JouRNAL oF BACTERIOLOGY, Sept. 1975, p. 1144-1149 Copyright Ot 1975 American Society for Microbiology

Vol. 123, No. 3 Printed in U.S.A.

Localization of Acid Phosphatase in Protoplasts from Saccharomyces cerevisiae H. J. M. VAN RIJN, W. A. M. LINNEMANS,* AND P. BOER

Laboratory for Physiological Chemistry, Biological Ultrastructure Research Unit, State University of Utrecht, Utrecht, The Netherlands Received for publication 16 April 1975

The localization of acid phosphatase (EC 3.1.3.2) in secreting protoplasts prepared from Saccharomyces cerevisiae is reported for the first time. Using a Gomori technique we were able to show acid phosphatase at those organelles in the protoplasts which are generally involved in the processes of biosynthesis and secretion of glycoproteins in eukaryotic cells. After incubation for acid phosphatase activity the Under favorable conditions yeast protoplasts are able to secrete newly formed cell wall protoplasts were again fixed for 30 min in a mixture of components, such as mannan, invertase (EC 3% glutaraldehyde, 2% formaldehyde, 1% acrolein, dimethylsulfoxide in 0.1 M sodium cacodylate 3.2.1.26), and acid phosphatase (EC 3.1.3.2) 2.5% (pH 7.4). Then the fixed material was rinsed thor(6, 10, 12, 23). In studies concerning the latter oughly in the 0.1 M buffer, pH 7.4, and postfixed 30 enzyme it has been shown that a small but min in 1% OSO4 in the same buffer. Fixation and reproducible amount of acid phosphatase is washings were carried out at room temperature. The strongly bound to secreting protoplasts (23). fixed material was dehydrated in graded acetone and We investigated this enzyme fraction because embedded in Araldite. Thin sections were cut on a it might have a function in the biosynthesis of Reichert OMU 2 microtome. Sections were stained the secreted acid phosphatase. About 70% of according to the method of Millonig (14). Both this protoplast-bound activity can be extracted stained and unstained sections were studied. Electron with 1% Triton X-100 (TE fraction). In tracer micrographs were taken with a Philips EM 200 or a EM 201 electron microscope. experiments, using a radioactive amino acid, we Philips Cytochemical of acid phosphatase. provided evidence for the existence of a precur- Acid phosphataselocalization activities in the protoplasts were sor-product relationship between the enzyme in located by a modified Gomori (7) method using this Triton extract and the secreted enzyme (3). para-nitrophenylphosphate or f-glycerophosphate as Whether this protoplast-bound acid phospha- a substrate. The prefixed protoplasts were incubated tase is localized within the plasmalemma or at room temperature for 30 min in 50 mM sodium outside the protoplast was not yet clear. An acetate (pH 5.5), containing 2.3 mM leadnitrate, 8.2 answer to -this question would provide us with mM substrate and 2.5% dimethylsulfoxide. The folmore insight in the secretory process of the lowing control experiments were carried out: (i) incubation of nonsecreting protoplasts (protoplasts susexoenzyme. We therefore decided to apply a pended in a medium containing 1 mM phosphate); cytochemical approach for the detection of acid (ii) incubation of protoplasts without substrate; and phosphatase with the electron microscope. (iii) addition of an enzyme inhibitor, NaF, to the Gunther et al. (8) and Bauer and Sigarlakie (2) incubation medium. demonstrated acid phosphatase in whole yeast cells. However, as far as we know this is the first RESULTS time that this enzyme is demonstrated in yeast protoplasts. The pH stability range of protoplast-bound enzyme is not very broad, namely, from pH 3.0 MATERIALS AND METHODS to 5.5 (3). Irreversible inactivation takes place Organism and cultivation. Protoplasts, secreting at lower and higher pH. After 30 min of fixation acid phosphatase, were prepared as described previ- with glutaraldehyde at pH 5.5 the remaining ously (23). activity is about 75 to 80%. Fixation below pH Electron microscopy. Protoplasts were prefixed for 5.5 gave a poor preservation of the protoplast 30 min in a mixture of 3% glutaraldehyde, 2.5% dimethylsulfoxide, and 12% (wt/vol) mannitol in 50 structure. Rinsing and incubation at pH 4.5 mM sodium acetate (pH 5.5). The fixed material was after glutaraldehyde fixation at pH 5.5 was rinsed shortly in 12% (wt/vol) mannitol, 2.5% dimeth- unreliable for ultrastructure preservation. The ylsulfoxide in 50 mM sodium acetate (pH 5.5). preservation of some subcellular organelles, like 1144

mitochondria, was rather poor due to the pH and the character of the buffer. Lead phosphate precipitates were found in particular regions in protoplasts, which were incubated in a modified Gomori medium. It is observed in vesicles or small vacuoles (Fig. 1 and 2A), in the central vacuole (Fig. 1, 2A, and 3A), in flat vesicles beneath the plasma membrane (Fig. 1, 2A, 2B, and 3C), in the endoplasmic reticulum (Fig. 1 and 3A), sometimes in the nuclear membrane (Fig. 1), in Golgi-like structures (Fig. 2A), and on the surface of the protoplast. The same protoplasmic structures, but lacking a lead phosphate precipitate, are found in proto-

plasts, in which synthesis of acid phosphatase is repressed (Fig. 2C, 3B, 3D) or in protoplasts incubated without substrate or with enzyme inhibitor. We found differences between the individual protoplasts with regard to extent and localization of acid phosphatase activity as visible by the presence of lead precipitate due to enzyme action. For instance, the central vacuole and the outer surface of the protoplast did not always show activity, nor did the nuclear membrane. To obtain an impression of the distribution of the acid phosphatase activity in the secreting protoplasts, a number of protoplasts were exam-

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9/ icvCV

FIG. 3. Details of stained sections of protoplasts of S. cerevisiae. A, Derepressed protoplast showing activity in the endoplasmic reticulum (ER) and in the central vacuole (CV). x52,000. B, Repressed protoplast. x52,000. C, Derepressed protoplast, showing activity in the flat vesicles (arrows). x52,000. D, Repressed protoplast; a flat vesicle (arrow) is shown. x52,000.

protoplasts show activity in both endoplasmic reticulum and flat vesicles. Hereward (9) described these flat vesicles as subsurface cisternae and related them with sites of protein synthesis. The endoplasmic reticulum plays a role in yeast cell wall synthesis (13), whereas in

plant cells these subsurface cisternae are also involved in the synthesis of material outside the plasmalemma (18). The endoplasmic reticulum is derived from the nuclear membrane (Fig. 2A arrow; see Matile et al. [13] for references). The enzyme

1148 VAN RIJN, LINNEMANS, AND BOER

J. BACTERIOL

ther research is being carried out to elucidate this point. The localization of acid phosphatase in the 80 endoplasmic reticulum in the small vesicles or 60 vacuoles, in a Golgi-like structure, and in flat vesicles underneath the plasmamembrane, 40 agrees with the view that the exocellular yeast glycoproteins are synthesized following a path20 way similar to that of glycoproteins of higher 0 eukaryotes (4, 11, 21, 22, 23). Although we are A B C D E F G not able to clarify the function and ontogeny of FIG. 4. Diagram representing the distribution of the diverse organelles with respect to glycoproacid-phosphatase in a population of protoplasts show- tein synthesis, we can now conclude that the ing enzyme activity (about 90% of the population). precursors of the exocellular acid phosphatase The values are the average of two counts in one par- (3) are located within the plasmalemma. ticular 100

experiment. In each counting about 100 protoplasts were involved. Ordinate: percentage of protoplasts showing acid phosphatase activity. Abcissa: (A) outside the plasma membrane, not attributable to remnants of cell wall material; (B) within the central vacuole; (C) in small vacuoles or vesicles; (D) in flat vesicles just beneath the plasma membrane; (E) in the endoplasmic reticulum; (F) in the nuclear membrane; and (G) in Golgi-like structures.

activity associated with this membrane is probably not due to glutaraldehyde activation as has been suggested by Bauer and Sigarlakie (2), because in our experiments aldehyde fixation never exceeded 30 min. We observed acid phosphatase in the central vacuole in about 50% of our active protoplasts, whereas Bauer and Sigarlakie (2) found no activity in this organelle. We were not able to exclude the possibility of intracellular degradation of acid phosphatase concomitant with its synthesis and excretion. The activity in the central vacuole, which is considered as a secondary lysosome (13), is in this respect very suggestive. Since it is known that the central vacuole is divided into a number of smaller vacuoles during mitosis (13), a number of active small vesicles could be derived from the central vacuole in such a way. We, however, found them also occurring together with the central vacuole. At this moment we are not able to determine the function of the small vesicles. We have found another characteristic structure in which acid phosphatase is located. This structure has some morphological resemblance with the Golgi apparatus (19, 20), whereas preliminary results indicate that thiamine pyrophosphatase (EC 3.6.1. ) activity, a marker for Golgi apparatus (5), is associated with the same structure. Since the occurrence of Golgi in yeast is controversial (1, 8, 13, 15, 16, 17) we tentatively describe this structure as Golgi-like. Fur-

ACKNOWLEDGMENTS We thank E. P. Steyn-Parvre and P. F. Elbers for their interest.

LITERATURE CITED 1. Bauer, H., and E. Sigarlakie. 1972. Ultrathin frozen sections of yeast cells. J. Ultrastr. Res. 40:197-204. 2. Bauer, H., and E. Sigarlakie. 1973. Cytochemistry on ultrathin sections of yeast cells. J. Microscr. (Oxford) 99:205-219. 3. Boer, P., H. J. M. van Rijn, A. Reinking, and E. P. Steyn-Parve. 1975. Biosynthesis of acid phosphatase of baker's yeast. Characterisation of a protoplast-bound fraction containing precursors of the exo-enzyme. Biochim. Biophys. Acta 377:331-342. 4. Cortat, M., P. Matile, and F. Kopp. 1973. Intracellular localization of mannan synthetase activity in budding baker's yeast. Biochem. Biophys. Res. Commun. 53: 482-489. 5. Dauwalder, M., J. E. Kephart, and W. G. Whalley. 1966. Phosphatase and the Golgi apparatus in differentiating cells. J. Cell Biol. 31:25A-26A. 6. Farkas, V., A. Svodoba, and S. Bauer. 1970. Secretion of cell wall glycoproteins by yeast protoplasts. Biochem. J. 118:755-758. 7. Gomori, G. 1952. Microscopic histochemistry. University of Chicago Press, Chicago. 8. Gunther, Th., W. Kattner, and H. J. Merker. 1966. Ueber das Verhalten und die Lokalisation der Saueren Phosphatase von Hefezellen bei Repression und Derepression. Exp. Cell Res. 45:133-147. 9. Hereward, F. V. 1974. Rough membranes in Schizosaccharomyces pombe protoplasts. Exp. Cell Res. 87: 213-218. 10. Islam, M. F., and J. 0. Lampen. 1962. Invertase secretion and sucrose fermentation by Saccharomyces cerevisiae protoplasts. Biochim. Biophys. Acta 58:294-302. 11. Lampen, J. O., S. C. Kuo, F. F. Cano, and J. Z. Tkacz. 1972. Structural features in synthesis of external enzymes by yeast, p. 819. In G. Terui (ed.), Fermentation technology today. Society of Fermentation Technology, Japan. 12. McLellan, W. L., and J. 0. Lampen. 1963. The acid phosphatase of yeast. Localization and secretion by protoplasts. Biochim. Biophys. Acta 67:324-326. 13. Matile, P., H. Moor, and C. Robinow. 1969. Yeast cytology, p. 219-302. In A. H. Rose and J. S. Harrison (ed.), The yeasts. vol. I. Academic Press Inc., New York. 14. Millonig, G. 1961. A modified procedure for lead staining

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of thin sections. J. Biophys. Biochem. Cytol. 11:736-739. Moor, H. 1967. Endoplasmic reticulum as the initiator of bud formation in yeast (S. cerevisiae). Arch. Mikrobiol. 57:135-146. Moor, H. 1967. Feinbau der Mikrotubuli in Hefe nach gefrieriitzung. Protoplasma 64:89-103. Moor, H., and K. Miihlethaler. 1963. Fine structure in frozen-etched yeast cells. J. Cell Biol. 17:609-628. Northcote, D. H., and F. B. P. Wooding. 1966. Development of sieve tubes in Acer pseudo platanus. Proc. R. Soc. London Ser. B. 163:524-537. Novikoff, A. B., E. Essner, S. Goldfisher, and M. Heus, 1962. Nucleoside phosphatase activities of cytomembranes. Symp. Int. Soc. Cell Biol. 1:149-192.

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20. Novikoff, A. B., E. Essner, and N. Quintana. 1964. Golgi apparatus and Iysosomes. Fed. Proc. Fed. Am. Soc. Exp. Biol. 23:1010-1025. 21. Roland, J. C. 1973. The relationship between the plasmalemma and plant cell wall. Int. Rev. Cytol. 36:45-92. 22. Sentandreu, R., and M. V. Elorza. 1973. The biosvnthesis pathway of veast mannan glycoproteins, p. 187. In J. R. Villanueva, I. Garcia-Acha, S. Gasc6n, and F. Uruburu (ed.), Yeast, mould and plant protoplast. Academic Press Inc., New York. 23. van Rijn. H. J. M., P. Boer, and E. P. Steyn-Parv&. 1972. Biosynthesis of acid phosphatase of baker's yeast. Factors influencing its production by protoplasts and characterization of the secreted enzyme. Biochim. Biophvs. Acta 268:431-441.

Localization of acid phosphatase in protoplasts from Saccharomyces cerevisiae.

JouRNAL oF BACTERIOLOGY, Sept. 1975, p. 1144-1149 Copyright Ot 1975 American Society for Microbiology Vol. 123, No. 3 Printed in U.S.A. Localization...
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