because the dimensions of the particles are above the range within which beam damage could produce a structure by producing holes in the specimen. Beam damage can well affect the shapes of targets of the size of globular protein molecules, which means perturbation at a level below that considered here, where mainly structural components in the 40-50-A range are considered. Theoretical Considerations

The need for using strong detergent action to solubilize the membrane proteins shows that the association of the components of the membranes is to a great ex-

tent due to nonpolar interaction. As a consequence, the membranes can be conceived of as predominantly nonpolar regions in the cell with water being excluded more or less completely from entering these regions. The cell could then be considered to be a two-phase system with an aqueous and a nonaqueous phase. When referring to this type of membranes as regions of a predominantly nonpolar nature, it is intended to emphasize the function of this type of membranes as sites for cell metabolism in contrast to the concept of membranes as boundary structures delimiting compartments in the cells. In the case of mitochondrial inner membranes or cristae membranes, the latter function is considered questionable on the basis of the observations reported in this paper. In other situations, the two functions, the metabolic function not associated with particular permeability properties and the boundary function, seem to be combined as in the rough surfaced cytomembranes. In still other cases, the boundary function is likely to be the dominant function of the membrane as in the case of at least certain types of plasma membranes. In the last case, the metabolism associated with the membrane might be confined to securing particular permeability properties of the membrane. The nonpolar character of the membranous regions would furnish special conditions for interaction between components in multienzyme systems. These conditions might well be necessary for the functioning of systems like the respiratory chain. The exclusion of water within these regions should favor charge interaction between substrate and enzyme and between components of multienzyme systems. The latter could also be of importance for the proper spatial arrangement of the components of such systems. The restricted exposure of protein molecules to an aqueous environment would furthermore reduce the restrictions in conformation of protein molecules imposed by water. It seems rea-

MITOCHONDRIAL MEMBRANE STRUCTURE

sonable to assume that this would favor reversible conformational changes. This fluctuation of the conformation could be of importance for the interaction of the molecules. It could furnish an important basis for a mechanism such as that proposed by Boyer (1974, 1977) and Rosing et al. (1977), according to which the coupling of phosphorylation to respiration in the respiratory chain would be mediated through conformational changes in proteins in the respiratory chain. Such conformational changes would thus be favored in the predominantly nonpolar environment of the membrane. At the same time, the absence of any electrical shielding effect of water molecules on charged sites at the surface of protein molecules in the membrane could favor conformational changes induced by changes in the distribution of charges within the membrane. The requirement for a nonpolar environment for the proper interaction between molecules in multienzyme systems would give a simple explanation why submitochondrial particles depend upon the presence of lipids to be functional. The membrane as a structure with predominantly extension in two dimensions will mean a suitable adaptation to the requirement of facilitated exchange of metabolites between the nonpolar and the aqueous phases by offering a large surface area for this exchange. By limiting the thickness of the membranes, that is, of the nonpolar region, water could exert a sufficient restriction on protein conformation to prevent too extensive conformational changes caused by the denaturing effect of a pure nonpolar environment. REFERENCES ANDERSSON-CEDERGREN, E. (1959)J. Ultrastruct. Res. Suppl. 1.

BOYER, P. D. (1974) in ERNSTER, ESTABROOK,AND SLATER (Eds.), Dynamics of Energy-Transducing

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Membranes, p. 289, Elsevier, Amsterdam. BOYER, P. D. (1977) in Trends in Biochemical Research, in press. CLELAND, K. W., AND SLATER,E. C. (1953) Biochem. J. 53, 547. DAEMS, W. TH., AND WISSE, E. (1966) J. Ultrastruct. Res. 16, 123. FLEISCHER, S., FLEISCHER,B., AND STOECKENIUS,W. (1965) Fed: Proc. 24, 296. LENARD, J., AND SINGER, S. J. (1968)J. CelIBiol. 37, 117. MUSCATELLO, U., GUAERIERA-BOBYLEVA,V., AND BUFFA, P. (1972) J. Ultrastruct. Res. 40, 235. PALADE, G. E. (1952) Anat. Rec. 114, 427. PALADE, G. E. (1953) J. Histochem. Cytochem. 1, 188. ROSING, J., KAYALAR,C., AND BOYER, P. D. (1977) J. Biol. Chem., in press. SJOSTRAND, F. S. (1948) J. Appl. Physics 19, 1188. SJOSTRAND, e. S. (1949) J. Ceil. Comp. Physiol. 33, 383. SJOSTRAND,f . S. (1953)Experientia 9, 68. SJOSTRAND, F. S. (1953) J. Cell. Comp. Physiol. 42, 45. SJOSTRAND, F. S. (1952)Nature (London) 171, 30 (submitted August 22, 1952). SJOSTRAND,F. S. (1954)Z. Wiss. Mikr. Mikr. Techn. 62, 65. SJOSTRAND, F. S. (1960) Rad. Res. Suppl. 2, 349. SJOSTRAND,F. S. (1967) Electron Microscopy of Cells and Tissues, Vol. 1, Academic Press, New York. SJOSTRAND,F. S. (1976)J. Ultrastruct. Res. 55, 271. SJOSTRAND, F. S., AND BARAJAS, L. (1968) J. Ultrastruct. Res. 25, 121. SJOSTRAND, F. S., AND BARAJAS, L. (1970) J. Ultrastruct. Res. 32, 293. SJOSTRAND, F. S., AND BERNHARD, W. (1976) J. Ultrastruct. Res. 56, 233. SJOSTRAND, F. S., AND HANZON, V. (1954) Exp. Cell Res. 7, 393. SJOSTRAND,f. S., AND KRETZER, F. (1975) J. Ultrastruct. Res. 53, 1. SJOSTRAND, F. S., AND RHODIN, J. (1953) Exp. Cell Res. 4, 426. SJOSTRAND, F. S., AND RHODIN, J. (1953) J. Appl. Phys. 24, 116. STOECKENIUS,W. (1959) J. Biophys. Biochem. Cytol. 5, 491. TANFORD, C., HAVENSTEIN, J. D., AND RANDS, D. (1955) J. Amer. Chem. Soc. 77, 6409. TANFORD, C., SWANSON, S. A., AND SHORE, W. S. (1955) J. Amer. Chem. Soc. 77, 6414. TANFORD, C., BUCKLEY, C. E., III, DE PARITOSH,K. K., AND LIVELY, E. P. (1962)J. Biol. Chem. 237, 1168.