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Modulation of a Cell Surface Glycoprotein in Yeast: Acid Phosphatase
Differentiat ion
Differentiation (1981) 19: 68-70
0 Springcr-Verlag 198 I
Preliminary Reports
Modulation of a Cell Surface Glycoprotein in Yeast: Acid Phosphatase M. ERNST SCHWEINGRUBER and ANNE-MARIE SCHWEINGRUBER Institute for General Microbiology, Altenbergrain 21, CH-3013 Bern, Switzerland Upon inorganic phosphate starvation the cell wall glycoprotein acid phosphatase of yeast Saccharomyces cerevisiae is derepressed. Purified acid phosphatase isolated from early log phase cells differs in reactivity and stability from acid phosphatase from late log phase cells indicating that the two enzymes are structurally different. This demonstrates that the yeast cell has not only the capacity to regulate the amount of acid phosphatase but also the ability to vary (modulate) the structure of the secreted enzyme. Modulation of acid phosphatase may be a mechanism which is involved in morphogenetic and behavioral differentiation of the yeast cell.
Introduction Changes in the surface properties of many eucaryotic cells occur during differentiation and at least some of these changes are known to be involved in cellular adhesion and cellular morphogenesis [11. To understand the molecular mechanisms involved in the regulation and differentiation of the cell surface structure, we are investigating the regulation of acid phosphatase of the yeast Saccharomyces cerevisiae. Acid phosphatase of yeast is a cell surface glycoprotein containing about equal amounts of mannose and protein [2-41. Upon starvation for inorganic phosphate acid phosphatase is derepressed [S, 61. Data indicating that derepression of acid phosphatase occurs substantially at the posttranslational level have been published recently [7]. Other data indicate regulation occurs at the transcriptional and translational level as well [S, 91. Many efforts have been made to elucidate the mechanisms by which the cell can regulate the amount of acid phosphatase. In contrast no data are available yet indicating whether acid phosphatase regulation has also a qualitative aspect. It is not known if the yeast cell has also the capacity to alter the structure (quality) of acid phosphatase. The date reported here indicate that the yeast cell is able to respond to changing nutritional conditions by altering the structure and thereby the reactivity and stability of acid phosphatase.
Methods Strain
The haploid strain H-42 [6] was kindly supplied by Dr. Y. Oshima. Media and Growth Conditiom
Yeast cells were grown in YEPD medium (1% Bacto-Yeast extract, 2% Bacto-Peptone, 2% dextrose autoclaved separately) from which inorganic phosphate had been removed by a slightly modified method of Rubin [lo]. This is called low phosphate medium. To prepare 2 I of a 5 times concentrated yeast extract-peptone solution for the low phosphate medium, the following protocol was used: l00g BactoYeast extract and 200 g Bacto-Peptone was dissolved in 1.5 I HzO.
Then 50 ml of 25% ammonia solution and 50 ml of 1M MgS04 were added to precipitate the inorganic phosphate. The solution was stirred 1 h and subsequently left at 4°C for approximately 16 h. The precipitate was collected by centrifugation and filtration. The pH of the supernatant was brought to 5.8 with conc. HCI. Water was added to give a fmal volume of 2 1. The orthophosphate concentration in this medium is not known. It is, however, low enough to derepress cells fully for acid phosphatase activity (Schweingruber, unpublished results). Cells were grown at 30°C in a 2-1 Erlenmeyer containing 500-ml medium on a rotary shaker (Pilot Shake RC2 SP) at a rotation speed of 100 rpm. Log phase cells were harvested at a cell density of about 3 x lo7celldml. Stationary phase cells had a cell density of about 2 x 108 cells. Determination of Enzyme Activity
Acid phosphatase activity was determined as follows: To an appropriate amount of enzyme solution or washed cells 1 ml of 0.2 M sodium acetate buffer pH 4.5 containing 1 mg nitrophenylphosphate (Merck) was added. The reaction was stopped after 5 min incubation at 37" C by the addition of 2 ml 0.5 N sodium hydroxide. The absorbancy of the yellow p-nitrophenol released was measured at 405nm against a control tube to which no enzyme had been added. One enzyme unit (U) was defined as the amount of enzyme required to produce 1 pmol of p-nitrophenol per minute at 37°C. Purification of Acid Phosphatase
Acid phosphatase from wildtype H-42 was purified as described by Dibenedetto and Cozzani [ll]. The purification procedure includes three steps: freeze-thawing, ammonium sulfate precipitation, and chromatography on Sepharose CL-6B. The criteria used for judging purity were polyacrylamide gel electrophoresis under denaturing and nondenaturing conditions as described [111. No other protein bands except those corresponding to the enzymatic activity were observed. For log phase cells the protein bands were somewhat broader than for stationary phase cells.
Results and Discussion Knowing that cell surface mannoproteins are responsible for cell morphology and cell-cell interactions [12, 131 and that growth conditions influence cell morphology [14], we were
0301-4681/81/0019/0068/$01.00
M. E. Schweingruber and A.-M. Schweingruber: Modulation of Acid Phosphatase of Yeast
69
Table 1. Purification of acid phosphatase of stationary phase cells and log phase cells. Acid phosphatase from step 5 is pure as judged by gel
electrophoresis under various conditions [ll] Purification step
1 Washed cells 2 Crude extract 3 Supernatant after freezing-thawing 4 Supernatant after (NH&S04 precipitation 5 Pooled eluate. from Sepharose-CL 6B column
Stationary phase cells
log phase cells
Activity
Spec. activity (U/mg protein)
Activity
Spec. activity (U/mg protein)
100
-
100 54
0.48
44
1.27 19
29.5 28 28 14.2
2.71 2.9
30 21
65
103.2
88
Table 2. Effect of pH on the activity of acid phosphatase purified from log phase cells and stationary phase cells. Acetate buffer with different
pHs was used to assay phosphatase activity. The maximal activity at pH 4.0 was taken as 100% PH
log phase enzyme
Stationary phase enzyme (%)
3.5 4.0 4.5 5.0 5.35
Fig.1. Lineweaver-Burk plots of acid phosphatase purified from log phase cells (0)and stationary phase cells (0).Nitrophenylphosphate was used as the substrates. Initial velocity v is plotted as the measured optical density at 405 nm. The K M for the early log phase enzyme is 1.80 mM. The KM for the late log phase enzyme is 0.43 mM. The corresponding K,,,s derived from measurements using whole cells are 1.5 m M and 0.3 mM
l
30
80
w
120
Inrtlvation thw ( m i d
Fig. 2. Heat inactivation of acid phosphatase purified from log phase cells (0)and stationary phase cells (0).The enzyme appropriately diluted in acetate buffer was preincubated at 55" C for the indicated period of time and was rapidly cooled in an ice bath. Enzyme activity without preincubation was scored as 100% (0.013U) and the remaining activities were expressed as a percentage of this value
100 100 74 52 21
98 100
90 75 51
interested in knowing if the yeast cell has the capacity to alter the structure of acid phosphatase. Therefore we compared purified acid phosphatase from cells grown under different physiological conditions, namely, from log phase cells and stationary phase cells. The purification protocol is given in Table 1. The two purified enzymes differ only slightly in their specific activities. However, they differ significantly in their affinity for nitrophenylphosphate. As illustrated in Fig. 1, the enzyme of stationary phase cells has greater affinity for nitrophenylphosphate than the enzyme of log phase cells. It can be argued that the differences of the substrate affinity are extraction artefacts. This possibility can be excluded since acid phosphatase is located at the surface of the cell and its activity can be measured directly in vivo using whole cells. Whole cells from the log phase and the stationary phase show a similar difference in substrate affinity as found for the pure enzymes (see Fig. 1). In addition to substrate affinity we compared stability at elevated temperatures and maximum initial velocity Vmax as a function of pH of the two enzymes. The data are given in Fig. 2 and Table 2. Acid phosphatase from stationary phase cells has a higher heat stability and a broader pH optimum than acid phosphatase of log phase cells. Since the enzymes used were pure, the differences in the tested properties have to reflect structural differences of the two enzymes. The easiest explanation for the structural differences in acid phosphatase of log phase cells and stationary phase cells is that exogenous signals such as nutrient availability are metabolically integrated by the cell and expressed in terms of differentially modulated acid phosphatase. It is conceivable that this differentially modulated acid phosphatase may in turn, together with other cell surface glycoproteins, influence cell morphology and cell behavior. Differential modulation may also be related to the cell cycle. It is not known if modulation affects intracellular location of acid
70
M. E. Schweingruber and A.-M. Schweingruber: Modulation of Acid Phosphatase of Yeast
phosphatase. Different distributions of the enzyme in the cell at various growth phases have been described [15]. The modulation mechanism is unknown. Differential expression of acid phosphatase isoenzymes however can probably be excluded. At least the major part of the repressible form of acid phosphatase seems to be coded for only by one gene, pho E [16].The most probable modulation mechanisms are covalent modifications of the polypeptide. The nature of the modifications is currently under investigation. Acknowledgements. This work was supported by the Swiss National Science Foundation.
References 1. Harmon RE (1978) Cell surface carbohydrate chemistry. Academic Press Inc, London, New York 2. Schmidt G, Bartsch G, Laumont M-C, Herman Th, Liss M (1%3) Acid phosphatase of Bakers’ yeast. An enzyme of the external surface. Biochemistry 2: 126 3. Linnemans WAM, Boer P, Elbers PF (1977) Localization of acid phosphatase in Sacchuromyces cerevisiue: a clue to cell wall formation. J Bacteriol 131:638 4. Boer P, Steyn-Pad EP (1966) Isolation and purification of an acid phosphatase from Baker’s yeast (Succharomyces cerevisiue). Biochim Biophys Acta 128: 400 5. Schurr A, Yagil E (1971) Regulation and characterization of acid and alkaline phosphatase in yeast. J Gen Microbiol 65: 291 6. Toh-e A, Ueda Y, Kakimoto S, Oshima Y (1973) Isolation and characterization of acid phosphatase mutants in Saccharomyces cerevisiue. J Bacteriol 113:727
Note udded in Proof: Since this report was submitted for publication we have found that mutants of the fission yeast Schizosucchuromyces pombe altered in the structure of acid phosphatase differ from wildtype in morphology and agglutination. This implies that modulation of acid phosphatase is a mechanism which is involved in morphogenetic and behavioural differentiation of the yeast cell.
7. Schweingruber ME,Schweingruber AM (1979) Posttranslational regulation of repressible acid phosphatase in yeast. Mol Gen Genet 173: 349 8. Elona MV, Rodriquez L, Villanueva JR, Sentandrea R (1978) Regulation of acid phosphatase synthesis in Sacchuromyces cerevisiae. Biochim Biophys Acta 521 : 342 9. Bostian KA, Lemire JM, Cannon LE, Halvorson HO (1980) In vitro synthesis of repressible yeast acid phosphatase: Identification of multiple mRNAs and products. Proc Natl Acad Sci 77: 4504 10. Rubin GM (1973) The nucleotide sequence of Succharomyces cerevisiue 5.8s ribosomal ribonucleic acid. J Biol Chem 248 : 3860 11. Dibenedetto G, Cozzani I (1975) Nonspecific acid phosphatase from Schizosacchuromyces pombe. Purification and physical chemical properties. Biochemistry 14: 2847 12. Ballou C (1976) Structure and biosynthesis of the mannan component of the yeast cell envelope.. Adv Microbiol Physiol 14: 93 13. Crandall M, Egel R, Mackay VL (1977) Physiology of mating in three yeasts. Adv Microbiol Physiol 15 : 307 14. Scherr GH, Weaver RH (1953) The dimorphism phenomenon in yeasts. Bacterial Rev 17: 51 15. Rainina EI, Zubatov AS, Buchwalow IB, Luzikov NV (1979) A cytochemical study of the localization of acid phosphatase in Succharomyces cerevisiue at different growth phases. Histochem J 11: 299 16. Toh-e A, Kakimoto S, Oshima Y (1975) Genes coding for the structure of the acid phosphatase in Succharomyces cerevisiue. Mol Gen Genet 143: 65
Received September 1980/Accepted January 1981
Differentiation (1981) 19: 68-70
0 Springcr-Verlag 198 I
Preliminary Reports
Modulation of a Cell Surface Glycoprotein in Yeast: Acid Phosphatase M. ERNST SCHWEINGRUBER and ANNE-MARIE SCHWEINGRUBER Institute for General Microbiology, Altenbergrain 21, CH-3013 Bern, Switzerland Upon inorganic phosphate starvation the cell wall glycoprotein acid phosphatase of yeast Saccharomyces cerevisiae is derepressed. Purified acid phosphatase isolated from early log phase cells differs in reactivity and stability from acid phosphatase from late log phase cells indicating that the two enzymes are structurally different. This demonstrates that the yeast cell has not only the capacity to regulate the amount of acid phosphatase but also the ability to vary (modulate) the structure of the secreted enzyme. Modulation of acid phosphatase may be a mechanism which is involved in morphogenetic and behavioral differentiation of the yeast cell.
Introduction Changes in the surface properties of many eucaryotic cells occur during differentiation and at least some of these changes are known to be involved in cellular adhesion and cellular morphogenesis [11. To understand the molecular mechanisms involved in the regulation and differentiation of the cell surface structure, we are investigating the regulation of acid phosphatase of the yeast Saccharomyces cerevisiae. Acid phosphatase of yeast is a cell surface glycoprotein containing about equal amounts of mannose and protein [2-41. Upon starvation for inorganic phosphate acid phosphatase is derepressed [S, 61. Data indicating that derepression of acid phosphatase occurs substantially at the posttranslational level have been published recently [7]. Other data indicate regulation occurs at the transcriptional and translational level as well [S, 91. Many efforts have been made to elucidate the mechanisms by which the cell can regulate the amount of acid phosphatase. In contrast no data are available yet indicating whether acid phosphatase regulation has also a qualitative aspect. It is not known if the yeast cell has also the capacity to alter the structure (quality) of acid phosphatase. The date reported here indicate that the yeast cell is able to respond to changing nutritional conditions by altering the structure and thereby the reactivity and stability of acid phosphatase.
Methods Strain
The haploid strain H-42 [6] was kindly supplied by Dr. Y. Oshima. Media and Growth Conditiom
Yeast cells were grown in YEPD medium (1% Bacto-Yeast extract, 2% Bacto-Peptone, 2% dextrose autoclaved separately) from which inorganic phosphate had been removed by a slightly modified method of Rubin [lo]. This is called low phosphate medium. To prepare 2 I of a 5 times concentrated yeast extract-peptone solution for the low phosphate medium, the following protocol was used: l00g BactoYeast extract and 200 g Bacto-Peptone was dissolved in 1.5 I HzO.
Then 50 ml of 25% ammonia solution and 50 ml of 1M MgS04 were added to precipitate the inorganic phosphate. The solution was stirred 1 h and subsequently left at 4°C for approximately 16 h. The precipitate was collected by centrifugation and filtration. The pH of the supernatant was brought to 5.8 with conc. HCI. Water was added to give a fmal volume of 2 1. The orthophosphate concentration in this medium is not known. It is, however, low enough to derepress cells fully for acid phosphatase activity (Schweingruber, unpublished results). Cells were grown at 30°C in a 2-1 Erlenmeyer containing 500-ml medium on a rotary shaker (Pilot Shake RC2 SP) at a rotation speed of 100 rpm. Log phase cells were harvested at a cell density of about 3 x lo7celldml. Stationary phase cells had a cell density of about 2 x 108 cells. Determination of Enzyme Activity
Acid phosphatase activity was determined as follows: To an appropriate amount of enzyme solution or washed cells 1 ml of 0.2 M sodium acetate buffer pH 4.5 containing 1 mg nitrophenylphosphate (Merck) was added. The reaction was stopped after 5 min incubation at 37" C by the addition of 2 ml 0.5 N sodium hydroxide. The absorbancy of the yellow p-nitrophenol released was measured at 405nm against a control tube to which no enzyme had been added. One enzyme unit (U) was defined as the amount of enzyme required to produce 1 pmol of p-nitrophenol per minute at 37°C. Purification of Acid Phosphatase
Acid phosphatase from wildtype H-42 was purified as described by Dibenedetto and Cozzani [ll]. The purification procedure includes three steps: freeze-thawing, ammonium sulfate precipitation, and chromatography on Sepharose CL-6B. The criteria used for judging purity were polyacrylamide gel electrophoresis under denaturing and nondenaturing conditions as described [111. No other protein bands except those corresponding to the enzymatic activity were observed. For log phase cells the protein bands were somewhat broader than for stationary phase cells.
Results and Discussion Knowing that cell surface mannoproteins are responsible for cell morphology and cell-cell interactions [12, 131 and that growth conditions influence cell morphology [14], we were
0301-4681/81/0019/0068/$01.00
M. E. Schweingruber and A.-M. Schweingruber: Modulation of Acid Phosphatase of Yeast
69
Table 1. Purification of acid phosphatase of stationary phase cells and log phase cells. Acid phosphatase from step 5 is pure as judged by gel
electrophoresis under various conditions [ll] Purification step
1 Washed cells 2 Crude extract 3 Supernatant after freezing-thawing 4 Supernatant after (NH&S04 precipitation 5 Pooled eluate. from Sepharose-CL 6B column
Stationary phase cells
log phase cells
Activity
Spec. activity (U/mg protein)
Activity
Spec. activity (U/mg protein)
100
-
100 54
0.48
44
1.27 19
29.5 28 28 14.2
2.71 2.9
30 21
65
103.2
88
Table 2. Effect of pH on the activity of acid phosphatase purified from log phase cells and stationary phase cells. Acetate buffer with different
pHs was used to assay phosphatase activity. The maximal activity at pH 4.0 was taken as 100% PH
log phase enzyme
Stationary phase enzyme (%)
3.5 4.0 4.5 5.0 5.35
Fig.1. Lineweaver-Burk plots of acid phosphatase purified from log phase cells (0)and stationary phase cells (0).Nitrophenylphosphate was used as the substrates. Initial velocity v is plotted as the measured optical density at 405 nm. The K M for the early log phase enzyme is 1.80 mM. The KM for the late log phase enzyme is 0.43 mM. The corresponding K,,,s derived from measurements using whole cells are 1.5 m M and 0.3 mM
l
30
80
w
120
Inrtlvation thw ( m i d
Fig. 2. Heat inactivation of acid phosphatase purified from log phase cells (0)and stationary phase cells (0).The enzyme appropriately diluted in acetate buffer was preincubated at 55" C for the indicated period of time and was rapidly cooled in an ice bath. Enzyme activity without preincubation was scored as 100% (0.013U) and the remaining activities were expressed as a percentage of this value
100 100 74 52 21
98 100
90 75 51
interested in knowing if the yeast cell has the capacity to alter the structure of acid phosphatase. Therefore we compared purified acid phosphatase from cells grown under different physiological conditions, namely, from log phase cells and stationary phase cells. The purification protocol is given in Table 1. The two purified enzymes differ only slightly in their specific activities. However, they differ significantly in their affinity for nitrophenylphosphate. As illustrated in Fig. 1, the enzyme of stationary phase cells has greater affinity for nitrophenylphosphate than the enzyme of log phase cells. It can be argued that the differences of the substrate affinity are extraction artefacts. This possibility can be excluded since acid phosphatase is located at the surface of the cell and its activity can be measured directly in vivo using whole cells. Whole cells from the log phase and the stationary phase show a similar difference in substrate affinity as found for the pure enzymes (see Fig. 1). In addition to substrate affinity we compared stability at elevated temperatures and maximum initial velocity Vmax as a function of pH of the two enzymes. The data are given in Fig. 2 and Table 2. Acid phosphatase from stationary phase cells has a higher heat stability and a broader pH optimum than acid phosphatase of log phase cells. Since the enzymes used were pure, the differences in the tested properties have to reflect structural differences of the two enzymes. The easiest explanation for the structural differences in acid phosphatase of log phase cells and stationary phase cells is that exogenous signals such as nutrient availability are metabolically integrated by the cell and expressed in terms of differentially modulated acid phosphatase. It is conceivable that this differentially modulated acid phosphatase may in turn, together with other cell surface glycoproteins, influence cell morphology and cell behavior. Differential modulation may also be related to the cell cycle. It is not known if modulation affects intracellular location of acid
70
M. E. Schweingruber and A.-M. Schweingruber: Modulation of Acid Phosphatase of Yeast
phosphatase. Different distributions of the enzyme in the cell at various growth phases have been described [15]. The modulation mechanism is unknown. Differential expression of acid phosphatase isoenzymes however can probably be excluded. At least the major part of the repressible form of acid phosphatase seems to be coded for only by one gene, pho E [16].The most probable modulation mechanisms are covalent modifications of the polypeptide. The nature of the modifications is currently under investigation. Acknowledgements. This work was supported by the Swiss National Science Foundation.
References 1. Harmon RE (1978) Cell surface carbohydrate chemistry. Academic Press Inc, London, New York 2. Schmidt G, Bartsch G, Laumont M-C, Herman Th, Liss M (1%3) Acid phosphatase of Bakers’ yeast. An enzyme of the external surface. Biochemistry 2: 126 3. Linnemans WAM, Boer P, Elbers PF (1977) Localization of acid phosphatase in Sacchuromyces cerevisiue: a clue to cell wall formation. J Bacteriol 131:638 4. Boer P, Steyn-Pad EP (1966) Isolation and purification of an acid phosphatase from Baker’s yeast (Succharomyces cerevisiue). Biochim Biophys Acta 128: 400 5. Schurr A, Yagil E (1971) Regulation and characterization of acid and alkaline phosphatase in yeast. J Gen Microbiol 65: 291 6. Toh-e A, Ueda Y, Kakimoto S, Oshima Y (1973) Isolation and characterization of acid phosphatase mutants in Saccharomyces cerevisiue. J Bacteriol 113:727
Note udded in Proof: Since this report was submitted for publication we have found that mutants of the fission yeast Schizosucchuromyces pombe altered in the structure of acid phosphatase differ from wildtype in morphology and agglutination. This implies that modulation of acid phosphatase is a mechanism which is involved in morphogenetic and behavioural differentiation of the yeast cell.
7. Schweingruber ME,Schweingruber AM (1979) Posttranslational regulation of repressible acid phosphatase in yeast. Mol Gen Genet 173: 349 8. Elona MV, Rodriquez L, Villanueva JR, Sentandrea R (1978) Regulation of acid phosphatase synthesis in Sacchuromyces cerevisiae. Biochim Biophys Acta 521 : 342 9. Bostian KA, Lemire JM, Cannon LE, Halvorson HO (1980) In vitro synthesis of repressible yeast acid phosphatase: Identification of multiple mRNAs and products. Proc Natl Acad Sci 77: 4504 10. Rubin GM (1973) The nucleotide sequence of Succharomyces cerevisiue 5.8s ribosomal ribonucleic acid. J Biol Chem 248 : 3860 11. Dibenedetto G, Cozzani I (1975) Nonspecific acid phosphatase from Schizosacchuromyces pombe. Purification and physical chemical properties. Biochemistry 14: 2847 12. Ballou C (1976) Structure and biosynthesis of the mannan component of the yeast cell envelope.. Adv Microbiol Physiol 14: 93 13. Crandall M, Egel R, Mackay VL (1977) Physiology of mating in three yeasts. Adv Microbiol Physiol 15 : 307 14. Scherr GH, Weaver RH (1953) The dimorphism phenomenon in yeasts. Bacterial Rev 17: 51 15. Rainina EI, Zubatov AS, Buchwalow IB, Luzikov NV (1979) A cytochemical study of the localization of acid phosphatase in Succharomyces cerevisiue at different growth phases. Histochem J 11: 299 16. Toh-e A, Kakimoto S, Oshima Y (1975) Genes coding for the structure of the acid phosphatase in Succharomyces cerevisiue. Mol Gen Genet 143: 65
Received September 1980/Accepted January 1981