Surface Structure of Yeast Protoplasts

EVA STREIBLOVA1 Laboratory for Electront Microscopy, Depairtmenit of Genieral Botaniy, Swiss Federal Ilistitiate of Technology. Zurich, Switzerl(and Received for publication 26 October 1967

The fine structure of the yeast cell wall during protoplast formation was studied by means of phase-contrast microscopy and the freeze-etching technique. The freeze-etching results indicated that at least in some cases the entire wall substance was not removed from the surface of the protoplasts. After a treatment of 30 min to 3 hr with 2% snail enzymes, an innermost thin wall layer as well as remnants of the fibrillar middle layer sometimes could be demonstrated.

The term "protoplast" means the living unit of of the original wall on the surface of the plasma the plant cell which lies within the cell wall and membrane. which in proper conditions can be plasmolyzed away from it. Kuster (11) has shown that the MATERIALS AND METHODS protoplast is able to survive if the cell wall is Cuiltuires anld clultivatioii. Saceharomyces cerevisiae, removed under proper conditions. He called Schizosaccharomyces pombe, Saceharomycodes Ilid- these cells from which the cell wall had been re- wigii, alnd Endomyces magntusii from the Collection of moved "gymnoplasts." In microbiology, the the Czechoslovak Academy of Sciencies were culti- term "protoplast" has been established to de- vated in the defined medium of Wickerham (28). scribe bacterial forms in which the cell wall is Preparatioln of protoplasts. Protoplasts were pre- entirely removed by the action of pared by the modified method of Eddy and William- enzymes (3). son (5), with the use of either of the following pre- For forms which retain remnants of the wall, the treatments. (i) Log-phase yeast cells were washed term "spheroplasts" is generally used (24). twice with 0.1 M 2-mercaptoethanol in tris(hydroxy- During the past decade, these terms have been methyl)aminomethane (Tris) buffer (pH 7.5). The generally accepted for bacteria as well as for mercaptoethanol was then removed by centrifugation yeasts. In the case of yeasts, evidence has not and decantation, and the resuspended cells were been presented that the cell wall is entirely absent washed twice with distilled water. (ii) Log-phase cells from the spherical units which are very sensitive without pretreatment were washed twice with distilled to osmotic shock. water. The technique of freeze-etching, developed by The cells obtained by both procedures were sus- pended in the digestion medium containing 0.9 M Moor et al. (14), makes it possible to examine the MgS04 7H20 in citrate-phosphate buffer (pH 5, 4), surface structure of cell membranes and or- to which dried Suc digestif d'Helix pomatia stabilis6 ganelles, whereas conventional methods of elec- (Industrie Biologique Francaise) at a concentration of tron microscopy did not allow this. Surface views 20 mg/ml was added. This mixture was incubated at of yeast plasma membrane were first published 28 C on a rotary shaker. by Moor and Muhlethaler (13). The present work Freeze-etchting method. Samples were removed from was undertaken to study the related problems of the incubation mixtures at various time intervals, cell wall architecture and of protoplast forma- collected by centrifugation, and washed three times tion. with 0.6 M KCI. A drop of concentrated suspension was placed on a copper disc and frozen in liquid In this report, the term "protoplast" is used for freon- 12 (dichlorodifluoromethane). This specimen yeast forms which in freeze-etchings are entirely was transferred to the Balzers apparatus and treated free from wall material, and the term "sphero- as described by Moor and Muhlethaler (13). Adhering plast" is used for forms which retain remnants cells were removed from the replica by treatment with 70', sulfuric acid, and were subsequently transferred 1On leave of absence from the Department of to distilled water and Eau de Javelle (a commercial Technical Microbiology, Institute of Microbiology, product containing a mixture of 5 g of calcium hypo- Czechoslovak Academy of Sciences, Prague, Czech- chlorite dissolved in 25 ml of water and 10 g of sodium oslovakia. carbonate dissolved in 200 ml of water). Finally, the 700 VOL. 95, 1968 SURFACE STRUCTURE OF PROTOPLASTS 701 replica was washed in distilled water, mounted on a after incubation for 90 min, almost all cells of the copper grid, and examined in a Zeiss EM 9 or a species investigated, excepting E. magnusii, be- Siemens Elmiscop I electron microscope. came spherical and osmotically fragile. In the presence of 2-mercaptoethanol, the conversion to RESULTS spheroplasts and protoplasts proceeds faster. Fre- It is known that yeasts reproduce in three dif- quently, the protoplasts do not emerge but remain ferent ways-by budding, by fission, and by a within the wall as it is being digested. Gradually, process between these two. In an earlier paper, I both the surface layer and the matrix are de- reported that different kinds of scars and dif- graded (Fig. 4). Next, the protective fibrillar net- ferent cell wall architectures arise, as a result of work is dissolved (Fig. 5), and, finally, the inner- structural changes produced in the wall, in asso- most wall layer is left behind (Fig. 6 and 7). ciation with cell division (22). Therefore, rep- Occasionally, protoplasts and spheroplasts are presentatives of budding yeast (S. cerevisiae), api- extruded through a hole in the wall. In the phase- culate yeast (S. Iudwigii), and two types of fission contrast microscope, delicate filaments connecting yeasts (S. pombe and E. magnusii) were studied. the emerging protoplast to the cell wall can often Figure 1 illustrates both the surface of the be observed, this is especially pronounced in E. plasmalemma and the fractured cell wall of an magnusii and S. pombe. These filaments probably exponentially growing cell of S. ludwigii and are identical with the innermost wall layer. Fila- reveals the essential characteristics, as reported ments arise when the innermost wall layer is by Moor and Muhlethaler (13). The surface of loosened from the wall in some places by enzyme. the cell wall is relatively smooth. The network of action. Only if the innermost wall layer remains fibrils is embedded in a finely granular matrix. attached to the rest of the cell wall or is ruptured The area between numerous invaginations of the and shed in the suspending medium are wall-free plasmalemma is sculptured by particles. The protoplasts obtained (Fig. 8). distribution pattern of these particles, as well as Freeze-etchings of fractured yeast spheroplasts their number, varies considerably in different and protoplasts have provided evidence that the yeast species. The particles have an average size surface of the plasmalemma remains almost un- of 60 to 150 A. The invaginations are frequently changed after the action of snail enzyme. The filled with homogeneous material derived from appearance and distribution of particles is ap- the splintered cell wall. In regions of the cyto- proximately the same as described for untreated plasmic membrane that are close to a perpendicu- cells. However, the shape and depth of the in- larly fractured wall, some fibrillar-like structures vaginations are affected by the molarity of the can be observed. Individual wall layers are never stabilizing medium. Even if the invaginations do separated by freeze-etching, since the fracturing disappear, areas free from particles indicate their process only provides sections across the cell original location. wall or the fracture plane follows the surface of This study provides evidence that a yeast the wall. population treated from 30 min to 3 hr with snail After incubation for 30 min in the digestion enzyme, in the presence or absence of 2-mer- medium, some of the cells appear to be un- captoethanol, consists of a mixture of wall-free changed as revealed by phase-contrast micros- protoplasts and of spheroplasts; the surfaces of copy; in other cells, the cytoplasm shrinks away the latter are still not entirely free from wall sub- from the cell wall. Spherical protoplast-like stances after this treatment. bodies susceptible to osmotic changes also occur. Even in cells which apparently do not show any DIscussION changes, the freeze-etchings suggest an alteration When considering the problem of structure in the ultrastructural integrity of the cell wall. An and formation of the yeast cell wall, the possi- innermost thin wall layer can be observed on the bility of obtaining a detailed cytological rep- surface of the plasmalemma covered by particles presentation of the innermost wall layer is of (Fig. 2). This layer has never been seen in un- great interest. Moreover, such information is treated cells. In shrunken protoplasts which re- equally desirable with regard to protoplast main within the wall, the replicas of oblique sec- formation. tions reveal the presence of the same thin layer The innermost wall layer is very thin and does closely adhering to the surface of the plasma- not contain any fibrillar material; its outer surface lemma (Fig. 3). is smooth and relatively free from particles. It The sensitivity to lytic snail enzymes of cells can never be separated from the adjacent wall from different species, as well as of individual substance by fracturing. In some cases, fractur- cells of the same species, varies greatly. However, ing with the freeze-etching procedure results in * biDww%-Ke-ffisslve - :^weC ;vf

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FIG. 1. Freeze-etched cell of Saccharomycodes ludwigii. Slirftice view of bothi the sairftice of the cell wall (cw) ancd the plasma membrane (pm), showing particles (p) alid iiivagintatiolns (i). Some ilivaginiatiolis retail? portioaIs oj the wall (w). X 63,000. FIG. 2. Freeze-etched cell ofSacch,aronmyces cerevisiae treated for 30 mniti with s,iail entzyme. The cell wall (cwv), the imiiiermost wall layer (w/l), alnd the plasma membrante (pm), showing itiumeroius particles. X 50,000. FIG. 3. Freeze-etched cell ofEndomyces mnagnusii treated fbr 30 mini with sniail en:Yme. Tlte spheroplaist shrilnks awayfrom the rest ofthe cell wall. Tlhefibrillcr layer (uf) is detached from the innermost wall laYer (wl), which is itself in close colitact with the plcasmiia mnembrane (pm11). X 68,000. 702 VOL. 95, 1968 SURFACE STRUCTURE OF PROTOPLASTS 703

FIG. 4. Freeze-etched cell ofSaccharomyces cerevisiae pretreated with 2-mercaptoethanol and then treatedfor 60 min with snail enzyme. The surface wall layer and the matrix are degraded and thefibrillar network (wf) appears. The plasma membrane (pm) shows invaginations (i) and particles (p). X 50,000. FIG. 5. Freeze-etched spheroplast ofSaccharomyces cerevisiae treatedfor 90 min with snail enzyme. The innermost wall layer (wl) as well as remnants offibrils, the plasma membrane (pm) showing in vaginations (i) andparti- cles (p), and the endoplasmic reticulum (er) adjacent to the plasma membrane are visible. X 80,000. some material from the innermost wall layer re- layer which remains behind after chemical treat- maining in the invaginations of the plasmalemma. ment was presented by Northcote and Horne However, if cells are treated with snail en- (18), but their opinion has not been accepted zyme, the innermost wall layer may be revealed. generally. In ultrathin sections, this innermost The innermost wall layer can be shed without the wall layer appears as an electron-dense structure loss of integrity of the protoplast. This layer is a with numerous invaginations into the cytoplasm common feature of the yeast cell wall, irrespec- (4, 12, 17, 23, 26). According to Vitols (26), this tive of the yeast species and mode of vegeta- material may be cell wall protein localized in this tive reproduction. region. Some "adherence or linkages" of the Cytological evidence for more than a single cytoplasmic membrane to the wall were de- 704 STREIBLOVA J. BACTERIOL.

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FIG. 6. Freeze-etched spheroplast of Schizosaccharomyces pombe treated for 60 miii with snail enzyme. The inn1ermost wall layer (wl) with remnants offibrils (wf), aiid the plasma membrane (pm) showing invaginiations (i) and particles (p) are to be seeii. X 56,000. FIG. 7. Freeze-etched spheroplasts of Schizosaccharomyces pombe treated for 90 miti with snail enzyme. The innermost wall layer (wl) with remnants offibrils (wf), the plasma membrane (pm), the endoplasmic reticulum (er) adjacent to the plasma membrane, and, on the right, a spheroplast (sp) surrounded by the innermost wall layer can be identified. X 28,000. VOL. 95, 1968 SURFACE STRUCTURE OF PROTOPLASTS 705

FIG. 8. Freeze-etched protoplast of Saccharomyces cerevisiae showing the frozeni medium-KCI (m) and the plasma membrane (pm) with invaginations (i) and particles (p). X 100,000. scribed by Alper et al. (1) in yeast treated with freeze-etching results indicate that, at least in ribonuclease. some cases, the entire wall substance has not been In S. pombe, S. ludwigii, and Sporobolomyces removed. The yeast populations studied repre- sp. (21), in Candida utilis, Oospora suaveolens, sent a mixture of spheroplasts and protoplasts. and Geotrichum lactis (7), and in S. pombe (8), The susceptibility of the innermost wall layer fine filaments connecting the protoplast and the and the rest of the cell wall with respect to attack cell wall have been observed by phase-contrast by Helix pomatia digestive juice seems to be very microscopy. Earlier attempts by electron micro- different. The reason for its higher resistance scopists to demonstrate these structures and their in S. cerevisiae may be as follows. It is gen- significance were without success. My observa- erally agreed that bud scars are more resistant tions suggest that the fine filaments represent to enzymatic digestion as well as to treatment remnants of the innermost wall layer which re- with chemicals than is the rest of the cell wall. main partly attached to the rest of the cell wall. Recently, it was reported that after treatment Ottolenghi (19) reported that annular rings of with snail enzymes a thin membrane remains bud scars are attached to a membranous matter attached to the bud scars (19). This thin mem- which remains behind after snail enzyme treat- brane is most probably the cell wall layer which ment. Although residues of bud scars have not remains behind after treatment with acid and yet been observed in freeze-etching micrographs, alkali, and which can be seen in the electron the membranous matter described by Ottolenghi micrographs of Northcote and Horne (18). (19) is most probably identical with the inner- Houwink and Kreger (10) showed that the cell most wall layer. wall of S. cerevisiae, after successive acid and During the past decade since protoplasts in alkali extractions, yielded a small residue of yeast were described (5, 15), the total absence of chitin, granular when examined in an electron the cell wall on the surface of the protoplasts has microscope. Recently, Bacon et al. (2) prepared been discussed. On the ultrastructural level, only residues of bud scars from the cell wall by com- ultrathin sections have been studied (6, 8, 16), plete extraction of the glucan; the scars were and no evidence of the presence of cell wall rem- found to contain only glucosamine, and traces of nants has been found. Recently, Webley et al. glucose and amino acids. They concluded that (27) described "a structure resembling a sphero- most of the chitin of the yeast cell wall is located plast" in S. cerevisiae cells lysed by the culture in the bud scar region. Their electron micrographs fluid of Cytophaga johnsonii. On the basis of light not only show bud scars but also thin membranes microscopy studies, Ottolenghi (19) considered which remain after the extraction procedure. It the possibility that in some cases remnants of the seems most probable that these membranes are cell wall remain attached to the protoplasts. The identical with the innermost wall layer revealed 706 STREIBLOVA J. BACTERIOL.

by freeze-etching. Furthermore, the cell wall- 2. BACON, J. S., E. D. DAVIDSON, D. JONES, AND I. F. hydrolyzing system of the snail enzyme is found TAYLOR. 1966. The location of chitin in the to have a relatively low proteolytic activity (9, yeast cell wall. Biochem. J. 101:36c-38c. the possibility cannot be entirely 3. BRENNER, S., F. A. DARK, P. GERHARDT, M. H. 20). However, JEYNES, 0. KANDLER, E. KELLENBERGER, E. excluded that, in addition to the glucosamine KELLENBERGER-NOBEL, M. MCQU[LLEN, M. polymeric complex which is very probably chitin, RUBIO-HUERTOS, M. R. J SALTON, R. E. polypeptides (i.e., proteins) could be present in STRANGE, J. ToMcsuK. AND C. WEIBULL. 1958. the innermost wall layer. Bacterial protoplasts. Nature 181:1713-1714. Protoplasts of yeasts have proved to be of great 4. CONTI, S. F., AND T. D. BROCK. 1965. Electron experimental value. Therefore, the availability of microscopy of cell fusion in conjugating Hani- a method for preparing protoplasts free from senula wingei. J. Bacteriol. 90:524-533. wall remnants is very important. The removal of 5. EDDY, A. A., AND D. H. WILLIAMSON. 1957. A method of isolating protoplasts from yeasts. the innermost wall layer by selective degradation Nature 179:1252-1253. by specific enzymes or by the use of wall lytic 6. ELBERS, P. F. 1961. Fixation of yeast protoplasts microbial enzymes should give the desired wall- for electron microscopy. Nature 191:1022-1023. free preparations. Another possibility which 7. GASC6N, S., A. G. OCHOA, AND J. R. VILLANEUVA. seems to be even more promising involves the 1965. Production of yeast and mold protoplasts rupture of the innermost wall layer by changing by treatment with the strepzyme of Micro- the osmotic conditions (19). monospora A S. Can. J. Microbiol. 11:573-580. At present, the criteria for defining protoplasts 8. HAVELKOVA, M. 1966. A comparative study of are loss of rigidity, resulting in a spherical form, submicroscopic structures of protoplasts of various yeast species. Folia Microbiol. (Prague) and osmotic fragility (25). However, both criteria 11:455-458. refer to spheroplasts as well as to protoplasts. 9. HOLDEN, M., AND M. V. TRACEY. 1950. A study Thus, it might be advisable to develop specific of enzymes that can break down tobacco-leaf methods for the identification of protoplasts. components. 2. Digestive juice of Helix on Although the freeze-etching method allows the defined substrates. Biochem. J. 47:407-414. demonstration of remnants of the cell wall on the 10. HouwINK, A. L., AND D. R. KREGER. 1953. Ob- surface of the plasmalemma, quantitative evalua- servations on the cell wall of yeasts. An electron tions of the presence of protoplasts and sphero- microscope and X-ray diffraction study. In most Antonie van Leeuwenhoek J. Microbiol. Serol. plasts seem to be rather problematic. 19:1-24. cases the innermost wall layer, as well as the 11. KUSTER, E. 1935. Die Pflanzenzelle. Gustav surface of the plasmalemma, appears on the Fischer, Jena. freeze-etchings; however, it may be that in some 12. LINDEGREN, C. C. 1963. Nucleoprotein layer of the cases the fracture plane follows the surface of the yeast cell. Nature 198:1325-1326. plasmalemma. Furthermore, the medium used 13. MOOR, H., AND K. MUJHLETHALER. 1963. Fine structure of frozen-etched yeast cells. J. Cell for the stabilization of protoplasts could give Biol. 17:609-627. rise to structures resembling the thin wall layer. 14. MOOR, H., K. MUHLETHALER, H. WALDNER, AND Studies concerning the porosity of the inner- A. FREY-WYSSLING. 1961. A new freezing most wail layer, its osmotic characteristics, and ultramicrotome. J. Biophys. Biochem. Cytol. the location of hydrolytic enzymes, associated 10:1-13. with the wall would be desirable. In addition, the 15. NECAS, 0. 1955. Vitability of cell fragments of yeasts. III. The regeneration of cells from eventual role of the innermost wall layer in the plasmatic formations. Folia Biol. (Prague) formation of the cell wall needs to be investi- 1:220-229. gated. These considerations make the existence 16. NECAS, 0. 1965. The mechanism of regeneration of the innermost wall layer described in this of yeast protoplasts. II. De novo formation of paper a matter of great interest. cell wall. Folia Biol. (Prague) 11:97-102. 17. NORTH, R. J. 1962. Method for revealing the ACKNOWLEDGMENTS membrane system of microorganisms. Nature 190:1215-1216. The author wishes to express her gratitude to A. 18. NORTHCOTE, D. H., AND R. W. HORNE. 1952. Frey-Wyssling. Grateful acknowledgments are made The chemical composition and structure of the to K. Muhlethaler, P. Matile, and H. Moor for their yeast cell wall. Biochem. J. 51:232-236. valuable help in preparing this paper for publication. 19. OTTOLENGHI, P. 1966. Do yeasts form true proto- This investigation was supported by the "Zentenar- plasts? Compt. Rend. Trav. Lab. Carlsberg fonds" of the Swiss Federal Institute of Technology. 35:363-368. 20. PHAFF, H. J. 1963. Cell wall of yeasts. Ann. Rev. LITERATURF CITED Microbiol. 17:15-30. 1. ALPER, R. E., J. L. DAINKO, AND F. SCHLENK. 21. ROST, K., AND H. VENNER. 1965. Enzymatische 1967. Properties of yeast cell ghosts obtained Zellwandverdauung bei Hefen durch Schneck- by ribonuclease action. J. Bacteriol. 93 :759-765. enenzym. Arch. Mikrobol. 51: 122-129. VOL. 95, 1968 SURFACE STRUCTURE OF PROTOPLASTS 707

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