JOURNAL OF BACTERIOLOGY, Feb. 1968, p. 700-707 Vol. 95, No. 2 Copyright © 1968 American Society for Microbiology Prinzted in U.S.A. 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 O,5,u - 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).