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Electronmicroscopic studies of isolated yeast mitochondria with negative staining and thin sectioning methods
Experimental
Cell Research
43, 301-310
301
(1966)
ELECTRONMICROSCOPIC STUDIES OF ISOLATED YEAST MITOCHONDRIA WITH NEGATIVE STAINING THIN SECTIONING METHODS Y. SHINAGAWA, Department
A. INOUYE,
T.
OHNISHI’
and
B. HAGIHARA
of Physiology, Medical School, Kyoto University, Department of Biochemistry, Medical School, Osaka University, Japan Received
January
AND
and
7, 1966
existence of mitochondria in yeast cells was demonstrated with the electron microscope by Agar and Douglas [1], Kawakami and Nehira [ll], Yotsuyanagi [30], Vitols, North and Linnane [29], and by Polakis, Bartley and Meek [17] and others by direct sectioning of cells. Mitochondrial structure in aerobically grown yeast cells was shown to be essentially similar to those in higher organisms, except that yeast mitochondria are a little smaller. Since yeast cells have a thick cell wall, they are rather resistant to the disruption by such usual mechanical procedures, as the use of the Nossal shaker [27] or the blending with glass beads [28]. Yeast mitochondria obtained by these disruption methods seem to be considerably damaged from the morphologic and physiologic view points. In 1959, a milder preparation procedure of yeast mitochondria, based on degradation of yeast cells to protoplasts by treatment with snail gut juice, was reported by Heyman and Blanchet [9, 331. Ohnishi and Hagihara [14, 151 developed this method further and isolated mitochondria from Saccharomyces carlsbergensis which would seem to be fairly undamaged. The biochemical properties of the isolated mitochondria were investigated [16]. Using a similar method, Duell, Inoue and Utter [S] have reported the isolation of mitochondria from spheroplasts of Saccharomyces cerevisiae. In the present communication, we wish to report the morphological aspects of mitochondria isolated from Saccharomyces carlsbergensis, as revealed by negative staining and thin sectioning methods. THE
MATERIALS
AND
METHODS
Preparation and assay.-Mitochondria from Saccharomyces carlsbergensis were prepared as previously described [16]. Respiration, respiratory control and P/O ratios were determined from polarographic tracings by the method of Chance and Williams 1 Present address: holm Va, Sweden.
Wenner-Gren
Institute,
University
of Stockholm,
Norrtullsgatan
Experimental
16, Stock-
Cell Research
43
I’. Shinagawa,
A. Inouye,
T. Ohnishi
and B. Hagihara
12, 41. These parameters were measured with every preparation of yeast mitochondria before electron microscopic examinations. EIectron microscopy.-Thin sectioning techniques: mitochondrial pellets were fixed with fresh buffered fixatives for 1 hr in the cold. The following fixatives were used: Osmium tetroxide; 1 per cent in 1 per cent dichromate buffer [5] (pH 6.5), containing 0.65 M (or 0.33 M) mannitol. Potassium permanganate; 1.2 per cent in 1 per cent dichromate buffer containing 0.65 M (or 0.33 M) mannitol. The dichromate solution was found to have a sufficient buffer capacity in the range of pH 6.5 to 7.4 [22]. After dehydration in an ascending ethanol series, the fixed pellets were immersed in Epon mixture [23] for 2 hr, transferred to capsules containing Epon mixture, and polymerized, first at 37” for IO hr, and then at 55” for 48 hr. Thin sections were cut with a microtome (JUM-5), stained with an aqueous solution of saturated uranyl acetate, and examined in a Hitachi HU-11 electron microscope. Negative staining techniques.-Isolated mitochondria were suspended in 0.025 M mannitol solution and diluted to approximately 0.05 per cent of protein concentration. The mitochondrial suspension was mounted directly (e.g., from a dropper) onto collodion filmed copper grids without carbon coating, since the carbon films made by usual evaporating unit frequently broke during electron microscope observation [lo]. Excess solution was absorbed by a filter paper which was placed beneath the grid. Then a 2 per cent aqueous solution of sodium phosphotungstate (pH 6.5) was further dropped onto the specimen grids. With this technique exposure of mitochondrial membranes to the reagent was minimized. Electron micrographs were made on a Japan Electron Optics JEM-7. Original magnifications were 10,000 and 20,000 and micrographs were further enlarged photographically. RESULTS Oxidative
and phosphorylative
properties
of yeast mitochondria
The mitochondria prepared from yeast protoplasts catalyze the aerobic oxidation of various members of the tricarboxylic acid cycle and tetramethylpara-phenylene diamine reduced by ascorbate just as do animal mitochondria. A characteristic property of yeast mitochondria is that they can also catalyze the aerobic oxidation of externally added NADH, of D- and I,lactate,and of ethanol. The respiratory rate with all of these substrates is regulated by inorganic phosphate or by phosphate acceptor, ADP. With succinate, or with tetramethyl-para-phenylene diamine reduced by ascorbate, as the substrate, a P/O ratio of about 1.8 and 1.0 respectively was observed, just as in case of animal mitochondria, while the P/O ratio with NADH or NAD-linked substrate was not higher than 2. Furthermore, the respiration with NADH or NAD-linked substrates was completely insensitive to rotenone and Amytal, in sharp contrast to that of animal mitochondria. Experimental
Cell Research
43
Electronmicroscopic
Fig. I.-Thin nitol. (Arrows 20 - 661807
section indicate
studies of isolated yeast mitochondria
of the mitochondrial fibers which might
fraction fixed be DNA.)
with
osmium
tetroxide
Experimental
303
in 0.33 M m..~:-
Cell Research
43
304
Y. Shinagawa,
Electron
A. Inouye,
T. Ohnishi
and B. Hagihara
microscopy
Figs. 1 and 2 show typical electron micrographs of the thin sections of the mitochondrial fraction fixed with osmium tetroxide. They indicate that the mitochondrial fraction is less contaminated by non-mitochondrial elements and fragments of mitochondria than those prepared by mechanical procedures, a fact concordant with our biochemical findings on this fraction. Detailed examination of the micrographs revealed, however, that the fraction is not completely free of certain contaminants such as membranous elements presumably originating from plasma membrane or the endoplasmic reticulum, and dense bodies which might be lipid granules. The isolated yeast mitochondria show a general resemblance in their appearance to those of higher organisms as well as to yeast mitochondria in situ as observed after sectioning the cells, as reported in our previous paper [16]. The average size of the isolated mitochondria is about 0.7 x 0.5 ,u. Electronmicrographs of the thin sections of mitochondrial pellet fixed by immersion in buffered osmium tetroxide indicate fairly good preservation of the form of mitochondria and the cristae mitochondriales. When the tonicity and pH of the hxative were adjusted to the physiologically optimal levels of yeast mitochondria (0.65 M sorbitol, pH 6.5), the maintenance of the tine structure of isolated mitochondria was also improved to some extent. The internal membranes have a tendency to be organized into vesiculotubular structures; the profiles of vesicles are about 1000 A in diameter. This tendency was also pointed out by Duell, Inoue and Utter [6]. Fibers which might represent DNA are seen in mitochondria (Fig. 1). Credence is given to this interpretation by the experiments by others. Yotsuyanagi [31] has demonstrated histochemically the presence of DNA fibers in yeast mitochondria. Schatz, Haslbrunner and Tuppy [21] have isolated DNA from yeast mitochondria. In addition, a number of other investigators, have claimed that DNA is present in miusing histochemical techniques, tochondria of various organisms [13, 20, 321. Permanganate fixation revealed the double membrane structure of mitochondria, but most of the cristae were disrupted as shown in Fig. 3. In a suspension of negatively stained yeast mitochondria, swollen mitochondria were observed (Fig. 4). They contain a large number of_[membra-
Fig. 2.-Thin nitol.
section
of the mitochondrial
Fig.
section
of mitochondria
3.-Thin
Experimental
Cell
Research
43
fraction fixed
with
fixed
with
permanganate
osmium
tetroxide
in 0.65 M man-
in 0.65 M mannitol.
Electronmicroscopic
studies of isolated yeast mitochondria
Experimental
305
Cell Research
43
306
I’. Shinagawa, A. Inouye, T. Ohnishi and B. Hagihara
Fig. Experimental
Cell Research
4.--Negatively 43
stained
yeast
mitochondria.
Electronmicroscopic
Fig.
studies of isolated yeast mitochondria
5.-Negatively
stained
cristae
307
fragments.
nous fragments-presumably fragments of cristae-of various size. On the surfa ce of these cristae, knob-like structures are observed. By the hypot onic treat1 nent, however, most mitochondria have been disrupted (Fig. 5) and fracti .onated cristae have floated out. Ribbon-like and plate-like image :s of Experimental
Cell Reseal rh 43
308
Y. Shinagawa,
A. Inouye,
T. Ohnishi
and B. Hagihara
cristae occur with about the same frequency. The electron micrographs in Figs. 4 and 5 were taken from the same grid. The knob-like structures appear more distinct on the cristal fragments floated out from the mitochondria than on those inside the swollen mitochondria. These knob-like structures are distributed less densely than in mammalian heart or liver mitochondria [7, 181. The particles on the stalks seem to be rather non-spherical and their mean size is about 80 x 60 A, i.e., somewhat smaller than those in other organisms hitherto reported [7, 18, 24, 26 1. At higher magnification it was found that particles were attached to both sides of the double membranes of cristae, with narrow stalks of 40 to 50 A of length. The thickness of the double membrane of cristae is 40 to 50 A, which corresponds to that of osmium fixed specimens in the thin sections.
DISCUSSION
The electron micrographs of thin sectioned specimens of yeast mitochondrial fraction presented in this paper demonstrated a better preservation of their internal structure and less contamination than those reported hitherto [6, 28, 331. This suggests that the preparation method applied in the present experiment is milder than that using a mechanical disintegration of the yeast cells or than those with enzymatic treatment so far reported. It is known that the nature of the fixatives and of the embedding media considerably modify the preservation of internal structure of the specimens. The vesiculotubular profiles of the cristae mitochondriales may be caused by specimen damage due to the osmium fixation. On the other hand, the permanganate fixation scarcely showed such a vesicular structure but disrupted most of the cristae. As a whole, preservation of mitochondrial structure seemed better in osmium fixed specimens, since the picture of cristae in the negatively stained mitochondria is concordant with that of osmium fixed sample in thin sections. Especially the use of buffered (pH 6.5) isotonic (0.65 M mannitol) fixatives, a medium very close to that of the physiologically optimal for the isolated yeast mitochondria, appeared to be effective in preserving mitochondrial fine structure. When yeast mitochondria were exposed to very hypotonic medium and stained with phosphotungstic acid, knob-like structures were observed on the surface of the cristae. The distribution of these structures was less dense and more irregular than reported for mitochondria from other organisms such as beef heart [a], rat liver [la], insect flight muscle [24], or Neurospora crassa [26], although less dense spacing of the knobs has also been observed Experimental
Cell
Research
43
Electronmicroscopic
studies of isolated yeast mitochondria
309
with certain types of animal mitochondria, e.g., ascaris and bee [3]. However, the negatively stained knobs of yeast mitochondria were of a smaller size than that of other mitochondria. Since the function of the knob-like structures is not yet settled [3, 12, 18, 19, 251, it is difficult at the present time to assess the implications of these differences.
SUMMARY
Yeast mitochondria isolated by treatment of yeast cells, S. carlsbergensis, with snail gut juice were studied with the negative staining and thin sectioning methods. The mitochondrial fraction was little contaminated by nonmitochondrial fractions, and the mitochondrial structure seemed to be fairly undamaged. With the negative staining method, knob-like structures on the surface of cristae were clearly observed; the particles on the stalks were rather nonspherical and their mean size was about 80 x 60 A, i.e., somewhat smaller than those reported in other organisms. We wish to express our gratitude to Dr L. Ernster and Dr B. A. Afzelius, WennerGren Institute, for having read this manuscript and their instructive discussions. Thanks are also due to Dr Y. Yotsuyanagi in Laboratory of Physiological Genetics of C.N.R.S. in France for his helpful discussions. We are grateful to Miss H. Komatsu for the excellent technical assistance. REFERENCES D. and DOUGLAS, H. C., J. Bact. 73, 365 (1957). B.. in G. E. W. WOLSTENHOLME and C. M. O’CONNOR (eds.), Ciba Foundation S$nposium on the regulation of cell metabolism, p. 19. J. & A.‘Ch&hill, Ltd., London, 1959. CHANCE, B., PARSON, D. F. and WILLIAMS, G. R., Science 142, 1176 (1963). CHANCE, B. and WILLIAMS, G. R., Advan. Enzymol. 17, 65 (1956). DALTON, A. J., Anat. Rec. 121, 281 (1955). DUELL, E. A., INOUE, S. and UTTER, M. F., J. Bact. 88, 1762 (1964). FERNANDEZ-MORAN, H., Circulation 26, 1039 (1962). FERNANDEZ-MORAN, H., ODA, T., BLAIR, P. V. and GREEN, D. E., J. Cell Rio!. 22, 63 (1964). HEYMAN-BLANCHET, T., ZAJDELA, F. and CHAIX, P., Biochim. Bivphys. Acta 36, 569 (1959). INOUYE, A., SHINAGAWA, Y., MASUMURA, S. and DATE, Y., J. Electronmicroscopy 12, 192
1. AGAR,
H.
2. CHANCE.
3. 4. 5. 6. 7. 8. 9. 10.
(1963). 11. 12. 13. 14.
15. 16. 17. 18.
KAWAKAMI,
LBw,
NEHIRA, T., J. Ferm. Technology 36, 393 (1958). B. A., Expfl Cell Res. 35, 431 (1964). S. and AFZELIUS, B. A., Exptl Cell Res. 37, 516 (1965). and HAGIHARA, B., J. Biochem. 56, 484 (1964). VI Int. Congr. Biochem. New York, 1964. KAWAGUCHI, K. and HAGIHARA, B., J. Biol. Chem. In press. S., BARTLEY, W. and MECK, G. A., Biochem. J. 90, 369 (1964). F., Science 140, 985 (1963).
N.
and
H. and AFZELIUS, M. M. K., NASS,
NASS, OHNISHI, T. Abstracts OHNISHI, T., POLAKIS, E. PARSONS, D.
Experimental
Cell Research
43
310 19.
20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.
Y. Shinagawa, RACKER,
A. lnouye,
T. Ohnishi
and B. Hagihara
E., TYLER, D. D., ESTABROOK, R. W., CONOVER, T. E., PARSONS, D. F. and CHANCE, B., An international symposium on oxidases and related oxidation reduction systems, Amherst, Mass. 1964. RIS, H., Funktionelle und morphologische Organisation der Zelle, p. 1. Springer-Verlag 0 H G, Berlin, Gottingen, Heidelberg, 1963. SCHATZ, G., HASLBRUNNER, E. and TUPPY, H., Biochem. Biophys. Res. Comm. 15, 127 (1964) SHINAGAWA, Y. and SHINAGAWA, Y., J. Elrctronmicroscopy 14, 56 (1965). SHINAGAWA, Y. and UCHIDA, Y., J. Electronmicroscopy 10, 86 (1961). SMITH, D. S., J. Cell Biol. 19, 115 (1963). STASNY, J. T. and CRANE, F. L., J. Cell Biol. 22, 49 (1964). STOCKENIUS, W., J. Cell Biof. 17, 443 (1963). UTTER, M. F., KEECH, D. B. and NOSSAL, P. M., Biochem. J. 68, 431 (1958). VITOLS, E. and LINNANE, A. W., J. Biophys. Biochem. Cyfof. 9, 701 (1961). VITOLS, E., NORTH, R. J. and LINNANE, A. W., J. Biophys. Biochem. Cyfof. 9, 689 (1961). YOTSUYANAGI, Y., Compf. Rend. Acad. Sci. Paris 248, 474 (1959). ~ Personal communication. YOTSUYANAGI, Y. and GUERRIER, C., Compf. Rend. Acad. Sci. Paris 260, 2234 (1965). ZAJDELA, F., HEYMAN-BLANCHET, T. and CHAIX, P., Compf. Rend. Acad. Sci. Paris 235, 1268 (1961).
Erperimenfal
Cell
Research
43
Cell Research
43, 301-310
301
(1966)
ELECTRONMICROSCOPIC STUDIES OF ISOLATED YEAST MITOCHONDRIA WITH NEGATIVE STAINING THIN SECTIONING METHODS Y. SHINAGAWA, Department
A. INOUYE,
T.
OHNISHI’
and
B. HAGIHARA
of Physiology, Medical School, Kyoto University, Department of Biochemistry, Medical School, Osaka University, Japan Received
January
AND
and
7, 1966
existence of mitochondria in yeast cells was demonstrated with the electron microscope by Agar and Douglas [1], Kawakami and Nehira [ll], Yotsuyanagi [30], Vitols, North and Linnane [29], and by Polakis, Bartley and Meek [17] and others by direct sectioning of cells. Mitochondrial structure in aerobically grown yeast cells was shown to be essentially similar to those in higher organisms, except that yeast mitochondria are a little smaller. Since yeast cells have a thick cell wall, they are rather resistant to the disruption by such usual mechanical procedures, as the use of the Nossal shaker [27] or the blending with glass beads [28]. Yeast mitochondria obtained by these disruption methods seem to be considerably damaged from the morphologic and physiologic view points. In 1959, a milder preparation procedure of yeast mitochondria, based on degradation of yeast cells to protoplasts by treatment with snail gut juice, was reported by Heyman and Blanchet [9, 331. Ohnishi and Hagihara [14, 151 developed this method further and isolated mitochondria from Saccharomyces carlsbergensis which would seem to be fairly undamaged. The biochemical properties of the isolated mitochondria were investigated [16]. Using a similar method, Duell, Inoue and Utter [S] have reported the isolation of mitochondria from spheroplasts of Saccharomyces cerevisiae. In the present communication, we wish to report the morphological aspects of mitochondria isolated from Saccharomyces carlsbergensis, as revealed by negative staining and thin sectioning methods. THE
MATERIALS
AND
METHODS
Preparation and assay.-Mitochondria from Saccharomyces carlsbergensis were prepared as previously described [16]. Respiration, respiratory control and P/O ratios were determined from polarographic tracings by the method of Chance and Williams 1 Present address: holm Va, Sweden.
Wenner-Gren
Institute,
University
of Stockholm,
Norrtullsgatan
Experimental
16, Stock-
Cell Research
43
I’. Shinagawa,
A. Inouye,
T. Ohnishi
and B. Hagihara
12, 41. These parameters were measured with every preparation of yeast mitochondria before electron microscopic examinations. EIectron microscopy.-Thin sectioning techniques: mitochondrial pellets were fixed with fresh buffered fixatives for 1 hr in the cold. The following fixatives were used: Osmium tetroxide; 1 per cent in 1 per cent dichromate buffer [5] (pH 6.5), containing 0.65 M (or 0.33 M) mannitol. Potassium permanganate; 1.2 per cent in 1 per cent dichromate buffer containing 0.65 M (or 0.33 M) mannitol. The dichromate solution was found to have a sufficient buffer capacity in the range of pH 6.5 to 7.4 [22]. After dehydration in an ascending ethanol series, the fixed pellets were immersed in Epon mixture [23] for 2 hr, transferred to capsules containing Epon mixture, and polymerized, first at 37” for IO hr, and then at 55” for 48 hr. Thin sections were cut with a microtome (JUM-5), stained with an aqueous solution of saturated uranyl acetate, and examined in a Hitachi HU-11 electron microscope. Negative staining techniques.-Isolated mitochondria were suspended in 0.025 M mannitol solution and diluted to approximately 0.05 per cent of protein concentration. The mitochondrial suspension was mounted directly (e.g., from a dropper) onto collodion filmed copper grids without carbon coating, since the carbon films made by usual evaporating unit frequently broke during electron microscope observation [lo]. Excess solution was absorbed by a filter paper which was placed beneath the grid. Then a 2 per cent aqueous solution of sodium phosphotungstate (pH 6.5) was further dropped onto the specimen grids. With this technique exposure of mitochondrial membranes to the reagent was minimized. Electron micrographs were made on a Japan Electron Optics JEM-7. Original magnifications were 10,000 and 20,000 and micrographs were further enlarged photographically. RESULTS Oxidative
and phosphorylative
properties
of yeast mitochondria
The mitochondria prepared from yeast protoplasts catalyze the aerobic oxidation of various members of the tricarboxylic acid cycle and tetramethylpara-phenylene diamine reduced by ascorbate just as do animal mitochondria. A characteristic property of yeast mitochondria is that they can also catalyze the aerobic oxidation of externally added NADH, of D- and I,lactate,and of ethanol. The respiratory rate with all of these substrates is regulated by inorganic phosphate or by phosphate acceptor, ADP. With succinate, or with tetramethyl-para-phenylene diamine reduced by ascorbate, as the substrate, a P/O ratio of about 1.8 and 1.0 respectively was observed, just as in case of animal mitochondria, while the P/O ratio with NADH or NAD-linked substrate was not higher than 2. Furthermore, the respiration with NADH or NAD-linked substrates was completely insensitive to rotenone and Amytal, in sharp contrast to that of animal mitochondria. Experimental
Cell Research
43
Electronmicroscopic
Fig. I.-Thin nitol. (Arrows 20 - 661807
section indicate
studies of isolated yeast mitochondria
of the mitochondrial fibers which might
fraction fixed be DNA.)
with
osmium
tetroxide
Experimental
303
in 0.33 M m..~:-
Cell Research
43
304
Y. Shinagawa,
Electron
A. Inouye,
T. Ohnishi
and B. Hagihara
microscopy
Figs. 1 and 2 show typical electron micrographs of the thin sections of the mitochondrial fraction fixed with osmium tetroxide. They indicate that the mitochondrial fraction is less contaminated by non-mitochondrial elements and fragments of mitochondria than those prepared by mechanical procedures, a fact concordant with our biochemical findings on this fraction. Detailed examination of the micrographs revealed, however, that the fraction is not completely free of certain contaminants such as membranous elements presumably originating from plasma membrane or the endoplasmic reticulum, and dense bodies which might be lipid granules. The isolated yeast mitochondria show a general resemblance in their appearance to those of higher organisms as well as to yeast mitochondria in situ as observed after sectioning the cells, as reported in our previous paper [16]. The average size of the isolated mitochondria is about 0.7 x 0.5 ,u. Electronmicrographs of the thin sections of mitochondrial pellet fixed by immersion in buffered osmium tetroxide indicate fairly good preservation of the form of mitochondria and the cristae mitochondriales. When the tonicity and pH of the hxative were adjusted to the physiologically optimal levels of yeast mitochondria (0.65 M sorbitol, pH 6.5), the maintenance of the tine structure of isolated mitochondria was also improved to some extent. The internal membranes have a tendency to be organized into vesiculotubular structures; the profiles of vesicles are about 1000 A in diameter. This tendency was also pointed out by Duell, Inoue and Utter [6]. Fibers which might represent DNA are seen in mitochondria (Fig. 1). Credence is given to this interpretation by the experiments by others. Yotsuyanagi [31] has demonstrated histochemically the presence of DNA fibers in yeast mitochondria. Schatz, Haslbrunner and Tuppy [21] have isolated DNA from yeast mitochondria. In addition, a number of other investigators, have claimed that DNA is present in miusing histochemical techniques, tochondria of various organisms [13, 20, 321. Permanganate fixation revealed the double membrane structure of mitochondria, but most of the cristae were disrupted as shown in Fig. 3. In a suspension of negatively stained yeast mitochondria, swollen mitochondria were observed (Fig. 4). They contain a large number of_[membra-
Fig. 2.-Thin nitol.
section
of the mitochondrial
Fig.
section
of mitochondria
3.-Thin
Experimental
Cell
Research
43
fraction fixed
with
fixed
with
permanganate
osmium
tetroxide
in 0.65 M man-
in 0.65 M mannitol.
Electronmicroscopic
studies of isolated yeast mitochondria
Experimental
305
Cell Research
43
306
I’. Shinagawa, A. Inouye, T. Ohnishi and B. Hagihara
Fig. Experimental
Cell Research
4.--Negatively 43
stained
yeast
mitochondria.
Electronmicroscopic
Fig.
studies of isolated yeast mitochondria
5.-Negatively
stained
cristae
307
fragments.
nous fragments-presumably fragments of cristae-of various size. On the surfa ce of these cristae, knob-like structures are observed. By the hypot onic treat1 nent, however, most mitochondria have been disrupted (Fig. 5) and fracti .onated cristae have floated out. Ribbon-like and plate-like image :s of Experimental
Cell Reseal rh 43
308
Y. Shinagawa,
A. Inouye,
T. Ohnishi
and B. Hagihara
cristae occur with about the same frequency. The electron micrographs in Figs. 4 and 5 were taken from the same grid. The knob-like structures appear more distinct on the cristal fragments floated out from the mitochondria than on those inside the swollen mitochondria. These knob-like structures are distributed less densely than in mammalian heart or liver mitochondria [7, 181. The particles on the stalks seem to be rather non-spherical and their mean size is about 80 x 60 A, i.e., somewhat smaller than those in other organisms hitherto reported [7, 18, 24, 26 1. At higher magnification it was found that particles were attached to both sides of the double membranes of cristae, with narrow stalks of 40 to 50 A of length. The thickness of the double membrane of cristae is 40 to 50 A, which corresponds to that of osmium fixed specimens in the thin sections.
DISCUSSION
The electron micrographs of thin sectioned specimens of yeast mitochondrial fraction presented in this paper demonstrated a better preservation of their internal structure and less contamination than those reported hitherto [6, 28, 331. This suggests that the preparation method applied in the present experiment is milder than that using a mechanical disintegration of the yeast cells or than those with enzymatic treatment so far reported. It is known that the nature of the fixatives and of the embedding media considerably modify the preservation of internal structure of the specimens. The vesiculotubular profiles of the cristae mitochondriales may be caused by specimen damage due to the osmium fixation. On the other hand, the permanganate fixation scarcely showed such a vesicular structure but disrupted most of the cristae. As a whole, preservation of mitochondrial structure seemed better in osmium fixed specimens, since the picture of cristae in the negatively stained mitochondria is concordant with that of osmium fixed sample in thin sections. Especially the use of buffered (pH 6.5) isotonic (0.65 M mannitol) fixatives, a medium very close to that of the physiologically optimal for the isolated yeast mitochondria, appeared to be effective in preserving mitochondrial fine structure. When yeast mitochondria were exposed to very hypotonic medium and stained with phosphotungstic acid, knob-like structures were observed on the surface of the cristae. The distribution of these structures was less dense and more irregular than reported for mitochondria from other organisms such as beef heart [a], rat liver [la], insect flight muscle [24], or Neurospora crassa [26], although less dense spacing of the knobs has also been observed Experimental
Cell
Research
43
Electronmicroscopic
studies of isolated yeast mitochondria
309
with certain types of animal mitochondria, e.g., ascaris and bee [3]. However, the negatively stained knobs of yeast mitochondria were of a smaller size than that of other mitochondria. Since the function of the knob-like structures is not yet settled [3, 12, 18, 19, 251, it is difficult at the present time to assess the implications of these differences.
SUMMARY
Yeast mitochondria isolated by treatment of yeast cells, S. carlsbergensis, with snail gut juice were studied with the negative staining and thin sectioning methods. The mitochondrial fraction was little contaminated by nonmitochondrial fractions, and the mitochondrial structure seemed to be fairly undamaged. With the negative staining method, knob-like structures on the surface of cristae were clearly observed; the particles on the stalks were rather nonspherical and their mean size was about 80 x 60 A, i.e., somewhat smaller than those reported in other organisms. We wish to express our gratitude to Dr L. Ernster and Dr B. A. Afzelius, WennerGren Institute, for having read this manuscript and their instructive discussions. Thanks are also due to Dr Y. Yotsuyanagi in Laboratory of Physiological Genetics of C.N.R.S. in France for his helpful discussions. We are grateful to Miss H. Komatsu for the excellent technical assistance. REFERENCES D. and DOUGLAS, H. C., J. Bact. 73, 365 (1957). B.. in G. E. W. WOLSTENHOLME and C. M. O’CONNOR (eds.), Ciba Foundation S$nposium on the regulation of cell metabolism, p. 19. J. & A.‘Ch&hill, Ltd., London, 1959. CHANCE, B., PARSON, D. F. and WILLIAMS, G. R., Science 142, 1176 (1963). CHANCE, B. and WILLIAMS, G. R., Advan. Enzymol. 17, 65 (1956). DALTON, A. J., Anat. Rec. 121, 281 (1955). DUELL, E. A., INOUE, S. and UTTER, M. F., J. Bact. 88, 1762 (1964). FERNANDEZ-MORAN, H., Circulation 26, 1039 (1962). FERNANDEZ-MORAN, H., ODA, T., BLAIR, P. V. and GREEN, D. E., J. Cell Rio!. 22, 63 (1964). HEYMAN-BLANCHET, T., ZAJDELA, F. and CHAIX, P., Biochim. Bivphys. Acta 36, 569 (1959). INOUYE, A., SHINAGAWA, Y., MASUMURA, S. and DATE, Y., J. Electronmicroscopy 12, 192
1. AGAR,
H.
2. CHANCE.
3. 4. 5. 6. 7. 8. 9. 10.
(1963). 11. 12. 13. 14.
15. 16. 17. 18.
KAWAKAMI,
LBw,
NEHIRA, T., J. Ferm. Technology 36, 393 (1958). B. A., Expfl Cell Res. 35, 431 (1964). S. and AFZELIUS, B. A., Exptl Cell Res. 37, 516 (1965). and HAGIHARA, B., J. Biochem. 56, 484 (1964). VI Int. Congr. Biochem. New York, 1964. KAWAGUCHI, K. and HAGIHARA, B., J. Biol. Chem. In press. S., BARTLEY, W. and MECK, G. A., Biochem. J. 90, 369 (1964). F., Science 140, 985 (1963).
N.
and
H. and AFZELIUS, M. M. K., NASS,
NASS, OHNISHI, T. Abstracts OHNISHI, T., POLAKIS, E. PARSONS, D.
Experimental
Cell Research
43
310 19.
20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33.
Y. Shinagawa, RACKER,
A. lnouye,
T. Ohnishi
and B. Hagihara
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