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

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

Localization of Acid Phosphatase in Protoplasts fromSaccharomyces 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 protoplastsprepared from Saccharomyces cerevisiae is reported for the first time. Using aGomori technique we were able to show acid phosphatase at those organelles inthe protoplasts which are generally involved in the processes of biosynthesis andsecretion of glycoproteins in eukaryotic cells.

Under favorable conditions yeast protoplastsare able to secrete newly formed cell wallcomponents, such as mannan, invertase (EC3.2.1.26), and acid phosphatase (EC 3.1.3.2)(6, 10, 12, 23). In studies concerning the latterenzyme it has been shown that a small butreproducible amount of acid phosphatase isstrongly bound to secreting protoplasts (23).We investigated this enzyme fraction because

it might have a function in the biosynthesis ofthe secreted acid phosphatase. About 70% ofthis protoplast-bound activity can be extractedwith 1% Triton X-100 (TE fraction). In tracerexperiments, using a radioactive amino acid, weprovided evidence for the existence of a precur-sor-product relationship between the enzyme inthis Triton extract and the secreted enzyme (3).Whether this protoplast-bound acid phospha-

tase is localized within the plasmalemma oroutside the protoplast was not yet clear. Ananswer to -this question would provide us withmore insight in the secretory process of theexoenzyme. We therefore decided to apply acytochemical approach for the detection of acidphosphatase with the electron microscope.Gunther et al. (8) and Bauer and Sigarlakie (2)demonstrated acid phosphatase in whole yeastcells. However, as far as we know this is the firsttime that this enzyme is demonstrated in yeastprotoplasts.

MATERIALS AND METHODSOrganism and cultivation. Protoplasts, secreting

acid phosphatase, were prepared as described previ-ously (23).

Electron microscopy. Protoplasts were prefixed for30 min in a mixture of 3% glutaraldehyde, 2.5%dimethylsulfoxide, and 12% (wt/vol) mannitol in 50mM sodium acetate (pH 5.5). The fixed material wasrinsed shortly in 12% (wt/vol) mannitol, 2.5% dimeth-ylsulfoxide in 50 mM sodium acetate (pH 5.5).

After incubation for acid phosphatase activity theprotoplasts were again fixed for 30 min in a mixture of3% glutaraldehyde, 2% formaldehyde, 1% acrolein,2.5% dimethylsulfoxide in 0.1 M sodium cacodylate(pH 7.4). Then the fixed material was rinsed thor-oughly in the 0.1 M buffer, pH 7.4, and postfixed 30min in 1% OSO4 in the same buffer. Fixation andwashings were carried out at room temperature. Thefixed material was dehydrated in graded acetone andembedded in Araldite. Thin sections were cut on aReichert OMU 2 microtome. Sections were stainedaccording to the method of Millonig (14). Bothstained and unstained sections were studied. Electronmicrographs were taken with a Philips EM 200 or aPhilips EM 201 electron microscope.

Cytochemical localization of acid phosphatase.Acid phosphatase activities in the protoplasts werelocated by a modified Gomori (7) method usingpara-nitrophenylphosphate or f-glycerophosphate asa substrate. The prefixed protoplasts were incubatedat room temperature for 30 min in 50 mM sodiumacetate (pH 5.5), containing 2.3 mM leadnitrate, 8.2mM substrate and 2.5% dimethylsulfoxide. The fol-lowing control experiments were carried out: (i) incu-bation of nonsecreting protoplasts (protoplasts sus-pended in a medium containing 1 mM phosphate);(ii) incubation of protoplasts without substrate; and(iii) addition of an enzyme inhibitor, NaF, to theincubation medium.

RESULTS

The pH stability range of protoplast-boundenzyme is not very broad, namely, from pH 3.0to 5.5 (3). Irreversible inactivation takes placeat lower and higher pH. After 30 min of fixationwith glutaraldehyde at pH 5.5 the remainingactivity is about 75 to 80%. Fixation below pH5.5 gave a poor preservation of the protoplaststructure. Rinsing and incubation at pH 4.5after glutaraldehyde fixation at pH 5.5 wasunreliable for ultrastructure preservation. Thepreservation of some subcellular organelles, like

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YEAST ACID PHOSPHATASE 1145

mitochondria, was rather poor due to the pHand the character of the buffer.Lead phosphate precipitates were found in

particular regions in protoplasts, which wereincubated in a modified Gomori medium. It isobserved in vesicles or small vacuoles (Fig. 1 and2A), in the central vacuole (Fig. 1, 2A, and 3A),in flat vesicles beneath the plasma membrane(Fig. 1, 2A, 2B, and 3C), in the endoplasmicreticulum (Fig. 1 and 3A), sometimes in the nu-clear membrane (Fig. 1), in Golgi-like structures(Fig. 2A), and on the surface of the protoplast.The same protoplasmic structures, but lacking alead phosphate precipitate, are found in proto-

plasts, in which synthesis of acid phosphatase isrepressed (Fig. 2C, 3B, 3D) or in protoplasts in-cubated without substrate or with enzyme in-hibitor.We found differences between the individual

protoplasts with regard to extent and localiza-tion of acid phosphatase activity as visible bythe presence of lead precipitate due to enzymeaction. For instance, the central vacuole and theouter surface of the protoplast did not alwaysshow activity, nor did the nuclear membrane.To obtain an impression of the distribution ofthe acid phosphatase activity in the secretingprotoplasts, a number of protoplasts were exam-

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FIG. 2. Unstained sections of protoplasts of S. cerevisiae. A, Derepressed protoplast lacking activity in thenuclear membrane (NM), endoplasmic reticulum (ER), and in the flat vesicles (FV). x26,000. B, Detail of aderepressed protoplast incubated for acid phosphatase, showing fusion of a flat vesicle with the plasmalemma(arrow). x58,200. C, Detail of a repressed protoplast, showing Golgi-like structure (G) lacking precipitate.x33,600.

ined, and the number of times that a certainlocation was observed was noted (Fig. 4). About10% of the protoplasts show no enzyme activity.

DISCUSSIONConsidering the distribution of acid phospha-

tase activity in secreting protoplasts (Fig. 4),the enzyme appears to be located mainly withinthe protoplast. Some activity is found on thesurface of protoplasts which is due either toabsorption of enzyme from lysed protoplasts orto cell wall synthesis.

According to Bauer and Sigarlakie (2) acidphosphatase is located in the cell wall, in smallvesicles or vacuoles, and in the nucleus of wholeyeast cells. These results are in agreement withthe observations of Gunther et al. (8). We foundacid phosphatase associated with the endoplas-mic reticulum, the flat vesicles and sometimeswith the nuclear membrane. Although Matile etal. (13) state that these flat vesicles belong tothe endoplasmic reticulum we made a distinc-tion between these structures, because we ob-served a difference in activity: only 14% of the

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FIG. 3. Details of stained sections of protoplasts of S. cerevisiae. A, Derepressed protoplast showing activityin 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; aflat vesicle (arrow) is shown. x52,000.

protoplasts show activity in both endoplasmicreticulum and flat vesicles. Hereward (9) de-scribed these flat vesicles as subsurface cister-nae and related them with sites of proteinsynthesis. The endoplasmic reticulum plays arole in yeast cell wall synthesis (13), whereas in

plant cells these subsurface cisternae are alsoinvolved in the synthesis of material outside theplasmalemma (18).The endoplasmic reticulum is derived from

the nuclear membrane (Fig. 2A arrow; seeMatile et al. [13] for references). The enzyme

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1148 VAN RIJN, LINNEMANS, AND BOER

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FIG. 4. Diagram representing the distribution ofacid-phosphatase in a population of protoplasts show-ing enzyme activity (about 90% of the population).The values are the average of two counts in one par-

ticular experiment. In each counting about 100 proto-plasts were involved. Ordinate: percentage of proto-plasts showing acid phosphatase activity. Abcissa:(A) outside the plasma membrane, not attributable toremnants of cell wall material; (B) within the centralvacuole; (C) in small vacuoles or vesicles; (D) in flatvesicles just beneath the plasma membrane; (E) in theendoplasmic reticulum; (F) in the nuclear membrane;and (G) in Golgi-like structures.

activity associated with this membrane isprobably not due to glutaraldehyde activationas has been suggested by Bauer and Sigarlakie(2), because in our experiments aldehyde fixa-tion 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 toexclude the possibility of intracellular degrada-tion of acid phosphatase concomitant with itssynthesis and excretion. The activity in thecentral vacuole, which is considered as a sec-

ondary lysosome (13), is in this respect verysuggestive.

Since it is known that the central vacuole isdivided into a number of smaller vacuolesduring mitosis (13), a number of active smallvesicles could be derived from the central vacu-ole in such a way. We, however, found them alsooccurring together with the central vacuole. Atthis moment we are not able to determine thefunction of the small vesicles.We have found another characteristic struc-

ture in which acid phosphatase is located. Thisstructure has some morphological resemblancewith the Golgi apparatus (19, 20), whereas pre-liminary results indicate that thiamine pyro-phosphatase (EC 3.6.1. ) activity, a marker forGolgi apparatus (5), is associated with the samestructure. Since the occurrence of Golgi in yeastis controversial (1, 8, 13, 15, 16, 17) we tenta-tively describe this structure as Golgi-like. Fur-

ther research is being carried out to elucidatethis point.The localization of acid phosphatase in the

endoplasmic reticulum in the small vesicles orvacuoles, in a Golgi-like structure, and in flatvesicles underneath the plasmamembrane,agrees with the view that the exocellular yeastglycoproteins are synthesized following a path-way similar to that of glycoproteins of highereukaryotes (4, 11, 21, 22, 23). Although we arenot able to clarify the function and ontogeny ofthe diverse organelles with respect to glycopro-tein synthesis, we can now conclude that theprecursors of the exocellular acid phosphatase(3) are located within the plasmalemma.

ACKNOWLEDGMENTS

We thank E. P. Steyn-Parvre and P. F. Elbers for theirinterest.

LITERATURE CITED1. 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 ofbaker's yeast. Characterisation of a protoplast-boundfraction containing precursors of the exo-enzyme. Bio-chim. Biophys. Acta 377:331-342.

4. Cortat, M., P. Matile, and F. Kopp. 1973. Intracellularlocalization of mannan synthetase activity in buddingbaker'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 differentiatingcells. J. Cell Biol. 31:25A-26A.

6. Farkas, V., A. Svodoba, and S. Bauer. 1970. Secretion ofcell wall glycoproteins by yeast protoplasts. Biochem. J.118:755-758.

7. Gomori, G. 1952. Microscopic histochemistry. Universityof Chicago Press, Chicago.

8. Gunther, Th., W. Kattner, and H. J. Merker. 1966. Ueberdas Verhalten und die Lokalisation der Saueren Phos-phatase von Hefezellen bei Repression und Derepres-sion. Exp. Cell Res. 45:133-147.

9. Hereward, F. V. 1974. Rough membranes in Schizosac-charomyces pombe protoplasts. Exp. Cell Res. 87:213-218.

10. Islam, M. F., and J. 0. Lampen. 1962. Invertase secretionand sucrose fermentation by Saccharomyces cerevisiaeprotoplasts. 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 en-zymes by yeast, p. 819. In G. Terui (ed.), Fermentationtechnology today. Society of Fermentation Technology,Japan.

12. McLellan, W. L., and J. 0. Lampen. 1963. The acidphosphatase of yeast. Localization and secretion byprotoplasts. Biochim. Biophys. Acta 67:324-326.

13. Matile, P., H. Moor, and C. Robinow. 1969. Yeastcytology, p. 219-302. In A. H. Rose and J. S. Harrison(ed.), The yeasts. vol. I. Academic Press Inc., NewYork.

14. Millonig, G. 1961. A modified procedure for lead staining

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of thin sections. J. Biophys. Biochem. Cytol.11:736-739.

15. Moor, H. 1967. Endoplasmic reticulum as the initiator ofbud formation in yeast (S. cerevisiae). Arch. Mikrobiol.57:135-146.

16. Moor, H. 1967. Feinbau der Mikrotubuli in Hefe nachgefrieriitzung. Protoplasma 64:89-103.

17. Moor, H., and K. Miihlethaler. 1963. Fine structure infrozen-etched yeast cells. J. Cell Biol. 17:609-628.

18. Northcote, D. H., and F. B. P. Wooding. 1966. Develop-ment of sieve tubes in Acer pseudo platanus. Proc. R.Soc. London Ser. B. 163:524-537.

19. Novikoff, A. B., E. Essner, S. Goldfisher, and M. Heus,1962. Nucleoside phosphatase activities of cytomem-branes. Symp. Int. Soc. Cell Biol. 1:149-192.

20. Novikoff, A. B., E. Essner, and N. Quintana. 1964. Golgiapparatus and Iysosomes. Fed. Proc. Fed. Am. Soc.Exp. Biol. 23:1010-1025.

21. Roland, J. C. 1973. The relationship between the plasma-lemma and plant cell wall. Int. Rev. Cytol. 36:45-92.

22. Sentandreu, R., and M. V. Elorza. 1973. The biosvnthesispathway of veast mannan glycoproteins, p. 187. InJ. 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 andcharacterization of the secreted enzyme. Biochim.Biophvs. Acta 268:431-441.

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