Are there knobs on energy transducing membranesin situ?

JOURNAL

OF

ULTRASTRUCTURE

RESEARCH

93, 138-l

43 (1985)

Are There Knobs on Energy Transducing Membranes in Situ? W. W. WAINIO Department of Biochemistry, Faculty of Arts and Sciences and Cook College, and the Bureau of Biological Research, Rutgers- The State University of New Jersey. Busch Campus, Piscataway, New Jersey 08854 Received December IO, 1985, and in revised form February 19, I986 It is argued here that the projections which are frequently seen on the matrix side of the mitochondrial inner membranes and which are characterized as globules or spherical projections or as knob-on-stalks are an artifact of the preparation and/or of the staining of the submitochondrial particles or mitochondria. In sectioned or freeze-fractured preparations of intact cells or mitochondria, the externalized spheres are rarely seen on the membranes. They are, however, almost always seen on fragmented preparations, especially if they have been negatively stained with 0 1985 Academic Press, Inc. phosphotungstate.

A recent publication by Telford et al. ( 1984) prompts me to make, and to support, the assertion that the knobs-on-stalks which are claimed by many to be a structural feature of the surface of mitochondria, chloroplasts, and some bacterial membranes are an artifact of the preparation of the particles and/or of negative staining. The particles studied by Telford et al. were either submitochondrial particles prepared from bovine heart muscle mitochondria by sonic oscillation in the presence of pyrophosphate, or ASU vesicles, i.e., submitochondrial particles prepared from bovine heart mitochondria by sonic oscillation in ammonia, passed through Sephadex, and treated with urea. The diameter of ASU vesicles is from 50 to 300 nm or l/20 to l/3 that of a typical rat liver mitochondrion. The vesicles are thus modified mitochondria, in that the cristae have become spherical membranes without invaginations due to the sonic oscillation. The matrix (inner) surface of the cristae is no longer protected by the outer membrane and is fully exposed to the aqueous medium and to any negative stain added. Since F,, the soluble ATPase, can be isolated as spheres approximately 90 %, in diameter and having a molecular weight of 350 kDa, it may be assumed that the spheres

are embedded in the matrix surface of the inner membrane ready to be “popped out” by sonic oscillation and/or dilution of the medium and/or negative staining. When the conditions are not too drastic, the spheres may remain attached to the mitochondrial ribbons or fragments by “stalks” which may be assumed to be the hydrophobic component of the larger (550 kDa) oligomycinsensitive ATPase. Otherwise, being hydrophilic, the spheres become soluble. Following the claims of Fernandez-Moran et al. (1962) that bovine heart muscle mitochondria have distinct arrays of particles on both sides of the membrane fragments, SjGstrand et al. (1964) had this to say: “The fact that edge-bound particles are not seen or very rarely seen in fresh preparations and that they are found very frequently after treatment with distilled water or Tyrode’s solution, or after incubation in distilled water at 37°C makes it justifiable to conclude that these structures are formed artificially in connection with the deterioration of the mitochondrial structure and that they do not represent the normal in viva structural organization of mitochondrial material.” Sjiistrand et al. were able to demonstrate that there was a direct relationship between the length of time of exposure of rat heart muscle mitochondria to distilled

138 0022-S320/85 $3.00 Copyright 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

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TRANSDUCING

MEMBRANES

139

water or Tyrode’s solution or 0.44 M su- enough out from the surface of the inner crose and the frequency with which edge- membranes to account for the stalked inner bound particles appeared. membrane particles of Fem&ndez-Moran.” Over a period of several years there was Instead, the matrix surface is shown to be much apparent support for the existence of relatively smooth due partially to a rather dense arrangement of particles having flat projecting particles with stalks (Stoeckenius, 1963; Smith, 1963; Parsons, 1963; Fer- surfaces. The dimensions of these particles n&ndez-Moran et al., 1964; Greville et al., exceed those of single protein molecules 1965), with only Malhotra and E&in (1967) which suggests to the authors that there are multimolecular aggregates. as another dissenting voice. They concluded that: “The presence of stalked particles may Only in two instances, where the mitobe dependent upon the physiological state chondria were clearly damaged, were proof the mitochondria and the procedure of jections seen in sectioned preparations. negative staining. Thus the apparent pres- Sjostrand (1968) found that rat intestinal ence or absence of stalked elementary par- epithelial cell mitochondria in which the inticles in electron micrographs may not in ner membrane had been transformed into itself have a physiological significance.” vesicular and tubular structures by aging and Malhotra and Eakin found that if mitoexposure to osmotic changes had projecchondria of Neurospora crassa were iso- tions on the surfaces of the tubules. Telford lated from 0.25 M sucrose containing 1 x and Racker ( 1973) fixed bovine brain mi1O-3 M ethylene-diamine tetraacetic acid tochondria with glutaraldehyde and OsO,, (EDTA), the mitochondria had no stalked and then stained with uranyl acetate and particles after staining with phosphotunglead citrate before thin sectioning, and notstate, even though they were disrupted. ed that there were rows of spheres on the Omission of the EDTA from the sucrose damaged mitochondria. In only one out of three freeze-fractured solution permitted the demonstration of preparations were there particles on the marows of particles on ribbon-like membranes. trix surface (Vail et al., 1972). Bovine heart The membrane fragments to which par- mitochondria were frozen in 30% glycerol ticles are seen to be attached, resemble the and fractured. The authors noted that the mitochondria exhibited large amplitude normal membrane only remotely. Many fragments are irregular in shape and the swelling, and damage, as a consequence of placement of knobs is far from uniform on their complete permeability to glycerol. these. Other fragments are characterized as In Table I, Part 3, there are listed 15 pubbeing ribbon-like and it is on these that the lications where stalked particles or knobspacing of the knobs is more regular. like structures were observed. Alkaline The most compelling evidence that ar- phosphotungstate was the negative stain algues against the existence of stalked “knobs” most without exception, In only four pubor “lollipops” is the absence of such pro- lications was it reported that the projections jecting structures in intact isolated mitowere not seen on these fragmented prepachondria or in mitochondria in intact cells. rations. When Ehrlich ascites cell mitoWhen mitochondria are either sectioned chondria were treated with phosphotung(Table I, Part 1) or freeze-fractured (Table state after the mitochondria were separated I, Part 2) and viewed in the electron microout of0.5 Msucrose (Mitchell, 1967a) there scope there were usually no projecting were no projections. It may be that the high “knobs” attached to either side of the inner concentration of sucrose condensed the mimitochondrial membrane. As Sjostrand and tochondria and prevented disruption of the Cassel (1978) stated, “There are no indiouter membrane and consequently alteracations that any particles are extending far tion of the inner membrane by the stain. In

140

W.W.WAINIO TABLE I KNOBS

Are there knobs?

ON FRAGMENTED,

Preparation

No

Bovine heart mitochondria

No

Mouse kidney

No No

Beef heart mitochondria Mouse pancreas

No

Mouse liver

No

Mouse cerebellar cortex, medulla oblongata, cerebral cortex Rat kidney

No No

Brown adipose tissue of newborn rabbit

Yes

Rat intestine mitochondria

No

Rat kidney

Yes

Bovine brain mitochondria

No

Rat heart

No

Mouse cerebellum

Yes

Bovine heart mitochondria

SECTIONED,

Treatment

AND

FREEZE-FRACTURED

PREPARATIONS

Observations

PART 1: SECTIONED PREPARATIONS Os04 fixed at low temUniform round or polyperature; sectioned hedral particles within the membrane In vivo fixation with perGlobular components in manganate or Os04; the mitochondria postfixed with many1 membrane separated acetate; thin-sectioned by septa 0~0~ fixed; Pb(OHb Spheres in membrane, stained, sectioned none projecting Liquid Nz; lyophilized; Rows of globules in miOs04 fixed; sectioned; tochondrial membrane; many1 acetate stain no projections Liquid Nr; acetone conQuintuple-layered pattern taming 0~0~; sectioned; membrane lead citrate or many1 acetate stain Liquid NZ; acetone conQuintuple-layered pattern taining OsOd sectioned; membrane lead citrate or many1 acetate stain Sectioned, many1 acetate No knobs on mitochonand lead citrate, or dria; no room for phosphotungstate stain knobs Glutaraldehyde fixed; Globular structures in sectioned, 0~0~ postcristae [no knobs] fixed phosphotungstate stain Fixed with 1% 0~0~ in Projections on intramito0.4 M sucrose-Krebs chondrial vesicles and Ringer’s tubules (a clear indication of damage to inner membrane) Perfused with glutaraldeGlobular structures withhyde; tissue pieces dein mitochondrial memhydrated with ethylene branes [no knobs] glycol; sectioned Glutaraldehyde and 0~0, Rows of spheres [with fixed, uranyl acetate clear evidence of damand lead citrate stain, age to mitochondria] thin-sectioned Perfused with glutaraldeGlobular structures withhyde; tissue dehydratin the membranes [no ed, sectioned knobs] Rapid freezing; freeze Typical cristae [no projections] substitution in 0~0~; sectioned PART 2: FREUE-FRACTURED PREPARATIONS 30% glycerol; freeze-fracParticles on matrix surtured face; large amplitude swelling with complete permeability to glycerol

Reference Femandez-Moran, 1962 Sjiistrand,

1963

Femartdez-Moran et al., 1964 Sjijstrand and Elfvin, 1964 Malhotra and Van Herreveld, 1965 Malhotra,

1966

Pease, 1966 Lindberg et al., 1967 Sjiistrand, 1968

SjGstrand and Barajas, 1969 Telford and Racker, 1973 Sjiistrand,

1977

Malhotra and Sikerwar, 1983

Vail

et al., 1972

KNOBS ON ENERGY

TRANSDUCING

141

MEMBRANES

TABLE I CONTINUED

Are there knobs?

Preparation

Treatment

Observations

No

Rat heart mitochondria

Freeze-fractured

No

Rat kidney tubule cell

Yes

Bovine heart mitochondria

Yes

Neurospora crassa mitochondria Calliphora muscle sarcosomes

Freeze-fractured through mitochondria PART 3: FRAGMENTED PALEPARATIONS In buffered phosphoDistinct arrays of partitungstate cles on both sides of membrane fragments Spherical particles with Phosphotungstate added to grid stalks Particles with head and Disrupted with water; stained with phosphostalk tungstate Mitochondria on a needle Projecting subunits havdipped into phosphoing head and stem tungstate Microdroplet cross-sprayA repeating particle: ing employing phoshead, stalk, and base photungstate Diluted with water; Particles on edges of stained with phosphostrands tungstate Particles on edges of Diluted with water 2 days before staining strands with phosphotungstate Tubular elements studSonicated, stained with phosphotungstate ded stalked particles Distilled water; bovine Knob-like subunits on serum albumin; phosedges of fragments photungstate stain Phosphotungstate to miNo projections tochondria out of 0.5 M sucrose Phosphotungstate to miDisrupted mitochondria tochondria changed with knobs from 0.125 M sucrose to 0.063 M sucrose Knobs Mitochondria spread on layer of phosphotungstate Inner membrane particles French press; phosphotungstate stain 10% water in acetone; Breaks in membrane; phosphotungstate stain particulate matter on membrane; densitometer trace shows no knobs Typical knobs Sonicated, stained with phosphotungstate

Yes

Yes

Eleven types of mitochondrla

Yes

Bovine heart mitochondria

Yes

Rat kidney cortex mitochondria

Yes

Rat heart mitochondria

Yes

Callifora muscle sarcosomes Bovine heart mitochondria

Yes No

Ehrlich ascites cell mitochondria

Yes

Ehrlich ascites cell mitochondria

Yes

Ehrlich ascites cell mitochondria

No

Bovine heart mitochondria Bovine heart mitochondria

No

Yes

Rat liver mitochondria

Matrix surface fairly smooth, dense array of particles with flat surfaces Fairly smooth matrix surface

Reference Sjdstrand and Cassel, 1978 Sjostrand, 1980

Femfurdez-Moran, 1962 Stoeckenius, 1963 Smith, 1963

Parsons, 1963 Fem&ndez-Moran et al., 1964 Sjiistrand et al., 1964 Sjostrand et al., 1964 Greville et aI., 1965 Cunningham et al., 1967 Mitchell,

1967a

Mitchell,

1967a

Mitchell,

1967b

Fleischer et al., 1967 Fleischer et al., 1967

Silver and Hall, 1967

142

W. W. WAINIO TABLE I CONTINUED

Are there knobs?

Preparation

No

Neurospora crassa mitochondria

Yes

Neurospora crassa mitochondria

No

Tubuli of mitochondria of Tetrahymena pyriformis

Yes

Fragments of ghosts of Mycobacterium phlei Thylakoids from spinach Thylakoids from spinach Thylakoids from spinach

Yes No Yes

Treatment

Observations

Isolated in sucrose and EDTA, phosphotungstate stain Isolated in sucrose without EDTA, phosphotungstate stain Fixed in glutaraldehyde; postfixed in 0~0,; stained with phosphotungstate or molybdate Phosphotungstate in 0.03% sucrose Phosphotungstate

stain

Silicotungstate; phosphotungstate stain Stained with phosphotungstate; deep-freeze etched

another instance (Fleischer et al., 1967), bovine heart mitochondria were extracted with 10% water in acetone before the phosphotungstate was applied. There was particulate matter on the membranes, but a densitometer trace across the membranes revealed no knobs. The tubuli of the mitochondria of Tetruhymena when finally stained with ammonium molybdate had no stalked particles, even though the particles were seen after final staining with phosphotungstate (Krebs et al., 1972). In the fourth instance, treatment with silicotungstate before phosphotungstate, prevented the appearance of particles on thylakoids from spinach (Garber and Steponkus, 1974). The narrowness of the matrix space in several kinds of mitochondria also makes it difficult to accept the presence of unseen stalked particles on both faces of the opposing membranes. In a study of isolated rat heart muscle mitochondria (Sjostrand, 1977), the total thickness of a crista (two inner membranes) was 250 A, while the width of the matrix space was 100 A. How

Reference

No stalked particles even though disrupted

Malhotra and Eakin, 1967

Rows of elementary partitles on ribbon-like membranes Phosphotungstate-some stalked particles Molybdate-no stalked particles Sphere-like structures attached by a stalk

Malhotra and Eakin, 1967

Numerous particles tuberating from edges No particles Numerous particles

Krebs et ai., 1972

Asano et al., 1973 Garber and Steponkus, 1974 Garber and Steponkus, 1974 Garber and Steponkus, 1974

could opposing rows of particles having diameters of 85-90 8, each and attached to stalks 30-50 A long be accommodated in a 100 8, space? The same argument applies to mitochondria in rat kidney proximal tubule cells (Maunsbach, 1966) and in the flight muscle of the blowfly, Calliphora erythrocephalu (Diptera) (Pressman, 1965). If the knobs were to extend into the matrix, it may also be asked whether they could then make contact with the enzymes of the respiratory chain from whence flows the free energy for the synthesis of ATP? Altematively, for what purpose would the ATP hydrolase/synthetase be so far removed from the other enzyme complexes of the inner membrane? If the particles are indeed artifacts of preparation and/or negative staining, it should be of interest to the authors of textbooks of biochemistry to set the record straight. Without exception the books carry such statements as “particles that resemble lollipops” (Rahn, 1983), “protruding structures called elementary bodies, or inner

KNOBS

ON

ENERGY

TRANSDUCING

membrane particles” and “spherical knoblike projections” (Zubay, 1983). Only one author states that “the enzyme molecules that synthesize ATP from ADP and phosphate are embedded in the inner membrane” (Lehninger, 1982), although he, as well as others, have figures or photographs showing the projecting knobs. Supported by a grant from the Charles and Johanna Busch Memorial Fund to the Bureau of Biological Research.

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J. Ultrastruct. Res. 38, 605-607. LEHNINGER, A. L. (1982) Principles of Biochemistry, Worth, New York. LINDBERG, O., DE PIERRE, J., RYLANDER, E., AND AFZELIUS, B. A. (1967) J. Cell Biol. 34,293-310. MALHOTRA, S. K. (1966) J. Ultrastruct. Rex 15, 1437. MALHOTRA, S. K., AND EAKIN, R. T. (1967) J. Cell Sci. 2,205-212. MALHOTRA, S. K., AND SIKERWAR, S. S. (1983) Trends

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S. K., AND VAN HARREVELD,

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MAUNSBACH, A. B. (1966) J. Ultrastruct. Res. 15,283309. MITCHELL, R. F. (1967a) J. Ultrastruct. Rex 18, 257276. MITCHELL, R. F. (1967b) J. Ultrastruct. Rex 18, 277286. PARSONS, D. F. (1963) Science 140,985-987. PEASE. D. C. (1966) J. Ultrastruct. Rex 14, 356-378. PRESSMAN, B. C. (1965) American Chemical Society, Abstracts of the Meeting, Sept., p. 3 1C. RAHN, J. D. (1983) Biochemistry, Harper and Row, New York. SILVER, B. B., AND HALL, J. C. (1967) In (ARCENEAUX, C. J., Ed.), 25th Annual Electron Microscopic Society Meeting, p. 160, Claitor’s Book Store, Baton Rouge, La. SIBSTRAND, F. S. (1963) J. Ultrastruct. Res. 9, 340361. SJ~STRAND, F. S. (1968) In (DALTON, A. J., AND HAGUENAU, F., Eds.), Ultrastructure in Biological Systems, Vol. 4, The Membranes, pp. 15 l-2 10, Academic Press, Orlando, Fla. SJBSTRAND, F. S. (1977) J. Ultrastruct. Rex 59, 292319. SJBSTRAND, F. S. (1980) J. Ultrastruct. Res. 72, 174188. SJBSTRAND, F. S., ANDERSSON-CEDERGREN, E., AND KARLSSON, U. (1964) Nature (London) 202, 10751078. SJBSTRAND, F. S., AND BARNAS, L. (1969) J. Ultrastrut. Res. 25, 121-155. SJBSTRAND, F. S., AND CASSEL, R. 2. (1978) J. Ultrastruct. Rex 63, 111-137. SJBSTRAND, F. S., AND ELF-VIN, L.-G. (1964) J. Ultra-

struct. Rex 10, 263-292. SMITH, D. S. (1963) J. Cell Biol. 19, 115-138. STOECKENIUS, W. (1963) J. Cell Biol. 17,443-454. TELFORD, N. N., LANGWORTHY, T. A., AND RACKER, E. (1984) J. Bioenergetics Biomembr. 16, 335-35 1. TELFORD, J. N., AND RACKER, E. (1973) J. Cell Biol. 57,580-586. VAIL, W. J., RILEY, R. K., AND WILLIAMS, C. H. (1972)

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ZUBAY, G. Reading.

(1983)

Biochemistry,

Addison-Wesley,