A speci c alkaline phosphatase from Saccharomyces cerevisiae with protein phosphatase activity

FEMS Microbiology Letters 161 (1998) 139^144 A speci c alkaline phosphatase from Saccharomyces cerevisiae with protein phosphatase activity Boriyana Tuleva, Evgenia Vasileva-Tonkova, Danka Galabova * Department of Microbial Biochemistry and Biosynthesis, Institute of Microbiology, Bulgarian Academy of Sciences, Acad. G. Bonchev str., bl 26, 1113 So a, Bulgaria Abstract Received 1 December 1997; revised 31 January 1998; accepted 4 February 1998 In this paper, specific PHO13 alkaline phosphatase from Saccharomyces cerevisiae was demonstrated to possess phosphoprotein phosphatase activity on the phosphoseryl proteins histone II-A and casein. The enzyme is a monomeric protein with molecular mass of 60 kda and hydrolyzes p-nitrophenyl phosphate with maximal activity at ph 8.2 with strong dependence on Mg 2 ions and an apparent K m of 3.6U10 35 M. No other substrates tested except phosphorylated histone II-A and casein were hydrolyzed at any significant rate. These data suggest that the physiological role of the p-nitrophenyl phosphate-specific phosphatase may involve participation in reversible protein phosphorylation. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Keywords: Saccharomyces cerevisiae; Protein phosphatase activity; Speci c alkaline phosphatase 1. Introduction Yeast cells produce a group of enzymes that are involved in phosphate uptake and utilization including di erent phosphatases. These enzymes are functionally similar in that they catalyze the same biological reaction but they are di erent proteins with optima at acid or alkaline ph, and with di erent metabolic roles in the cell. Alkaline phosphatases (ALPases) in Saccharomyces cerevisiae are the products of two structural genes (PHO8 and PHO13) [1]. PHO8 alkaline phosphatase (EC 3.1.3.1) is a Mg 2 / Zn 2 -dependent dimeric protein [2] similar to the ALPase in Escherichia coli and in mammalian cells * Corresponding author. Tel.: +359 (2) 700 106; Fax: +359 (2) 700 109; E-mail: dgal@bgearn.acad.bg [3]. The enzyme product of PHO13 is a monomeric protein and is speci c to p-nitrophenyl phosphate (pnpp) [4,5] or histidinyl phosphate [6]. The physiological role of this enzyme is not clear and its natural substrate has not been identi ed. In the past few years much attention has been focused on the nature and physiological role of the protein phosphatases which reverse the actions of protein kinases [7,8]. Reversible phosphorylation of serine and threonine residues accounts for more than 97% of the protein-bound phosphate in cells. Demonstrated similarities between yeast and mammalian protein phosphatases make it possible to use the advantages of yeast in delineating the physiological roles of di erent phosphatases [9]. In this work a speci c alkaline phosphatase in S. cerevisiae 257 is characterized in terms of its phys- 0378-1097 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V. PII S0378-1097(98)00065-2

140 B. Tuleva et al. / FEMS Microbiology Letters 161 (1998) 139^144 icochemical and catalytic properties. Our results demonstrated that the enzyme possesses an activity on phosphorylated proteins which has not been described so far. To avoid any interference of acid phosphatases, a strain defective in constitutive and repressible forms of acid phosphatases was used. 2. Materials and methods 2.1. Yeast and growth conditions S. cerevisiae strain 257 was kindly provided by Prof. P. Venkov from the Institute of Molecular Biology (IMB), Bulgarian Academy of Science (IMB culture collection 257). It was originally obtained from the collection of Prof. A. Hinnen (AH:220, MATa leu2-3 leu2-112 his3 trp1 pho3 pho5). The strain was grown in 500-ml Erlenmeyer asks with 100 ml medium under slow rotary agitation at 28³C on YEPD medium (4% (w/v) Bacto-yeast extract, 2% (w/v) Bacto-peptone and 2% (w/v) glucose). 2.2. Preparation of phosphoprotein substrates Histone II-A (Sigma) was phosphorylated by incubating with [Q- 32 P]ATP in the presence of campdependent protein kinase as described in [10] with some modi cations. The incubation mixture ( nal volume 4 ml) contained 20 mm Tris-HCl ph 7.4, 90 units of protein kinase (Sigma), 2 mg histone II-A, 20 WC i (2U10 6 cpm) of [Q- 32 P]ATP (3000 C i / mmol), 100 Wmol MgCl 2. The reaction was carried out at 30³C for 4 h and terminated by the addition of 100 nmol unlabeled ATP and 20 Wmol EDTA. The reaction mixture was dialyzed at 4³C rst against 20 mm Tris-HCl ph 7.4 containing 5 mm EDTA for 4 h, and then against the bu er without EDTA overnight. Unlabeled phosphohistone II-A and phosphocasein were prepared as described by Fig. 1. Elution pro les of ALPase from DEAE-cellulose column. The sample loaded was as described in Section 2. I, start of the elution with 20 mm Tris-HCl ph 8.6; II, start of the salt gradient (0^0.6 M NaCl, 60 ml) in 20 mm Tris-HCl ph 8.6. All activities were determinated in absence of Mg 2 ions. Symbols: solid line, OD 280 ; b, activity on pnpp; a, activity on phosphocasein; O, activity on unlabeled phosphohistone. Inset, activity of the ALPase on Q- 32 P-labeled histone.

B. Tuleva et al. / FEMS Microbiology Letters 161 (1998) 139^144 141 Fig. 2. Molecular mass determination of ALPase. a: Non-denatured PAGE of ALPase. The earlier peak eluted at 0.2^0.3 M NaCl from DEAE-cellulose column (sequences 50^60) stained for protein (lane 2) and developed for phosphatase activity (lane 3). Molecular mass markers (lane 1): bovine serum albumin (67 kda) and chymotripsinogen (25 kda). b: Gel ltration on a Sephadex G-100 column of the ALPase eluted from the DEAE-cellulose column (earlier peak). Symbols: solid line, OD 280 ; a, activity on pnpp. Molecular mass markers: 1, bovine serum albumin (67 kda); 2, ovalbumin (45 kda); 3, trypsin (23 kda); 4, cytochrome c (12.4 kda). A, ALPase. Meisler and Langan [11] and Hemmings [12], respectively. 2.3. Alkaline phosphatase assay ALPase activity was assayed in 20 mm Tris-HCl bu er ph 8.2 using pnpp as substrate as described earlier [13]. The enzyme activity was calculated using a molar absorption coe cient of 16 200 M 31 cm 31 for the absorbance of the p-nitrophenylate ion (pnp) at 410 nm. One enzyme unit (U) was de ned as the amount of enzyme releasing 1 nmol pnp per min at 37³C. When other substrates were tested the method of Bencini et al. [14] was used. One unit of enzyme activity was de ned as 1 nmol of inorganic phosphate liberated from the substrate per min at 37³C. The molar absorption coe cient for the phosphomolybdate complex at 350 nm was 7200 M 31 cm 31. Determination of the ph optimum of the enzyme was performed using pnpp as substrate in 50 mm bu ers, including Tris-HCl bu er (ph 7.2^9.3), and carbonate-bicarbonate bu er (ph 10). Protein content was estimated as described by Bradford with BSA as a standard. 2.4. Protein phosphatase assay Dephosphorylation of the [Q- 32 P]phosphohistone was carried out according to the procedure described by Matsumoto et al. [10]. When unlabeled phosphohistone II-A and phosphocasein were used as substrates the assay mixture contained 100 Wl enzyme solution, 100 Wl 20 mm Tris-HCl ph 8.0 and 100 Wl Fig. 3. In uence of Mg 2 ions on ALPase activity. The enzyme (dialyzed against distilled water for 15 min, a; and non-dialyzed, b) was pre-incubated at 25³C for 30 min in the presence of increasing concentrations of Mg 2 ions in 20 mm Tris-HCl ph 8.2. The hydrolytic activity was measured with pnpp as described in Section 2. Data are means of three determinations þ S.E.M.

142 B. Tuleva et al. / FEMS Microbiology Letters 161 (1998) 139^144 Table 1 Substrate speci city of the ALPase Substrate Speci c activity (nmol min 31 mg 31 ) Relative activity (%) pnpp 507.0 þ 55.7 100 K-Naphthyl phosphate 2.52 þ 0.03 6 1.0 o-phospho-dl-tyrosine 7.14 þ 087 6 2.0 o-phospho-dl-serine 7.62 þ 0.93 6 2.0 ATP 22.4 þ 2.52 4.4 ADP 7.62 þ 0.92 6 2.0 3P-AMP 12.00 þ 1.32 2.4 K-Glycerophosphate 0 0 L-Glycerophosphate 0 0 Phosphate released was determined as described in Section 2. Substrate and enzyme blanks were run for each sample. Mean values of three di erent experiments þ S.E.M. are given. phosphoprotein (30 nmol P i determined as alkalilabile phosphate as described in [10]). The phosphate released was determined by the method of Bencini et al. [14]. 2.5. Chromatography The cell-free extract of an exponentially grown culture (20 h) was prepared as described in a previous paper [15]. The supernatant uid after dialysis against 20 mm Tris-HCl ph 8.2 was loaded on a DEAE-cellulose column (1.8U10 cm) equilibrated with the same bu er. Elution of the ALPase was achieved by a linear NaCl gradient (0^0.6 M, 60 ml) in the initial bu er. Fractions of 1.2 ml were collected at a ow rate of 25 ml h 31. Active fractions were pooled, concentrated and loaded onto a Sephadex G-100 column. 2.6. Determination of molecular mass The molecular mass of the ALPase was estimated by gel ltration on a Sephadex G-100 column (1.2U30 cm) calibrated beforehand with the following molecular mass markers: bovine serum albumin (67 kda), ovalbumin (45 kda), trypsin (23 kda), and cytochrome c (12.4 kda). The column was equilibrated and eluted with 20 mm Tris-HCl ph 8.6 containing 0.1 M NaCl. Non-denaturing PAGE was performed on a 10% (w/v) polyacrylamide slab gel. Proteins were visualized by staining with Coomassie brilliant blue R-250. After the native PAGE, the gels were washed with deionized water and twice in 0.1 M Tris-HCl ph 8.2, containing 2 mm MgCl 2. For monitoring the phosphatase activity gels were incubated at 37³C for 15 min in the same bu er with 2 mm pnpp, washed and then incubated at 37³C in ammonium molybdate solution prepared according the method of Ames [16] for detection of inorganic phosphate. 3. Results and discussion 3.1. Chromatographic separation of phosphatase activities Two peaks with alkaline phosphatase activity were obtained upon anion exchange chromatography on DEAE-cellulose (Fig. 1). ALPase from the second peak was non-speci c enzyme and was characterized in a separate work. ALPase from the rst peak was eluted at 0.2^0.3 M NaCl and was used for the present study. Table 2 In uence of Mg 2 ions on the protein phosphatase activity of the ALPase Substrate Speci c activity (nmol min 31 mg 31 ) without Mg 2 with Mg 2 (10 mm) Histone II-A 42.04 þ 4.62 40.4 þ 4.84 Casein 4.10 þ 0.48 3.8 þ 0.45 pnpp 25.30 þ 2.74 531.3 þ 58.21 Phosphate released was determined as described in Section 2. Mean values of three determinations þ S.E.M. are given.

Table 3 Similarities between the S. cerevisiae 257 ALPase and pnpp-speci c phosphatase from S. cerevisiae described in [5] Enzyme property B. Tuleva et al. / FEMS Microbiology Letters 161 (1998) 139^144 143 pnpp-speci c alkaline phosphatase from: S. cerevisiae 257 S. cerevisiae [5] ph optimum 8.2 8.0^8.2 Molecular weight (kda) 60 60 Stability stable at 50³C; inactivated at 70³C stable at 50³C; inactivated at 70³C Dependence on metal ions the activity was increased 20^25-fold by 10 mm Mg 2 ions the activity was increased 10^12-fold by 20 mm Mg 2 ions Substrate speci city hydrolyzes only pnpp hydrolyzes only pnpp K m 36 WM 37 WM Protein phosphatase activity yes no data 3.2. Properties of the ALPase When a sample of concentrated active fractions was subjected to native PAGE, only one band with pnppase activity was observed, corresponding to the molecular mass of 60 kda (Fig. 2a, lane 3). The molecular mass of the partially puri ed ALPase was estimated to be 60 kda also by gel ltration on Sephadex G-100 (Fig. 2b). ALPase activity was maximal at ph 8.2 with pnpp as substrate (not shown). The enzyme was strongly activated by Mg 2 ions. As can be seen in Fig. 1, the pnppase activity in the absence of Mg 2 was almost undetectable. Maximal activity was registered at 10 mm Mg 2 ions with about 26-fold increased activity (Fig. 3). After dialysis against distilled water, the enzyme activity was increased by Mg 2 but to a lesser degree than with non-dialyzed sample. The enzyme was relatively thermostable: 67 and 20% of the maximum activity remained at 50 and 60³C for 10 min, respectively. Mg 2 ions decreased the heat denaturation of the enzyme at 50 and 60³C, but not at 70³C (not shown). 3.3. Substrate speci city and K m The study of the speci city of ALPase revealed a strong preference for pnpp. The activity of the enzyme on other compounds tested was very low (Table 1). The high speci city of the enzyme to pnpp was con rmed by determination of the Michaelis constant by the method of Lineweaver and Burk. A mean value of 3.6U10 35 M was calculated. 3.4. Protein phosphatase activity ALPase showed activity on two natural proteins, casein and histone II-A. As shown in Fig. 1, peaks of enzyme activity on histone II-A and casein were identical with those of pnppase activity. This nding was strongly con rmed when labeled phosphohistone was used (Fig. 1, inset). In contrast to the strong dependence of pnppase activity on Mg 2 ions, protein phosphatase activity was not Mg 2 -dependent (Table 2). Similar di erential Mg 2 e ects have been observed for alkaline phosphatases of mammalian origin that could dephosphorylate phosphoproteins in vitro [17,18]. It was demonstrated that although the alkaline phosphatases are metalloenzymes and require divalent cations for optimal activity, the chelator EDTA had little or no e ect on the activity of the major cellular protein phosphatases in cell extracts [18]. In the present work a partially puri ed ALPase of S. cerevisiae 257 with a ph optimum at ph 8.2 and high speci c activity for pnpp has been described. Data obtained by us are summarized and compared with those of Attias and Bonnet [5] for the pnppspeci c ALPase from S. cerevisiae (Table 3). It is obvious that both enzymes are identical in almost all physicochemical and catalytic properties. This resemblance con rms the assumption that the enzyme described in this work is the pnpp-speci c ALPase. In the literature so far there are no data about the possible natural substrates of this enzyme. The newly demonstrated property of protein serine/threonine phosphatase activity may provide a clue to elucidate the unclear physiological role of the enzyme.

144 B. Tuleva et al. / FEMS Microbiology Letters 161 (1998) 139^144 Acknowledgments We thank Prof. P. Venkov (Institute of Molecular Biology, So a) for providing strain 257, and Dr. A. Todorova (Bulgarian Medical Academy) for help with the labeling of the protein substrates. This work was supported by Grant K444 from the National Fund for Scienti c Investigations, Bulgarian Ministry of Education and Technologies. References [1] Vogel, K. and Hinnen, A. (1990) The yeast phosphatase system. Mol. Microbiol. 4, 2013^2017. [2] Onishi, H.R., Tkacz, J.S. and Lampen, J.O. (1979) Glycoprotein nature of yeast alkaline phosphatase. Formation of active enzyme in the presence of tunicamycin. J. Biol. Chem. 254, 11943^11952. [3] Janeway, C.M.L., Murphy, J.E., Chaidaroglou, A. and Kantrowitz, E.R. (1993) Magnesium in the active site of Escherichia coli alkaline phosphatase is important for both structural stabilization and catalysis. Biochemistry 32, 1601^1609. [4] Toh-e, A., Nakamura, H. and Oshima, Y. (1976) A gene controlling the synthesis of non speci c alkaline phosphatase in Saccharomyces cerevisiae. Biochim. Biophys. Acta 428, 182^192. [5] Attias, J. and Bonnet, J.L. (1972) A speci c alkaline p-nitrophenylphosphatase activity from baker's yeast. Biochim. Biophys. Acta 268, 422^430. [6] Gorman, J.A. and Hu, A.S.L. (1969) The separation and partial characterization of L-histidinol phosphatase and an alkaline phosphatase of Saccharomyces cerevisiae. J. Biol. Chem. 244, 1645^1650. [7] Cohen, P. (1989) Structure and regulation of protein phosphatases. Annu. Rev. Biochem. 58, 453^508. [8] Shenolikar, S. (1994) Protein serine/threonine phosphatasesnew avenues for cell regulation. Annu. Rev. Cell Biol. 10, 55^86. [9] Cohen, P., Schelling, D.L. and Stark, M.J.R. (1989) Remarkable similarities between yeast and mammalian protein phosphatases. FEBS Lett. 250, 601^606. [10] Matsumoto, K., Uno, I., Kato, K. and Ishikawa, T. (1985) Isolation and characterization of a phosphoprotein phosphatase-de cient mutant in yeast. Yeast 1, 25^38. [11] Meisler, M.H. and Langan, T.A. (1969) Characterization of a phosphatase speci c for phosphorylated histone II-A and protamine. J. Biol. Chem. 244, 4961^4968. [12] Hemmings, B.A. (1981) Reactivation of the phospho form of the NAD-dependent glutamate dehydrogenase by a yeast protein phosphatase. Eur. J. Biochem. 116, 47^50. [13] Galabova, D., Tuleva, B. and Balasheva, M. (1993) Phosphatase activity during growth of Yarrowia lipolytica. FEMS Microbiol. Lett. 109, 45^48. [14] Bencini, D.A., Wild, J.R. and O'Donovan, G.A. (1983) Linear one-step assay for the determination of orthophosphate. Anal. Biochem. 132, 254^258. [15] Galabova, D., Tuleva, B. and Spasova, D. (1996) Permeabilization of Yarrowia lipolytica cells by triton X-100. Enzyme Microb. Technol. 18, 18^22. [16] Ames, B.N. (1966) Assay of inorganic phosphate, total phosphate and phosphatases. Methods Enzymol. 8, 115^118. [17] Swarup, G., Cohen, S. and Garbers, D.L. (1981) Dephosphorylation of proteins containing phosphotyrosine by alkaline phosphatases. J. Biol. Chem. 256, 8197^8201. [18] Foulkes, J.G., Erikson, E. and Erikson, R.L. (1983) Multiple phosphotyrosyl- and phosphoseryl-protein phosphatases from chicken brain. J. Biol. Chem. 258, 431^438.