Vol. 140, No. 1
JOURNAL OF BACTERIOLOGY, Oct. 1979, p. 289-293 0021-9193/79/ 10-0289/05$02.00/0
NOTES Scanning Electron Microscope Study of Saccharomyces cerevisiae Spheroplast Formation ALASTAIR T. PRINGLE,'t JOHN FORSDYKE,- AND ANTHONY H. ROSE*
Z7ymology Laboratory, School of Biological Sciences,' and Electron Optics Uni t,2 UnWiersity of Bath, Clatverton Doun, Bath BA2 7AY, Avon, England Received for publication 31 May 1979
A suspension of Saccharomyces ceretisiae NCYC366 in buffered 1.2 M sorbitol containing Zymolyase-5000 (a ,/-glucanase-containing preparation) showed maximum osmotic sensitivity after 30 min of incubation at 30°C. A scanning electron microscope study of spheroplast formation, using a very high resolution (4-nm) machine, revealed several new morphological features. The surface of the plug in bud scars on intact cells appeared warty. The wall, which assumed a beady appearance as digestion proceeded, ultimately sloughed off to reveal the furrowed surface of the plasma membrane. Bud scars were resistant to digestion and, as incubation proceeded, they became surrounded by an outer annulus, which may be the secondary septum. Wall material was completely removed from the majority of cells only after 60 min of digestion. The surface of spheroplasts was studded with particles, about 25 to 30 nm in diameter. Many spheroplasts had a single large indentation, which may be in that part of the plasma membrane originally underlying the birth scar.
Spheroplasts can be prepared from strains of Saccharomyces cereL'isiae by digesting cells with /8-glucanase-containing preparations, such as snail gut juice or various microbial enzymes (15, 19). Strains of this yeast differ in their susceptibility to /8-glucanase digestion (9, 17) and, with refractory strains, spheroplast formation can be accelerated by treating cells with sulfhydryl compounds (3, 7) or proteolytic enzymes (20). Several transmission electron microscope studies of spheroplast formation from S. ceretuisiae have been published (e.g., ref. 6), but so far no detailed examination has been reported involving use of the scanning electron microscope. The present communication reports such a study on S. cereLisiae NCYC366, a strain that is particularly susceptible to digestion with /Bglucanase (1, 8). The study used, as a source of ,8-glucanase, Zymolyase-5000, a preparation obtained from filtrates of Arthrobacter luteus cultures (11, 13). Zymolyase is reported to be free from ,B- (1--2), 13- (1--*4), and ,B- (1--6) glucanases and phosphomannanase (12). It contains an endo-,8-(1-.*3) glucanase which is active on some but not all /8-(1-*3) glucans, and it has been suggested that it may have a preference for glucans possessing a firm molecular aggregation t Present address: Department of Biochemistry, University of Kansas Medical Center, Kansas City, KS, 66103.
due to hydrogen bonding (12). The present study was carried out with a scanning electron microscope that has an extremely high resolving power (about 4 nm), with the result that several hitherto unreported features of spheroplast formation and of the topography of the plasmamembrane surface were revealed. In this study, the term "spheroplast" is used to describe structures from which all, or almost all, of the wall has been removed. MATERIALS AND METHODS Experimental cultures. S. cerevisiae NCYC366 was maintained on slopes of malt extract-yeast extract-glucose-peptone-agar medium (25). Cultures were grown aerobically in 1-liter batches at 30°C as described by Hunter and Rose (10), except that the mixture of vitamins in the medium was replaced by yeast extract (Oxoid Ltd.; 0.1'S, wt/vol). Cells were harvested from late-exponential-phase cultures (0.22 to 0.24 mg, dry wt per ml) by centrifugation at 12,250 x g and 4°C for 1 min, and washed three times in
imidazole-hydrochloride (10 mM)-MgCl2 (10 mM) buffer (pH 6.0) containing 1.2 M sorbitol. Preparation of spheroplasts. Spheroplasts were prepared from freshly harvested cells by suspending them (6 x 108/ml) in imidazole-hydrochloride (10 mM) buffer (pH 6.0) containing 1.2 M sorbitol and Zymolyase-5000 (0.4 mg/6 mg, dry wt of cells). The suspension in a conical flask was incubated at 30°C in a shaker water bath at 100 oscillations per min. Forma289
290
J. BA('TE'KIOL.
NO'T'ES
tion of osmoticallt sensitive structures Ws nmonitored l) c,dldding 0.1 ml of suspension to 2.9 tmil of water oi 2.09mnl of buffered 1.2 M sorl)itol, gently shakinig the (iltnted suspensions, and mieasuring the extinct ion of S ()0( thr suspesliOns (EHI at I m) inIna t'iucami' spe t rophotometer I ). Scanning electron microscopy. Portionls of the Susp)ension of cells in Zvnmolvase-cont aining btiffered sorbitol werr removed at intervt!als anid rapirlly centriftigerl at 4C( in precooled tutibes, andI thte Strc'Ll't oLIres w-''ere gent lV washed twN'ice in iimli(dazoioh h(yro(hlortide hUffer (pH b.5) containing 12 M soirhitol. T'hey wNere thein suLspended in thte same buffer isul)I)lenmente(t with :l', (wt/vol) glutaraldehvde, nld the suspensiO)n as agitated occasionally'v over a I-h petiod. Fixedi stFLmCtUres were wsashed t wice in inildazole-h dvrochlorlide buffer containing 1.2 M sorbitol, and suspended in the samie bufferedl solrition containing 2)' (\ t/vol) otsmiuLlm tetroxide for a furtther hour. Aftetr centtifugation, thte StrLuctotres were dehydrated fot 1f) nin in 5fGi (vol, vol ) and then 70' (vol/vol) et hanol, and( finallY thre e times for- 15 mmiin abSOlute ethaInol. Thev wer-e thenl susplende(d in acetonie and rlrit(l in a P olaron H 5000 critical-point (irier P'olaron lEAl-lipment I t d-., 'Nat ford, Herts, England). A small portion of the (tlie(i mnaterial was attache(d to a conlductive stub with (lrouble-sitled cellulose tape an(i spritter coated with gotl in the p)resence of argon. They wer-e examinlet(l in a fJEl`0 1 00 CX electron microsrope fromii JEOL ([ TK.) Ltdl., JEOL HouLse, Girove Park, London, Englandl, having (a tunlgsten filtament ancl f itte(l wvith a tJEO)LI ASII) highresolution scanning device. Ani accelerating voltage of 20 kV was used. All photographs were taken on Ilforcl HP 4 film. Chemicals. All chemicals used were AnalaR or of the highest purity available commerciall'. Zvmolvase5000 was purchased from the Kirin Brewery Co. Ltd., 'I'akasaka, Gumma Pref., Japan.
RESULTS AND DISCUSSION Maximum osmotic sensitivity in populations of cells incubated in Zymolvase-containing brifferedi sorbitol was observed after :30 nmim of incubation (Fig. 1). Scanning electron micrographs showed that S. ceret,isiac NQYC:36(i has spherical cells with a smooth but slightly humpy surface (Fig. 2). When viewed at an acute angle, the surface of the plug and the raised rim in bhiul scars a)p)eared warty, a mlorphological feature not previously reported for 5S. cere 'isiae (Fig. 3). Unscarred wall nmaterial rapidly assumed a beady appearance as digestion proceeded (Fig. 4), which suggests that the 1/3l(13) glucaiis hydrolvsed by Zymolvase are probably not uniforrmlv accessible to the enzvme, and rmay therefore not be present as uniform shells within the wall as suggeste(l by Kopecka' et al. (14). Af'ter fu'Lrther digestion, fissures al)pearerl in the wall, exposing the furrowed surface of the uLnderlying l)lasma membrane (Fig. 4). Spheroplasts appeared to be produced after the f'ormation of fissures and sloughing off of partially digested wall nmaterial. There was never any indir-ation
thfat they appearetl 1y emer-giIng fr-onm a hole that formed in the wall. Both tvl)es of' spheroplast formation have p)reviously been observed with yeasts (24), but it was not surprising to encounter the less common type of formation in S. (ceretlisiae NCYC$366 in view of its extreme sUseptibility to digestion with /3-glucanase. 'I'he reaction of' huld scars ancl stirrourinding wNall to Zvmnolvase cligestiorn was of' partirrilar initer-est. I)DLring the first If) to :30 tnin of digestion, the raised rimi an( plugm in bri,l scars lost their surf'are layers, inclUdrling warts, which suggests that they or the wall mnaterial beneath themII miayv be composed, in p)art at least, of/'(1-;3) glucans (5). Thereafter, the raised rims anrl plugs in burl s(ars unrlerwent little (hange in
appearance.
There was no evidence that the base of the Ktid scar was digested away to reveal the surface of' the utlnerlving plasma membrane. As incu-
bation protrededle Iresistant bud scat's becamiie nlb an outer annulus (Fig. 5). 'T'his surrounlded sitructure, mlacre prot)minent after the removal of overlying wall material, is rontceivablv the wirIer, eler tron-dense secondary sep)tum which is fOrmed in 1oth the claughter and mother c ell beneath the plrimatr septtli (4). Seichertovii et al. (21) observel a sinmilar strIucture to the anniLltus af'ter treatment of S. ccieretisiac walls with boiling 2%' HCl. The resistanre of the base of the bhti sr-ar w hir-h inclrres the l)pimary septrIM (4) to digestion with Zvmolvase is pmrobablv explained 1w the presence of' chitin in the septuim (2, 5), brt it is clear, too, that the annulxis is otanle tl) of' material resistant to Zvmnolvase, possibly also chitin. Chitinase activity has not been detecterl in vrn)olvase (12). After
p)rt)-
longed rdigestion, tl)e bu(d srar ancl exposed an-
itltus, with sonme Srirrounrling wall material, slotigherl oftf the sUif'ace of the p)lasma utiemibianie.
were
Spheroplast f'ormation was nIot svnnchronorIS antI, even aftei0 3) miin of' incubation, a tinme which roinciderl with maximuLinm osn)otic senisitivitv itn the cell stispension (Fig. I), only about 25%i of the strutCtUries in the suspension were spheroplasts. The majority of the remaindeir wrere partlv rovererl with wall matetial (Fig. 4) and correspond to the "plrospheroplasts" clesc ribnerl Da-ling et al. ((). After 60 mmn of he inrtubation, abouLt 95'% Of the rells had been converted inito spherloplasts. 'IThe presence of a
high proportion of prospheroplasts in popular)f cells in /3-glucanase-containing buffered s0r1itol at a time when the suLspension showed ftaxinitit ost)motic sensitivity has imlp)ortant ini)tions
plication)s for- workers who prepare isolatedl pllasna mlermibranies of S. cr-rt'iisiaze byv osntl)ti( 1Ysis of sp)heroplasts. t.TIless sp)herolp)last foI-nia-
VOL. 140, 1979
~.
NOTES
291
:.....
FiG.. 1. Time course of formation of osmotically sensitive structures duiring incubation of S. ceretvisiae NCYC366 in buffered sorbitol containing Zymolyase-5000. Absorbance of portions of the cell suspension diluted into water (0) or buffer-ed sorbitol W@. FIG. 2. Intact cells of S. cercu,isiae NCYC.366, showing thc smzooth bumipy sur-face. Bar indicates 2.0 jimi. FIG. 3. Surface morphology, of bud( scars on S. cereLisiae NCYC366 showing warty appearance of plug (P) and raised rim 'R,). Bar- indicates 1.0 jimz. FIG. 4. Partly digested wall (W) of S. cerevisiae NCYC366 after- 45 nuin of digestion in buffered sorbitolZymolyase, shouwing the furrouwed nature of the plasma-membrane surface (M,). Bar indicates 1.0 jim.
292
NOTES
,J. BAt'TF'RIOL.
scar on S. cereL'isiae NCYC366 after 30 min digestion in buffered annulus (A) surrounding the remains of a raised rim (R) and plug (P,).
FIG. 5. Surface appearance of bud
sorbitol-7Zymolyase showing Bar- indicates 0.5 ~tm.
an outer
FiG. (3. Spheroplast of S.. (creu,siac ACYC'366 tirtiioll-v /1-cc /x-toi aodher'ing no/i iniatcr'ial. Baoc ind(icatcs I. 0 "nin. Fin;. 7. Fine stralctarc nf the fivrroncd snr/acc of 0 sphcrnplast nf S. ccrcu,isiac AVYC.366. Bar, indicates 0.25 omti. FiG. 8. Spheroplast of S. cerei'isiae NCYC366, showing a smiall atmnonit of adherinitg nollimaterilal (U1) anid .sn. an.in(lntation (I). Bar indicates 1.0
VOL. 140, 1979
tion has been followed by scanning electron microscopy, membranes prepared in this way may contain unacceptably large amounts of wall material. Streiblova (22) made a similar observation after her freeze-etch study of protoplast formation in S. cereuisiae. The furrowed appearance of the plasma-membrane surface, which was first reported by Moor and Muhlethaler (18) and later by others (22, 23) in freeze-fractured S. cerevisiae, and which is evident from the micrographs published by Miegeville and Morin (16), was visualized in finer detail in the present study (Fig. 6). Because, during spheroplast formation, the plasma membrane is probably subjected to a variety of tensions as the wall is gradually removed, it would seem that the furrowed surface structure of the plasma-membrane surface is stable and not easily disrupted. It is difficult to reconcile this finding with the suggestion made by Takeo et al. (23) that the furrows are channels of lipid which might be expected to be disrupted during spheroplast formation. Our observations also differ from those of Takeo et al. (23), who reported the presence of short rather than long furrows on the outer surface of the plasma membrane in exponentially-grown, freeze-fractured S. ceretisiae. Moreover, particles seen lying on the outside of the plasma membrane in the present study (Fig. 7) were about 25 to 30 nm in diameter, which is almost twice the value reported for these particles by Takeo et al. (23). These differences are, conceivably, the result of using two different techniques for exposing the outer surface of the yeast plasma membrane. Many spheroplasts were observed to have a single large indentation (Fig. 8). That this indentation was not observed on all spheroplasts could have been because on some spheroplasts it was located on that part of the surface not visible in the micrographs. This structural feature, which has not previously been reported, could be, in view of its size and because no spheroplast was observed to possess two of them, the area of plasma membrane underlying the birth scar. Retention of this indentation and of furrows after spheroplast formation suggests that the plasma membrane can retain a mosaic-type infrastructure in certain domains, which conceivably may be related to the presence of intracellular microfibrils. This research was supported by grants fronm the Science Research Council (England), for which we express thanks. We are also grateful to J. A. Hossack for timely assistance in spheroplast preparation. LITERATURE CITED 1. Alterthum, F., and A. H. Rose. 1973. Osmotic lysis of sphaeroplasts from Saccharomvces cer etutsiae grown anaerobically in media containing different unsaturated fatty acids. ,J. Gen. Microbiol. 77:371-382.
NOTES
293
2. Bacon, J. S. D., E. D. Davidson, D. Jones, and I. F. Taylor. 1966. The location of chitin in the yeast cell wall. Biochem. J. 101:36-38. 3. Burger, M., E. E. Bacon, and J. S. D. Bacon. 1961. Some observations on the form and location of invertase in the yeast cell. Biochem. J. 78:504-511. 4. Cabib, E. 1975. Molecular aspects of yeast morphogenesis. Annu. Rev. Microbiol. 29:191-214. 5. Cabib, E., and B. Bowers. 1971. Chitin and yeast budding. Localization of chitin in yeast bud scars. J. Biol. Chem. 246:152-159. 6. Darling, S., J. Theilade, and A. Birch-Andersen. 1969. Kinetic and morphological observations on Saccharomvces cereuisiae during spheroplast formation. ,J. Bacteriol. 98:797-810. 7. Duell, E. A., S. Inoue, and M. F. Utter. 1964. Isolation and properties of intact mitochondria from spheroplasts of yeast. J. Bacteriol. 88:1762-1773. 8. Eddy, A. A. 1958. The structure of the yeast cell wall. II. Degradative studies with enzymes. Proc. R. Soc. London Ser. B 149:425-440. 9. Holter, H., and P. Ottolenghi. 1960. Observations on yeast protoplasts. C. R. Trav. Lab. Carlsberg 31:409422. 1t). Hunter, K., and A. H. Rose. 1972. Lipid composition of Saccharomyces cereuislae as influenced by growth temperature. Biochim. Biophys. Acta 260:639-653. 11. Kaneko, T., K. Kitamura, and Y. Yamamoto. 1973. Susceptibilities of yeasts to yeast cell wall lytic enzymes of Arthrobacter luteus. Agr. Biol. Chem. 37:2295-2302. 12. Kitamura, K., and Y. Yamamoto. 1972. Purification and properties of an enzyme, Zymolyase, which lyses viable yeast cells. Arch. Biochem. Biophys. 153:403406. 13. Kitamura, K., T. Kaneko, and Y. Yamamoto. 1971. Lysis of viable yeast cells by enzymes of Arthrobacter luteus. Arch. Biochem. Biophys. 145:4(12-404. 14. Kopecka, M., H. J. Phaff, and G. H. Fleet. 1974. Demonstration of a fibrillar component in the cell wall of the yeast Saccharomyces ceretisiae and its chemical nature. J. Cell Biol. 62:66-76. 15. Kuo, S.-C., and S. Yamamoto. 1975. Preparation and growth of protoplasts. Methods Cell Biol. 11:169-183. 16. Miegeville, M., and 0. Morin. 1977. Nouvelle contribution de la microscopic electronique a bayalage a l'tude des protoplastes de levures. C. R. Acad. Sci. Paris. 284: 1935-1938. 17. Millbank, J. W., and R. M. Macrae. 1964. Degradation of yeast cell wall by fractionated snail gut enzyme. Nature (London) 201:1347. 18. Moor, H., and K. Muhlethaler. 1963. Fine structure in frozen-etched yeast cells. J. Cell Biol. 17:609-628. 19. Phaff, H. J. 1977. Enzymatic yeast cell wall degradation. Adv. Chem. Ser. 160:244-282. 20. Russell, I., I. F. Garrison, and G. G. Stewart. 1973. Studies on the formation of spheroplasts from stationary phase cells of Saccharomyces cereuisiae. J. Inst. Brew. London 79:48-54. 21. Seichertova, O., K. Beran, and J. Ludvik. 1969. Contribution to the structure of the cell wall in the area of the bud scar in Saccharomyces cereuisiae. Antonie van
Leeuwenhoek,J. Microbiol. Serol. 35:(Suppl. B)13. 22. Streiblova, E. 1968. Surface structure of yeast protoplasts. J. Bacteriol. 95:700-707. 23. Takeoe, K., M. Shigeta, and Y. Takagi. 1976. Plasma membrane ultrastructural differences between exponential and stationary phases of Saccharomyces cereL'isiae as revealed by freeze-etching. J. Gen. Microbiol. 97:323-329. 24. Villanueva, J. R. 1966. Protoplasts of fungi, p. 3-62. In G. C. Ainsworth and A. F. Sussman, The fungi, vol. 2. Academic Press Inc., New York. 25. Wickerham, L. J. 1951. Taxonomy of yeasts. U.S. Dep. Agric. Tech. Bull. 1029:1-56.
JOURNAL OF BACTERIOLOGY, Oct. 1979, p. 289-293 0021-9193/79/ 10-0289/05$02.00/0
NOTES Scanning Electron Microscope Study of Saccharomyces cerevisiae Spheroplast Formation ALASTAIR T. PRINGLE,'t JOHN FORSDYKE,- AND ANTHONY H. ROSE*
Z7ymology Laboratory, School of Biological Sciences,' and Electron Optics Uni t,2 UnWiersity of Bath, Clatverton Doun, Bath BA2 7AY, Avon, England Received for publication 31 May 1979
A suspension of Saccharomyces ceretisiae NCYC366 in buffered 1.2 M sorbitol containing Zymolyase-5000 (a ,/-glucanase-containing preparation) showed maximum osmotic sensitivity after 30 min of incubation at 30°C. A scanning electron microscope study of spheroplast formation, using a very high resolution (4-nm) machine, revealed several new morphological features. The surface of the plug in bud scars on intact cells appeared warty. The wall, which assumed a beady appearance as digestion proceeded, ultimately sloughed off to reveal the furrowed surface of the plasma membrane. Bud scars were resistant to digestion and, as incubation proceeded, they became surrounded by an outer annulus, which may be the secondary septum. Wall material was completely removed from the majority of cells only after 60 min of digestion. The surface of spheroplasts was studded with particles, about 25 to 30 nm in diameter. Many spheroplasts had a single large indentation, which may be in that part of the plasma membrane originally underlying the birth scar.
Spheroplasts can be prepared from strains of Saccharomyces cereL'isiae by digesting cells with /8-glucanase-containing preparations, such as snail gut juice or various microbial enzymes (15, 19). Strains of this yeast differ in their susceptibility to /8-glucanase digestion (9, 17) and, with refractory strains, spheroplast formation can be accelerated by treating cells with sulfhydryl compounds (3, 7) or proteolytic enzymes (20). Several transmission electron microscope studies of spheroplast formation from S. ceretuisiae have been published (e.g., ref. 6), but so far no detailed examination has been reported involving use of the scanning electron microscope. The present communication reports such a study on S. cereLisiae NCYC366, a strain that is particularly susceptible to digestion with /Bglucanase (1, 8). The study used, as a source of ,8-glucanase, Zymolyase-5000, a preparation obtained from filtrates of Arthrobacter luteus cultures (11, 13). Zymolyase is reported to be free from ,B- (1--2), 13- (1--*4), and ,B- (1--6) glucanases and phosphomannanase (12). It contains an endo-,8-(1-.*3) glucanase which is active on some but not all /8-(1-*3) glucans, and it has been suggested that it may have a preference for glucans possessing a firm molecular aggregation t Present address: Department of Biochemistry, University of Kansas Medical Center, Kansas City, KS, 66103.
due to hydrogen bonding (12). The present study was carried out with a scanning electron microscope that has an extremely high resolving power (about 4 nm), with the result that several hitherto unreported features of spheroplast formation and of the topography of the plasmamembrane surface were revealed. In this study, the term "spheroplast" is used to describe structures from which all, or almost all, of the wall has been removed. MATERIALS AND METHODS Experimental cultures. S. cerevisiae NCYC366 was maintained on slopes of malt extract-yeast extract-glucose-peptone-agar medium (25). Cultures were grown aerobically in 1-liter batches at 30°C as described by Hunter and Rose (10), except that the mixture of vitamins in the medium was replaced by yeast extract (Oxoid Ltd.; 0.1'S, wt/vol). Cells were harvested from late-exponential-phase cultures (0.22 to 0.24 mg, dry wt per ml) by centrifugation at 12,250 x g and 4°C for 1 min, and washed three times in
imidazole-hydrochloride (10 mM)-MgCl2 (10 mM) buffer (pH 6.0) containing 1.2 M sorbitol. Preparation of spheroplasts. Spheroplasts were prepared from freshly harvested cells by suspending them (6 x 108/ml) in imidazole-hydrochloride (10 mM) buffer (pH 6.0) containing 1.2 M sorbitol and Zymolyase-5000 (0.4 mg/6 mg, dry wt of cells). The suspension in a conical flask was incubated at 30°C in a shaker water bath at 100 oscillations per min. Forma289
290
J. BA('TE'KIOL.
NO'T'ES
tion of osmoticallt sensitive structures Ws nmonitored l) c,dldding 0.1 ml of suspension to 2.9 tmil of water oi 2.09mnl of buffered 1.2 M sorl)itol, gently shakinig the (iltnted suspensions, and mieasuring the extinct ion of S ()0( thr suspesliOns (EHI at I m) inIna t'iucami' spe t rophotometer I ). Scanning electron microscopy. Portionls of the Susp)ension of cells in Zvnmolvase-cont aining btiffered sorbitol werr removed at intervt!als anid rapirlly centriftigerl at 4C( in precooled tutibes, andI thte Strc'Ll't oLIres w-''ere gent lV washed twN'ice in iimli(dazoioh h(yro(hlortide hUffer (pH b.5) containing 12 M soirhitol. T'hey wNere thein suLspended in thte same buffer isul)I)lenmente(t with :l', (wt/vol) glutaraldehvde, nld the suspensiO)n as agitated occasionally'v over a I-h petiod. Fixedi stFLmCtUres were wsashed t wice in inildazole-h dvrochlorlide buffer containing 1.2 M sorbitol, and suspended in the samie bufferedl solrition containing 2)' (\ t/vol) otsmiuLlm tetroxide for a furtther hour. Aftetr centtifugation, thte StrLuctotres were dehydrated fot 1f) nin in 5fGi (vol, vol ) and then 70' (vol/vol) et hanol, and( finallY thre e times for- 15 mmiin abSOlute ethaInol. Thev wer-e thenl susplende(d in acetonie and rlrit(l in a P olaron H 5000 critical-point (irier P'olaron lEAl-lipment I t d-., 'Nat ford, Herts, England). A small portion of the (tlie(i mnaterial was attache(d to a conlductive stub with (lrouble-sitled cellulose tape an(i spritter coated with gotl in the p)resence of argon. They wer-e examinlet(l in a fJEl`0 1 00 CX electron microsrope fromii JEOL ([ TK.) Ltdl., JEOL HouLse, Girove Park, London, Englandl, having (a tunlgsten filtament ancl f itte(l wvith a tJEO)LI ASII) highresolution scanning device. Ani accelerating voltage of 20 kV was used. All photographs were taken on Ilforcl HP 4 film. Chemicals. All chemicals used were AnalaR or of the highest purity available commerciall'. Zvmolvase5000 was purchased from the Kirin Brewery Co. Ltd., 'I'akasaka, Gumma Pref., Japan.
RESULTS AND DISCUSSION Maximum osmotic sensitivity in populations of cells incubated in Zymolvase-containing brifferedi sorbitol was observed after :30 nmim of incubation (Fig. 1). Scanning electron micrographs showed that S. ceret,isiac NQYC:36(i has spherical cells with a smooth but slightly humpy surface (Fig. 2). When viewed at an acute angle, the surface of the plug and the raised rim in bhiul scars a)p)eared warty, a mlorphological feature not previously reported for 5S. cere 'isiae (Fig. 3). Unscarred wall nmaterial rapidly assumed a beady appearance as digestion proceeded (Fig. 4), which suggests that the 1/3l(13) glucaiis hydrolvsed by Zymolvase are probably not uniforrmlv accessible to the enzvme, and rmay therefore not be present as uniform shells within the wall as suggeste(l by Kopecka' et al. (14). Af'ter fu'Lrther digestion, fissures al)pearerl in the wall, exposing the furrowed surface of the uLnderlying l)lasma membrane (Fig. 4). Spheroplasts appeared to be produced after the f'ormation of fissures and sloughing off of partially digested wall nmaterial. There was never any indir-ation
thfat they appearetl 1y emer-giIng fr-onm a hole that formed in the wall. Both tvl)es of' spheroplast formation have p)reviously been observed with yeasts (24), but it was not surprising to encounter the less common type of formation in S. (ceretlisiae NCYC$366 in view of its extreme sUseptibility to digestion with /3-glucanase. 'I'he reaction of' huld scars ancl stirrourinding wNall to Zvmnolvase cligestiorn was of' partirrilar initer-est. I)DLring the first If) to :30 tnin of digestion, the raised rimi an( plugm in bri,l scars lost their surf'are layers, inclUdrling warts, which suggests that they or the wall mnaterial beneath themII miayv be composed, in p)art at least, of/'(1-;3) glucans (5). Thereafter, the raised rims anrl plugs in burl s(ars unrlerwent little (hange in
appearance.
There was no evidence that the base of the Ktid scar was digested away to reveal the surface of' the utlnerlving plasma membrane. As incu-
bation protrededle Iresistant bud scat's becamiie nlb an outer annulus (Fig. 5). 'T'his surrounlded sitructure, mlacre prot)minent after the removal of overlying wall material, is rontceivablv the wirIer, eler tron-dense secondary sep)tum which is fOrmed in 1oth the claughter and mother c ell beneath the plrimatr septtli (4). Seichertovii et al. (21) observel a sinmilar strIucture to the anniLltus af'ter treatment of S. ccieretisiac walls with boiling 2%' HCl. The resistanre of the base of the bhti sr-ar w hir-h inclrres the l)pimary septrIM (4) to digestion with Zvmolvase is pmrobablv explained 1w the presence of' chitin in the septuim (2, 5), brt it is clear, too, that the annulxis is otanle tl) of' material resistant to Zvmnolvase, possibly also chitin. Chitinase activity has not been detecterl in vrn)olvase (12). After
p)rt)-
longed rdigestion, tl)e bu(d srar ancl exposed an-
itltus, with sonme Srirrounrling wall material, slotigherl oftf the sUif'ace of the p)lasma utiemibianie.
were
Spheroplast f'ormation was nIot svnnchronorIS antI, even aftei0 3) miin of' incubation, a tinme which roinciderl with maximuLinm osn)otic senisitivitv itn the cell stispension (Fig. I), only about 25%i of the strutCtUries in the suspension were spheroplasts. The majority of the remaindeir wrere partlv rovererl with wall matetial (Fig. 4) and correspond to the "plrospheroplasts" clesc ribnerl Da-ling et al. ((). After 60 mmn of he inrtubation, abouLt 95'% Of the rells had been converted inito spherloplasts. 'IThe presence of a
high proportion of prospheroplasts in popular)f cells in /3-glucanase-containing buffered s0r1itol at a time when the suLspension showed ftaxinitit ost)motic sensitivity has imlp)ortant ini)tions
plication)s for- workers who prepare isolatedl pllasna mlermibranies of S. cr-rt'iisiaze byv osntl)ti( 1Ysis of sp)heroplasts. t.TIless sp)herolp)last foI-nia-
VOL. 140, 1979
~.
NOTES
291
:.....
FiG.. 1. Time course of formation of osmotically sensitive structures duiring incubation of S. ceretvisiae NCYC366 in buffered sorbitol containing Zymolyase-5000. Absorbance of portions of the cell suspension diluted into water (0) or buffer-ed sorbitol W@. FIG. 2. Intact cells of S. cercu,isiae NCYC.366, showing thc smzooth bumipy sur-face. Bar indicates 2.0 jimi. FIG. 3. Surface morphology, of bud( scars on S. cereLisiae NCYC366 showing warty appearance of plug (P) and raised rim 'R,). Bar- indicates 1.0 jimz. FIG. 4. Partly digested wall (W) of S. cerevisiae NCYC366 after- 45 nuin of digestion in buffered sorbitolZymolyase, shouwing the furrouwed nature of the plasma-membrane surface (M,). Bar indicates 1.0 jim.
292
NOTES
,J. BAt'TF'RIOL.
scar on S. cereL'isiae NCYC366 after 30 min digestion in buffered annulus (A) surrounding the remains of a raised rim (R) and plug (P,).
FIG. 5. Surface appearance of bud
sorbitol-7Zymolyase showing Bar- indicates 0.5 ~tm.
an outer
FiG. (3. Spheroplast of S.. (creu,siac ACYC'366 tirtiioll-v /1-cc /x-toi aodher'ing no/i iniatcr'ial. Baoc ind(icatcs I. 0 "nin. Fin;. 7. Fine stralctarc nf the fivrroncd snr/acc of 0 sphcrnplast nf S. ccrcu,isiac AVYC.366. Bar, indicates 0.25 omti. FiG. 8. Spheroplast of S. cerei'isiae NCYC366, showing a smiall atmnonit of adherinitg nollimaterilal (U1) anid .sn. an.in(lntation (I). Bar indicates 1.0
VOL. 140, 1979
tion has been followed by scanning electron microscopy, membranes prepared in this way may contain unacceptably large amounts of wall material. Streiblova (22) made a similar observation after her freeze-etch study of protoplast formation in S. cereuisiae. The furrowed appearance of the plasma-membrane surface, which was first reported by Moor and Muhlethaler (18) and later by others (22, 23) in freeze-fractured S. cerevisiae, and which is evident from the micrographs published by Miegeville and Morin (16), was visualized in finer detail in the present study (Fig. 6). Because, during spheroplast formation, the plasma membrane is probably subjected to a variety of tensions as the wall is gradually removed, it would seem that the furrowed surface structure of the plasma-membrane surface is stable and not easily disrupted. It is difficult to reconcile this finding with the suggestion made by Takeo et al. (23) that the furrows are channels of lipid which might be expected to be disrupted during spheroplast formation. Our observations also differ from those of Takeo et al. (23), who reported the presence of short rather than long furrows on the outer surface of the plasma membrane in exponentially-grown, freeze-fractured S. ceretisiae. Moreover, particles seen lying on the outside of the plasma membrane in the present study (Fig. 7) were about 25 to 30 nm in diameter, which is almost twice the value reported for these particles by Takeo et al. (23). These differences are, conceivably, the result of using two different techniques for exposing the outer surface of the yeast plasma membrane. Many spheroplasts were observed to have a single large indentation (Fig. 8). That this indentation was not observed on all spheroplasts could have been because on some spheroplasts it was located on that part of the surface not visible in the micrographs. This structural feature, which has not previously been reported, could be, in view of its size and because no spheroplast was observed to possess two of them, the area of plasma membrane underlying the birth scar. Retention of this indentation and of furrows after spheroplast formation suggests that the plasma membrane can retain a mosaic-type infrastructure in certain domains, which conceivably may be related to the presence of intracellular microfibrils. This research was supported by grants fronm the Science Research Council (England), for which we express thanks. We are also grateful to J. A. Hossack for timely assistance in spheroplast preparation. LITERATURE CITED 1. Alterthum, F., and A. H. Rose. 1973. Osmotic lysis of sphaeroplasts from Saccharomvces cer etutsiae grown anaerobically in media containing different unsaturated fatty acids. ,J. Gen. Microbiol. 77:371-382.
NOTES
293
2. Bacon, J. S. D., E. D. Davidson, D. Jones, and I. F. Taylor. 1966. The location of chitin in the yeast cell wall. Biochem. J. 101:36-38. 3. Burger, M., E. E. Bacon, and J. S. D. Bacon. 1961. Some observations on the form and location of invertase in the yeast cell. Biochem. J. 78:504-511. 4. Cabib, E. 1975. Molecular aspects of yeast morphogenesis. Annu. Rev. Microbiol. 29:191-214. 5. Cabib, E., and B. Bowers. 1971. Chitin and yeast budding. Localization of chitin in yeast bud scars. J. Biol. Chem. 246:152-159. 6. Darling, S., J. Theilade, and A. Birch-Andersen. 1969. Kinetic and morphological observations on Saccharomvces cereuisiae during spheroplast formation. ,J. Bacteriol. 98:797-810. 7. Duell, E. A., S. Inoue, and M. F. Utter. 1964. Isolation and properties of intact mitochondria from spheroplasts of yeast. J. Bacteriol. 88:1762-1773. 8. Eddy, A. A. 1958. The structure of the yeast cell wall. II. Degradative studies with enzymes. Proc. R. Soc. London Ser. B 149:425-440. 9. Holter, H., and P. Ottolenghi. 1960. Observations on yeast protoplasts. C. R. Trav. Lab. Carlsberg 31:409422. 1t). Hunter, K., and A. H. Rose. 1972. Lipid composition of Saccharomyces cereuislae as influenced by growth temperature. Biochim. Biophys. Acta 260:639-653. 11. Kaneko, T., K. Kitamura, and Y. Yamamoto. 1973. Susceptibilities of yeasts to yeast cell wall lytic enzymes of Arthrobacter luteus. Agr. Biol. Chem. 37:2295-2302. 12. Kitamura, K., and Y. Yamamoto. 1972. Purification and properties of an enzyme, Zymolyase, which lyses viable yeast cells. Arch. Biochem. Biophys. 153:403406. 13. Kitamura, K., T. Kaneko, and Y. Yamamoto. 1971. Lysis of viable yeast cells by enzymes of Arthrobacter luteus. Arch. Biochem. Biophys. 145:4(12-404. 14. Kopecka, M., H. J. Phaff, and G. H. Fleet. 1974. Demonstration of a fibrillar component in the cell wall of the yeast Saccharomyces ceretisiae and its chemical nature. J. Cell Biol. 62:66-76. 15. Kuo, S.-C., and S. Yamamoto. 1975. Preparation and growth of protoplasts. Methods Cell Biol. 11:169-183. 16. Miegeville, M., and 0. Morin. 1977. Nouvelle contribution de la microscopic electronique a bayalage a l'tude des protoplastes de levures. C. R. Acad. Sci. Paris. 284: 1935-1938. 17. Millbank, J. W., and R. M. Macrae. 1964. Degradation of yeast cell wall by fractionated snail gut enzyme. Nature (London) 201:1347. 18. Moor, H., and K. Muhlethaler. 1963. Fine structure in frozen-etched yeast cells. J. Cell Biol. 17:609-628. 19. Phaff, H. J. 1977. Enzymatic yeast cell wall degradation. Adv. Chem. Ser. 160:244-282. 20. Russell, I., I. F. Garrison, and G. G. Stewart. 1973. Studies on the formation of spheroplasts from stationary phase cells of Saccharomyces cereuisiae. J. Inst. Brew. London 79:48-54. 21. Seichertova, O., K. Beran, and J. Ludvik. 1969. Contribution to the structure of the cell wall in the area of the bud scar in Saccharomyces cereuisiae. Antonie van
Leeuwenhoek,J. Microbiol. Serol. 35:(Suppl. B)13. 22. Streiblova, E. 1968. Surface structure of yeast protoplasts. J. Bacteriol. 95:700-707. 23. Takeoe, K., M. Shigeta, and Y. Takagi. 1976. Plasma membrane ultrastructural differences between exponential and stationary phases of Saccharomyces cereL'isiae as revealed by freeze-etching. J. Gen. Microbiol. 97:323-329. 24. Villanueva, J. R. 1966. Protoplasts of fungi, p. 3-62. In G. C. Ainsworth and A. F. Sussman, The fungi, vol. 2. Academic Press Inc., New York. 25. Wickerham, L. J. 1951. Taxonomy of yeasts. U.S. Dep. Agric. Tech. Bull. 1029:1-56.
