An inducible acid phosphatase from the yeast Pichia pastoris: characterization of the gene and its product

Gene, 163 (1995) 19 26 ©1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.50

19

GENE 09097

An inducible acid phosphatase from the yeast Pichia pastoris: characterization of the gene and its product (Secretion; PH01; pulse-chase; signal sequence; promoter)

William E. Payne, Pamela M. Gannon and Chris A. Kaiser Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Received by G.P. Livi: 6 February 1995; Accepted: 26 April 1995; Received at publishers: 29 May 1995

SUMMARY

To develop the budding yeast Pichia pastoris (Pp) as a model system for the study of protein secretion, we have characterized a secreted acid phosphatase (Pholp) from this yeast. Pholp can be induced 100-fold by starvation for phosphate. The enzyme was purified to homogeneity from a cell-wall extract by DEAE-Sepharose chromatography. We selected mutants that lacked extracellular phosphatase activity and the gene (PH01) encoding Pholp was isolated from a recombinant plasmid library of Pp DNA by complementation of the mutant defect. PHO1 encodes a protein of 468 amino acids (aa) with homology to repressible acid phosphatases from other yeast species. The sequence contains a 15-aa N-terminal signal sequence and six potential N-linked glycosylation sites. Antiserum to Pholp was used to show that Pholp transits the Pp secretory pathway in less than 5 min.

INTRODUCTION

Yeast cells utilize inorganic phosphate (Pi) as the preferred extracellular phosphate source. When Pi is depleted, extracellular phosphatases liberate Pi from organic substrates (reviewed in Johnston and Carlson, 1992). Acid phosphatases (Pho; orthophosphoricmonoester phosphohydrolase (acid optimum), EC 3.1.3.2) have been described in many yeast species, e.g., Candida

Correspondence to: Dr. C. A. Kaiser, Department of Biology, 68 534, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA. Tel. (1-617) 253-9804; Fax (1-617) 253-8699; e-mail: [email protected] Abbreviations: A, absorbance (1 cm); A., Aspergillus; aa, amino acid(s); Ab, antibody(ies); bp, base pair(s); DEAE, diethylaminoethyl; DTT, dithiothreitol; E., Escherichia; Endo Hf, recombinant form of endoglycosidase H (New England Biolabs, Beverly, MA, USA); ER, endoplasmic reticulum; HRP, horseradish peroxidase; Ig, immunoglobulin; IP, immunoprecipitation; kb, kilobase(s) or 1000 bp; low-Pi SD, SD prepared without phosphate; low-Pi YPD, phosphate-depleted YPD;

SSDI 0378-1119(95)00379-7

albicans (Odds and Hierholzer, 1973); Kluyveromyces lactis (Altikrete et al., 1984); Rhodotorulaflutinis (Trimble et al., 1981); S. cerevisiae (Suomalainen et al., 1960; Schmidt et al., 1963); Sz. pombe (Dibenedetto, 1972) and Yarrowia lipolytica (Ogrydziak et al., 1982). In S. cerevisiae the primary Pho (encoded by the PH05 gene) is a secreted glycoprotein that has been used to follow the function of the secretory pathway (Field and Schekman, 1980; Novick et al., 1980).

MES, 2-[N-morpholino]ethane sulfonic acid; nt, nucleotide(s); oligo, oligodeoxyribonucleotide;ORF, open reading frame; P., Pichia; Pi, inorganic phosphate; PA, polyacrylamide; PAGE, PA-gel electrophoresis; PARS1, Pp autonomously replicating sequence 1; Pho, acid phosphatase; Pho +, phenotype showing Pho activity; Pho , phenotype showing no Pho activity; PH01, gene encoding Pholp; Pholp, Pp Pho; PH05, gene encoding S. cerevisiae Pho5p; Pho5p, S. cerevisiae Pho; PMSF, phenylmethylsulfonyl fluoride; Pp, P. pastoris; S., Saccharomyces; SD, synthetic dextrose medium; SDS, sodium dodecyl sulfate; Sz., Schizosaccharomyces; UTR, untranslated region(s); UV, ultraviolet; wt, wild type; Xaa, any aa; YNB, yeast nitrogen base medium; YPD, rich medium.

20 TABLE I Yeast strains used in this study Straina

Relevant markers b

Source/CommenV

GS 115 GS190 CKY303 CKY304 CKY305 CKY306 CKY307 CKY308 CKY309 CKY310 CKY311 CKY312 CKY313

his4-1 arg4-1 arg4-1 phol-1 his4-1 phol-2 his4-1 phol-3 his4-1 phol-4 arg4-1/ARG4 HIS4/his4-1 PHO1/phol-2 arg4-1/ARG4 HIS4/his4-1 PHO1/phol-3 arg4-1/ARG4 HIS4/his4-1 PHO1/phoI-4 his-1/HIS4 ARG4/his4-1 PHO1/pho1-1 arg4-1/ARG4 HIS4/his4-1 phol-1/phol-2 arg4-1/ARG4 HIS4/his4-1 phoi-1/phol-3 arg4-1/ARG4 HIS4/his4-1 pho1-1/phol-4

NRRL NRRL This study This study This study This study This study; This study; This study; This study; This study; This study; This study;

GS190 x CKY304 GS190 x CKY305 GS190 x CKY306 GSll5 x CKY303 CKY303 x CKY304 CKY303 x CKY305 CKY303 x CKY306

a Strains GSll5 and GS190 were obtained from the Northern Regional Research Center (NRRL), Agricultural Research Service, US Department of Agriculture (Peoria, IL, USA). b Genetic nomenclature follows conventions set forth for S. cerevisiae, c Symbols x mark the haploid mating of strains to form diploids.

We are interested in using the budding yeast Pichia pastoris (Pp) for the study of the secretory pathway. In electron micrographs of S. cerevisiae cells, the Golgi apparatus appears dispersed within the cell and direct examination of transport vesicle budding and fusion processes have been difficult to achieve (Preuss et al., 1991; 1992). In constrast, the architecture of the Golgi stacks and budding regions of the ER are vividly apparent in Pp cells making this a particularly promising organism for the study of the morphological events associated with transport vesicle formation and fusion. In this paper we characterize a secreted Pp Pho (Pholp) for use as a marker for the function of the secretory pathway.

RESULTS AND DISCUSSION

(a) Secreted Pp acid phosphatase (Pho) The wild type (wt) Pp strain GS115 (Table I) was tested for extracellular Pho activity by assaying the ability of intact cells to hydrolyze p-nitrophenylphosphate. Cells grown in rich medium depleted of Pi (low-Pi YPD) (Rubin, 1974) expressed approx. 100-times more extracellular Pho activity than cells grown in YPD medium (Table II). The induced Pho had an acidic pH optimum, since activity was apparent when assayed at pH 5.0 but not when assayed at pH 8.0 (data not shown). The kinetics of induction of the enzyme was assayed after transfer of a Pp culture in exponential growth from YPD medium to low-Pi YPD medium by following the development of extracellular Pho activity (Fig. 1). New Pho activity was detected within 2 h after induction and rapidly reached a maximal rate in approx. 2 generations of cell growth (Fig. 1). Most Pho activity was associated with washed

cells but about 15% of the total activity was in the extracellular medium (Fig. 1). A similar pattern of Pho induction was observed in minimal medium (low-Pi SD), but new Pho activity was not detected until 4 h after induction (data not shown).

(b) Purification of the Pp Pho enzyme More than 95% of Pho was released from the Pp cell wall with lM,3-glucanase and the active Pho was completely precipitated from the cell-wall extract with ethanol. Pho was purified to homogeneity by chromatography on DEAE-Sepharose. The DEAE-Sepharose eluate resolved by SDS-PAGE and stained with Coomassie brilliant blue exhibits a single protein species that migrates as a diffuse band of approx. 80-100 kDa (Fig. 2A). The heterogeneous mobility of Pho is a result of N-linked glycosylation since treatment of the enzyme with endoglycosidase Hf (Endo Hf) results in a sharp protein band that migrates at 54 kDa on SDS-PAGE (Fig. 2A). The yield of purified Pho was 1 mg of protein from 6.5 x 103 A6oo units of cells. TABLE II Derepression of acid phosphatase (Pho) by phosphate starvation Enzyme source

Washed cells Medium

Pho activity a high-Pi medium units/A6oo (× 103)

low-Pi medium units/A6oo (x 103)

5.9 0.5

334 52

a pp strain GSll5 was grown for 6 h in low-Pi YPD medium and the Pho activity was determined under conditions described in the legend to Fig. 1.

21 (d) Kinetics of Pho secretion 300

.~ oo ~. ~ ".= om o o

250

.~¢'" .x •

2o0 -

~

150

~ "~

100

.~.

.~¢""

e-

0.

1

..x'" ..X"

. - X "°°

&

P"

x....x'"

0.1 .....0 ..................0

50 ..... •0 ............

o I

I

I

I

I

I

!

I

I

0

1

2

3

4

5

6

7

8

Time (h) Fig. 1. Kinetics of derepression of Pp Pho activity in Pp cells starved for Pi. Cells in exponential growth in YPD medium were suspended in low-Pi YPD medium. Pho was assayed in washed cells (closed circles) and in the culture supernatant (open circles). All activities are normalized to cell density and expressed as enzyme units per ml of cell culture at an A600 of 1.0. Growth (X) was determined as A6oonm. Yeast methods: Yeast media were prepared as described by Kaiser et al. (1994). YPD is a rich medium (1% Bacto-yeast extract/2% Bacto-peptone/2% glucose. Rich low-phosphate medium (low-Pi YPD) was prepared by precipitation of P~ as MgNH4PO 4 from YPD (Rubin, 1974). SD medium is a supplemented synthetic minimal medium. Low-P~ SD medium contains 20 mg KH2PO 4 per litre with the remaining K.phosphate replaced by KC1. SC (synthetic complete) medium was prepared as described (Kaiser et al., 1994). All Pp strains were grown at 30°C. Measurement and deteetion of Pho activity: Pho activity is assayed either with a suspension of intact cells or with growth medium after removal of the cells by centrifugation. The reaction mixture is comprised of 0.2 ml substrate solution containing 5 mg/ml p-nitrophenylphosphate (Sigma) in 20 m M Tris pH 7.5 and 0.8 ml of enzyme sample in 30 m M Na.acetate (pH 5). After incubation at 37°C for 15 min the assay is stopped by addition of 0.5 ml of 1 M Na2CO 3 (pH 10) and the A 4 2 0 n m of the liberated nitrophenol is measured. One Pho unit is defined as the amount of enzyme which catalyzes the release of 1 gmol of p-nitrophenol per min under the specified assay conditions.

The specific activity of the purified Pho is 1100 enzyme units/rag protein. Pho is inactive at pH 7.5 but has a broad pH optimum exhibiting > 90% of the full activity between pH 3.2 and 5.5 (data not shown). Salt concentration has little effect on Pho activity, as the enzyme shows 80% of full activity in 1 M NaC1. Pho apparently has no divalent cationic requirements since full activity was observed in the presence of 10 mM EDTA. The purified Pho was stable for months at 4°C when stored in 50 mM MES (pH 6.1)/300 mM NaC1. (c) Pho antiserum

Deglycosylated Pholp was used to elicit an immune response in rabbits. On Western blots the Pho serum reacts with a heterogeneous population of molecules ranging in size from approx. 80 to 100 kDa (Fig. 2B). Following the treatment of cell extracts with Endo H f only one protein species of approx. 54 kDa predominates (Fig. 2B).

Pulse-chase experiments were done to examine the kinetics of both glycosylation and the appearance of Pholp at the cell surface. Radiolabelled Pho was liberated from the cell wall by spheroplasting with J3-1,3-glucanase and then immunoprecipitated with antiPholp Ab. Labelled Pho was detectable at the cell surface following a 5-min pulse with [35S3methionine (Fig. 3; t = 0), indicating that Pholp was rapidly translocated into the ER, glycosylated and transported to the cell surface. After a 5-min chase period, the majority of labelled protein had arrived at the cell surface (Fig. 3). Following 10 min of chase, a further 25% increase in the abundance of the glycosylated Pho at the cell surface was observed. With increasing chase time the amount of protein at the cell surface plateaued and then slowly dissipated as the protein was released into the surrounding medium (data not shown). Pho was apparently quite stable at the cell surface, since 80% of the maximum signal obtained following initiation of the chase remained after a 2-h chase period (data not shown). (e) Isolation of PHO mutants

To provide a positive selection whereby the gene coding for Pholp could be identified genetically, we isolated mutants defective in Pho activity. Pp strains GS 115 and GS190 were mutagenized with UV light and were screened by the Pho plate assay for colonies that did not express active enzyme. Three PHO- mutants of strain GSll5 (CKY304, 305 and 306) and one PHO- mutant of strain GS190 (CKY303) were isolated. None of the PHO- mutant strains showed Pho activity above the background level of uninduced wt cells (Table III). When assayed by Western analysis for the presence of protein recognized by the anti-Pholp Ab, extracts from strains CKY304 and CKY306 showed no detectable Pholp after treatment with Endo Hf (data not shown). Strains CKY303 and CKY305 carrying the pho1-1 and phol-3 alleles respectively, had low levels of Pholp that was evident after treatment with Endo Hf (data not shown). All four mutants are recessive since diploids formed by mating the mutants to a wt strain express high levels of Pho activity (Table III). Comptementation tests were performed by mating the mutant derived from GS190 to each of the three mutants derived from GSll5. The resulting diploid strains were selected for by complementation of the Arg- and His- mutations. All heteroallelic diploids expressed low Pho levels indicating that all four mutations are in the same gene (Table III). We designate this gene as PH01 and the gene product as Pholp. (f) Isolation of the PHO1 gene

The PHO1 gene was isolated by rescuing the Phophenotype exhibited by CKY304 with a genomic Pp

22

A kDa

1

2

3

116 -

86-

66-

56-

41-

DNA library constructed in plasmid pYM8 carrying the S. cerevisiae HIS4 gene as a selectable marker and PARS1 (gift of Jim Cregg; Cregg et al., 1985). Approx. 6000 His + transformants were replica plated to low-Pi YPD and were screened for Pho activity by the plate assay. Two His+Pho + colonies were detected. Plasmid DNA was recovered from His ÷Pho ÷ yeast strains by transforming E. coli XL1-Blue with total yeast DNA. We isolated one plasmid, pPG31, that rescued the Pho- phenotype upon retransformation into CKY304 when tested by the Pho plate assay. This strain (called CKY314) was similar to wt strain GSll5 in levels and regulation of Pho activity by phosphate in the medium (Table III). Western blotting of cell extracts with anti-Pho lp Ab showed that Pho produced by the rescued strain was the same as the wt Pho ÷ (data not shown). We failed to isolate plasmid DNA from the second Pho ÷ transformant probably because the plasmid had integrated into the genome.

B kDa

1

2

3

4

116 -86--

66-56--

41--

Fig. 2. Purification and immunodetection of Pp Pho. (A) PAGE of Pp Pho. Purified Pho was deglycosylated enzymatically with Endo Hf in two steps as described below. Lanes: 1, 10 ~tg of purified Pho; 2, 10 txg of purified Pho following treatment with Endo Hf under nondenaturing conditions; 3, 10 ~tg of purified Pho following treatment with Endo Hf under denaturing conditions. The protein migrating at 71 kDa is Endo Hr. (B) Immunodetection of Pho in Pp cell extracts. Strain GSll5 was grown exponentially in YPD medium (lanes 1 and 2) or induced in low-Pi YPD for 6.5 h (lanes 3 and 4). Protein extracts were prepared, treated with Endo Hr (lanes 2 and 4), resolved by 8% PAGE, blotted to nitrocellulose and probed with anti-Pholp Ab. Purification of the Pp Pho protein: Pp strain GSll5 was grown to exponential phase in YPD at 30°C. The cells were suspended in 2-1itres of low-Pi YPD at an A60onm of 1.0 and Pho was induced for 6.5 h. The cells were washed once with 0.5 M Tris (pH 8), and suspended in 100 ml of spheroplast buffer (0.5 M Tris, pH 8/1.2 M NaC1/28 mM 13-mercaptoethanol). Spheroplasts were generated by the addition of 2x 104 units of 13-1,3-glucanase (prepared from E. coli expressing the Oerskovia xanthineolytica enzyme). Spheroplasts were removed by centrifugation at 5000 × g for 10 min and the cell-wall extract was chilled to 4°C. The Pho enzyme was precipitated from the cell-wall extract by the addition of 1.1 vol. of 95% ethanol followed by incubation on ice for 30 min (Boer and Steyn-Parve, 1966). The precipitated enzyme was collected by centrifugation at 12 000 x g for 10 min, and the pellet was suspended in 12 ml of 50 mM MES (pH 6.1). The suspension was clarified at 12000×g, and the supernatant was applied to a DEAE-Sepharose

column equilibrated in 50 mM MES (pH 6.1). The column was eluted with a linear gradient of zero to 600 mM NaCI. Pho eluted at approx. 300 mM NaCI. The peak fractions contained 1 mg of pure Pho and were stored at 4°C in elution buffer. Ab preparation: To produce Pho antiserum the purified protein was deglycosylated to expose epitopes and to reduce the potential for generating an immune response to the carbohydrate moiety of the glycoprotein. Removal of carbohydrate chains was carried out in two steps. First the protein was partially deglycosylated with Endo H r under nondenaturing conditions by incubating 700 ~tg of the protein with 10 000 units of Endo Hf (New England Biolabs) in 50mM MES (pH 6.1)/0.3 M NaC1 at 37°C for 2 h. This treatment removes some but not all of the N-linked chains (panel A). The partially deglycosylated protein remained enzymatically active, was diluted five-fold with water and was applied to a DEAE-Sepharose column equilibrated with 20mM MES (pH6.1). The column was washed with 20 mM MES (pH 6.1)/100 mM NaC1, and Pho was eluted with a linear NaC1 gradient. A single protein peak of Pho activity eluted at 650 mM NaC1. The peak fractions were pooled, brought to 1% SDS and the remaining carbohydrate was removed by treatment with Endo Hf following denaturation of the protein at 100°C for 5 min (panel A). New Zealand white rabbits were immunized subcutaneously with 100 ~tg of deglycosylated Pholp in Freund's complete adjuvant. At monthly intervals the rabbits were boosted by injection with 100 ~tg of Pholp in Freund's incomplete adjuvant. Serum was collected 10 days after the second boost. Non-specific Ab, primarily to carbohydratecontaining proteins, were removed by adsorption of serum to whole Pp cells by the method of Payne and Schekman (1985). Briefly, a one ml aliquot of whole serum was incubated with 20 A600 units of whole cells of the phol-2 mutant (CKY304) grown in YPD medium supplemented with 50mM K.phosphate. The adsorption was repeated three times. Preparation of Pp protein extracts and Western blotting: Pp protein extracts were prepared from 2 A6o0 units of induced cells in 30 I~1 ESB (60mM Tris.HC1, pH 6.8/100mM DTT/2% SDS/10% glycerol/0.001% bromphenol blue) in the presence of 100 ~tM PMSF (phenylmethylsulfonyl fluoride) by vigorous agitation with glass beads. Extracts were diluted with 70 I.tl of ESB and 20 lxl of each extract was resolved by 0.1% SDS-8% PAGE (Laemmli, 1970). Western analysis was carried out using standard procedures (Harlow and Lane, 1988). Western blots were developed using the ECL Western luminescence detection system (Amersham) with HRP-coupled sheep anti-rabbit Ig (Amersham) at 1:10000 dilution as the secondary Ab.

23 TABLE III Acid phosphatase (Pho) activity of wt, phol and heteroallelic diploid strainsa

250 11,

Strain

Relevant genotype b

200 e= &->, ,~,_ u)

150

6 vx

100 0

50

|

!

J

I

I

0

5

10

2O

30

Chase time (min)

Fig. 3. Pulse-chase analysis of Pho secretion. Pp strain GSII5 was derepressed for Pho synthesis at 30°C and pulse labeled with [35S]methionine for 5 min. Chase was initiated with the addition of excess unlabelled methionine. Extracellular P h o l p was liberated from the cell wall by treatment with 13-1,3-glucanase and was immunoprecipitated with anti-Pho l p Ab. The kinetics of Pho secretion was determined by quantification of the immunoprecipitated Pho following SDS-PAGE and fluorography using ImageQuaNT TM software on a Molecular Dynamics (Sunnyvale, CA, USA) Phosphoimager 445SI. Density units are arbitrary. Cell labeling and immunoprecipitation: Cells were grown to exponential phase in SC medium and transferred to low-Pi SD medium to induce Pholp. The induced culture was radiolabelled in low-Pi SD medium (without methionine and cysteine) with 30 ~tCi [3sS] methionine (Express protein labelling mix (New England Nuclear, Boston, MA, USA); spec. act. 1200 Ci/mmol) per A6o0 unit of cells at 30°C for 5 min. Chase was initiated with the addition of methionine to 270~tM (100x chase solution: 100mM (NH4)2SO4/25mM cysteine/27 mM methionine/10 mg cyclohexamide per ml). Labelled samples of 5 A6oo units of cells were collected into chilled tubes containing NaN 3(final concentration 20 mM). Cell-wall extracts were prepared as follows: Cells from each time-point were washed in water plus 1 mM PMSF and suspended in Tris/13-mercaptoethanol/PMSF (100raM Tris-HC1, pH 9.4/50 mM 13-mercaptoethanol/1 mM PMSF) and incubated at room temperature for 10 min. The samples were then washed once in 10 mM Tris, pH 7.4/1.2 M sorbitol and suspended in 100 gl of the same solution. Zymolyase (50 units; 100000 units/g; ICN Biomedicals, Costa Mesa, CA, USA) was added and cell walls were digested for 30 min at 30°C. Spheroplasts were removed at 2500 rpm and the supernatant was diluted with one ml IP buffer (50raM Tris-HCl, pH 7.4/150 mM NaC1/I% Triton X-100/0.2% SDS). Protein A binding proteins were removed by adsorption to 50 gl of 10% Staphylococcus aureus cells (Sigma, St. Louis, MO, USA) which were then removed by centrifugation at 12000xg for 5 min. Immune complexes were formed by incubation with 2 111 of 3-times adsorbed anti-Pholp Ab at 4°C for 12 h. Immune complexes were then collected by binding to protein A-Sepharose beads (20 lal of a 50% slurry; Pharmacia, Piscataway, NJ, USA) at 25°C for 2 h. The beads were washed twice with IP buffer and once with detergent-free IP buffer (50mM Tris.HCl pH7.4/150mM NaCI). Protein complexes were eluted into 30 gl ESB by heating to 100°C for 2 min. The sample (20 lal) was resolved by 0.1% SDS-8% PAGE and visualized on a phosphoimager.

GS115 GS190 CKY303 CKY304 CKY305 CKY306 CKY307 CKY308 CKY309 CKY310 CKY311 CKY312 CKY313 CKY314

PH01 PH01 phol-1 phol-2 phol-3 phol-4 PHOl/phol-2 PHOl/phol-3 PHO1/phol-4 PHO1/phol-1 phol-1/phol-2 phol-1/phol-3 phol-1/phol-4 CKY304+pPG31(PH01)

Enzyme activity units/A600 High-Pi (x 103)

Low-Ph (x 103)

14.9 8.9 8.1 8.9 9.5 10.5 4.3 4.8 4.4 4.5 5.4 4.4 4.3 2.5

219 240 11 10 11 9 162 176 190 147 13 12 12 174

"Pp strains were induced for 6-8 h in low-Pi SD medium and the Pho activity was determined under conditions described in the legend to Fig. 1. b pp genetic methods: Strains were mated by the method of Liu et al. (1992). Strains were transformed either by the spheroplast method (Cregg et al., 1985) or by electroporation (Rickey, 1990). Isolation of Pho mutants: Pp cells were mutagenized by ultraviolet irradiation as follows: Strains GS 115 and GS 190 were grown to exponential phase in YPD, washed twice in 50 mM EDTA to prevent clumping of cells, and suspended in sterile water. The cells were exposed to light from a germicidal lamp by swirling in a petri plate. The mutagenized cells were harvested by centrifugation and suspended in 25% glycerol for storage at - 70°C. A time of exposure to UV was selected that gave approx. 2% survival. Cells were plated onto YPD medium at a density of 300 colonies/plate. Colonies were grown for 4 days at 30°C and then replica plated to low-Pi YPD plates for overnight growth at 30°C. Colonies that did not express phosphatase activity were identified by the plate Pho assay. The plate Pho assay (Hansche and Beres, 1979) is carried out by replica plating well-spaced colonies onto plates containing low-phosphate medium (low-Pi YPD or low-Pi SD). After overnight growth to induce the enzyme the plate is overlayed with assay cocktail (30raM Na.acetate, pH 5/0.5mg Fast Garnet/0.2mg [3-napthylphosphate; all per ml) in 5 ml of molten 1% agarose at 45°C. Colonies expressing phosphatase activity develop a dark red color.

T h e PH01 g e n e w a s l o c a t e d i n p P G 3 1

by subcloning

different restriction fragments from the insert of pPG31 into

the

S. cerevisiae/E, coli s h u t t l e v e c t o r

(Sikorski and Hieter,

pRS313

1989) a n d s c r e e n i n g b y W e s t e r n

a n a l y s i s o f E. coli l y s a t e s for p r o t e i n f r a g m e n t s o f P h o l p that reacted with Pholp promoter

A b a f t e r i n d u c t i o n f r o m t h e lac

present on pRS313 (data not shown). By this

m e t h o d p a r t o f t h e PHOI c o d i n g s e q u e n c e w a s f o u n d t o be present on plasmid clone pPG34 that contains a 2.6-kb

ApaI-EcoRI f r a g m e n t o f g e n o m i c Pp D N A ( F i g . 4).

24

Produceo proleln fragment recognized by anUPholp anllbody

w

I

I

o

1

I 4

I 51~

) pPG31 pPG32 pPG34

+

pPG35 pPG36 pPG37

+

pPG38 pPG39

Fig. 4. Physical map of the PHOI locus. The coding region of PHOI is indicated by the large black arrow oriented in the direction of tran-

scription. Relevant restriction sites are noted. Regions of the PHO1 locus subcloned from pPG31 are shown schematically below the map. Each subclone was induced from the lac promoter present on the parent vector and protein extracts were analyzed by Western analysis using the anti-Pholp Ab. Subclones that expressed a polypeptide recognized by the anti-Pholp Ab are indicated. Strains: E. coli strains used for plasmid maintenance were TOP10F' I-F'TcR} mcrA A(mrr-hsdRMSmcrBC) 480 lacAM15 AlacX74 deoR fecAl araD139 A(ara,leu)7697 galU galK k rpsL endA1 nupG] (Invitrogen, San Diego, CA, USA) and XL1-BLUE [F'{proAB+laclqZAM15 Tnl0(TcR)} fecAl endA1 gyrA96 thi- 1 hsdR 17 supE44 relA 1 lac] ( Stratagene, La Jolla, CA, USA). Nucleic aeid techniques: Total yeast DNA was isolated as described by Hoffman and Winston (1987). Plasmid DNA was purified from E. coli by boiling and by alkaline lysis (Qiagen, Chatsworth, CA, USA).

T T C T T T G C G T A A C A C T C A A A G T A T A C CC C T G T T

-240

AGTC TTTATTCAC C T G T T G C T G C T T G G A A C A C T C A A A G T A T A C C CCTGTTAGTCTTTATTC AC CTGTTGCTGCT TGGTGCAGTTAC CAATTACTTGTTTC C A C T T G A A A A G C T T G T T T T TTTTTGATAGCACAGAAACGTGC~TCCGATAAGCTAAAC TTCAACGAGAATATAAAAGCTGAAAAGATTCTTGTCAAGAACTTGTACAACGAC CAATAAGTCTTTCAAGGCATCAGAC

T GC C CT C T C A G A G T G T G T C G C A G T A G A T C G A G T C ~ G T C

-120 -I

A T G TTT TCT CCT A T T C T A A G T C T G G A A A T T A T T CTC GCT T T G GCT A C T CTC C A A T C A GTC TTT G C G G T T GAG T T G C A G C A C GTT CTT G G A

90

IM

30

F

S

m

I

L

S

L

E

I

I

h

A

h

h I T

h

Q

S

V

F

i

V

E

L

Q

H

V

m

G

G T C AAC GAC A G A CCC T A T C C T C A G A G G A C A G A T G A T CAG TAC AAC ATT CTG A G A CAT C T G G G A GGC T T G GGC CCC TAC A T C G G T TAC A A T V N D R P Y P Q R T D D Q Y N I U R H L G G L O P Y I G Y N

180

G G A T G G G G A A T T G C T G C T G A G T C T G A A A T T G A A TCC TGT A C G A T T G A T CAG GCT C A T C T G T T G A T G A G A C A T G G A G A A A G A TAC C C A A G T G W G I A A E S E I E S C T I D Q A H L L M R H G E R Y P S

240 90

ACC A A T GTG G G G A A A C A A C T A G A A GCT T T G TAC C A G A A A C T A C T A GAT GCT G A T G T G G A A G T C CCT A C A G G A C C A T T G T C T TTC TTT C A A T N V G K Q L E A L Y Q K L L D A D V E V P T G P L S F F Q

330

G A C TAT G A T T A C TTC GTC T C T G A C GCC GCT T G G TAC GAG C A A G A A A C A A C T A A G GGT TTC TAC T C G G G G T T A AAC ACC G C T TTC G A T TTT D Y D Y F V S D A A W Y E Q E T T K G F Y S G L N T A F D F

420 150

G G T ACC A C T T T G A G A G A A C G A TAT GAC CAT TTG A T A AAC A C T AGC G A A G A A G G A A A G A A A C T T TCT G T T TGG G C T GGC TCT C A A G A A A G A G T T L R E R Y D H L I N T S E E G K K L S V W A G S Q E R

510

GTT GTT GAC ACC C-CA A A G TAC TTT G C T C A A G G A T T T A T G A A A TCT AAC TAC ACC G A T A T G G T T G A A GTC G T T G C T C T A G A A G A G GAG A A A V V D T A K Y F A Q G F M K S N Y T D M V E V V A L E E E K

600 210

TCC C A G G G A CTC A A C T C T C T A A C G GCT C G A A T T T C A T G T C C A AAC TAT AAC AGC CAT A T C TAC A A A G A T GGC GAC TTC CCC A A T GAC A T T S Q G L N S L T A R I S C P N Y N S H I Y K D G D F P N D I

690

G C T G A A A G A G A A GCT GAC A G A T T G A A C A C T CTT TCT C C A G G A TTT AAC A T T A C T G C A G A T G A T A T T C C A A C A A T T GCC C T A TAC TGT GGC A E R E A D R L N T L S P G F N I T A D D I P T I A L Y C G

780 270

TTT G A A C T A A A T G T A A G A G G T G A G T C A TCC TTC TGT GAC GTC TTG T C A A G A G A G G C T C T A C T G TAC A C T GCT T A T CTT A G A G A T T T G G G A F E L N V R G E S S F C D V L S R E A L L Y T A Y L R D L G

870 300

T G G TAT TAC A A T G T T G G A A A C G G G A A C C C A CTT G G A A A G A C A ATC GGC TAC GTC TAT CCC AAC GCC A C A A G A C A G C T G T T G G A A AAC A C A W Y Y N V G N G N P L G K T I G Y V Y A N A T R Q L L E N T

960

G A A GCT GAT CCT A G A G A T TAT C C T TTG TAC TTT TCC TTT A G T CAT GAT ACC GAT CTG C T T C A A G T A TTC A C T T C A CTC G G T CTT TTC A A C E A D P R D Y P L Y F S F S H D T D L L Q V F T S L G L F N

1050 360

G T G A C A G A T C T G C C A T T A GAC C A G A T T C A A TTC C A G ACC TCT TTC A A A TCT ACC G A A A T A G T T CCC A T G G G A G C A A G A T T G CTT ACC G A G V T D L P L D Q I Q F Q T S F K S T E I V P M G A R L L T E

1140 390

A G A T T A T T G TGT A C T G T T G A A G G T G A A G A A A A A TAC TAC GTT A G A ACT ATC CTC AAC G A T G C A GTC TTC C C A CTG A G T GAC TGT TCC T C T R L L C T V E G E E K Y Y V R T I L N D A V F P L S D C S S

1230 420

GGC CCT C-GA TTC T C T T G T C C G T T G AAC G A T TAT GTT T C T A G A CTT GAG G C A TTG AAC G A G GAC A G T GAC TTT G C G G A A A A C TGT G G A GTT G P G F S C P L N D Y V S R L E A L N E D S D F A E N C G V

1320 450

CCT A A A A A T G C T TCC TAC C C A C T T G A A C T A T C A TTC TTC T G G G A T GAC TTG T C A T A A P K N A S Y P L E L S F F W D D L S *

1377 468

A A A T G G T A A G G A A T G T T T T G C A T C A G A T A C GAGTT CAAAAC G A T T A A G A A G A G A A T G C TCTTTTTT TTGTTTC TATC CAATTCIGAC TATTTTCGTTTATTTTAAATAC~GTACAAC TTT A A C T A G A T G A T A T C T T C T T C T T C A A A C GATACCACTTCTCTCATAC T A G G T G G A G G T T C AATGGAT C C TACAAACTC CAACGATACAAATCCAACAC C A A C G A T A A C C A G T A A A A A C C C T A T C A G G C C G C G G A A A A T C G A A C C T C C A C T G A T C A A C A C TC C C A A A A A G A T G T A G A A G C C AC CT C TTCC C A A A A A T G A C A A C A A A A A C G A T A G A T A C G G C G T A A C T T T AGGTAC CAAq'?

1496

60

120

180

240

330

1615 1734

25 (g) The nt and aa sequence The complete 2.6-kb DNA sequence of clone pPG34 was determined, and revealed an ORF that encodes a protein with homology to other yeast Pho (see below). The ORF on clone pPG34 begins at the ApaI site (Fig. 4) and encodes a protein of 46 623 Da. This is the approximate molecular mass of the protein fragment expressed in E. coli from pPG34 that was detected with Pholp Ab. The sequence of the PH01 gene was completed utilizing clone pPG31 as template. The nt sequence of PH01 and the predicted aa sequence of Pholp are shown in Fig. 5. An ORF encoding a 468-aa protein (52632 Da) is identified. There is a region at the N terminus of the predicted protein that is similar in structure to the signal sequences found at the N terminus of secreted proteins. A 15-aa signal peptide would be removed during secretion if cleavage was to occur after the frequently observed recognition sequence of Ala-Xaa-Ala (Perlman and Halvorson, 1983). There are six potential sites for N-linked glycosylation (AsnXaa-Ser/Thr). The predicted Pholp sequence shows significant homology to Pho enzymes from S. cerevisiae (Pho5p), Sz. pombe (Pholp, Pho4p), and Aspergillus niger (PhyA). Pairwise alignments between Pp Pholp and each of these Pho proteins using BESTFIT revealed 30-34% identity and 50-55% conservative substitutions along the full length of the protein for each alignment. There are three regions of the proteins that share a high degree of homology (Fig. 6) and two of these contain conserved His residues previously implicated in catalysis. The first His region contains the R H G X R X P motif where His acts as the nucleophilic acceptor in the attack of the phospho group of the substrate (Bazan et al., 1989; Ostanin et al., 1992). The second region contains a His residue in a conserved 'HD' dipeptide that is believed to donate a proton to the substrate leaving group during catalysis (Ostanin et al., 1992). (h) Conclusions (1) We outline a simple procedure for the purification of Pp Pholp, liberated from the cell wall by 13-1,3-glucanase and precipitated with ethanol followed

aa

SpPholp sp Pho4p Pp Pholp Sc An

Pho5p Phyap

58

* * **sl

58

CAIKQVHLLQRHGSRNPTGDDT

73

CTIDQAHLL

MRHGER

64

CEHKQLQMVGRHGERYPTVSLA

71

CRVTFAQVL

314

"1"[STNVG*

YP

HGARYPTDSKG

VFFA;THDAN I I PVET AL~FF

334 339

LYFSFSiHDTDLLQVFTSLGLF

331 355

LYADFSi~NGIISILFALGLY

387

KYYVRHLVNEEVFPLSDCGFGPSNT

397

KYYVRHLVNQQVYPLTDCGYGPSGA

401

KYYVRTILNDAVFPLSDCSSGPGFS

391

..YVRYVINDAVVPIETCSTGPGFS

421

..LVRVLVNDRVVPLHGCPVDA...

Fig. 6. Comparison of Pho proteins. Alignment of the predicted catalytic domains by the method of Feng and Do•little (1987) is shown. A solid dot above a position indicates aa identity among all sequences examined. A asterisk above a position indicates a conservative aa substitution at that position in 4 out of 5 sequences. Consensus motifs are enclosed by a box. The conserved His residues involved in enzyme catalysis are shown in bold. Gaps introduced to optimize the alignment are indicated by dots. Computer analysis: Sequence homology searches were carried out at the National Center for Biotechnology Information using the BLAST network service (Altschul et al., 1990). Analysis of nt and aa sequences was performed with GCG software (version 7.3; Genetics Computer Group, Madison, WI, USA). The similarities among the different Pho proteins were determined by using BESTFIT. The multiple sequence alignment was created by the progressive alignment method of Feng and Do•little (1987) using PILEUP.

by DEAE-Sepharose chromatography. A 2-1itre culture of induced Pp cells yields > 1 mg of pure Pholp. (2) Pholp is a glycoprotein of approx. 80-100 kDa, and the sequence of the PHOI gene indicates that the protein size is 51 kDa and has six N-linked carbohydrate chains. PH01 encodes a putative signal sequence with a predicted signal peptidase cleavage site after the conserved Ala-Xaa-Ala at aa 15 (Perlman and Halvorson, 1983). (3) The Pp Pholp sequence shows significant homology to repressible Pho from other yeast species (S. cerevisiae, Sz. pombe) and the filamentous fungus A. niger var.

Fig. 5. The nt sequence of PHOI and deduced aa sequence of Pholp. Numbers start with the ATG start codon. The putative signal sequence is boxed. The stop codon is marked by an asterisk. Potential N-glycosylation sites (Asn-Xaa-Ser/Thr) are heavy underlined. Regions of homology between Pho proteins (see section g) are light underlined. The GenBank accession No. for the PHO1 nt sequence is U28658. Sequenee analysis of PHOI: A 4.0-kb region of genomic Pp DNA containing the PHO1 locus from ClaI to EcoRI (Fig. 4) was sequenced by the dideoxy chain termination method (Sanger et al., 1977) using the Sequenase T7 DNA polymerase system (US Biochemical, Cleveland, OH, USA). The sequence of the noncoding strand of P H 0 1 was determined by constructing a series of nested deletions by unidirectional digestion of pPG34 with exonuclease III (Henikoff, 1987). The sequence of the coding strand was completed by sequencing from the polylinker of appropriate subclones and by oligo primer walking following the synthesis of primers based upon the sequence of the noncoding strand. The sequence of the entire coding region was determined for both strands. The sequence of the 5' U T R containing the PHOI promoter was determined on only one strand.

26

awamori. The Pholp sequence also shows homology to Pho enzymes from E. coli (Touati and Danchin, 1987), rat (Himeno et al., 1989) and human (Pohlmann et al., 1988), but only in the highly conserved catalytic domains of the enzyme. (4) Pholp is translocated into the ER, glycosylated and transported to the cell surface in about 5 min. Thus the kinetics of secretion in Pp are similar to that in S. cerevisiae. (5) The regulated PH01 promoter will be useful for heterologous gene expression in P. pastoris. The most commonly used promoter for the expression of heterologous proteins in Pp is the regulated promoter of the alcohol oxidase-encoding gene (AOX1). While the AOX1 promoter provides a high level of expression, induction is carried out by growth on methanol as a carbon source which leads to dramatic changes in cell morphology and physiology. The PH01 promoter has the advantage that it can be induced about 100-fold under conditions that do not significantly alter cell growth or morphology.

REFERENCES Altikrete, H., Kouri, M., Charpentier, C., Lematre, J. and Bonaly, R.: Extracytoplasmic phosphatases of selected species of Saccharomyces, Kluyveromyces and Rhodotorula. Phytochemistry 23 (1984) 1551-1555. Altschul, S.F., Gish, W., Miller, W, Myers, E.W. and Lipman, D.J.: Basic local alignment search tool. J. Mol. Biol. 215 (1990) 403 410. Bazan, J.F., Fletterick, R.J. and Pilkis, S.J.: Evolution of a bifunctional enzyme: 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Proc. Natl. Acad. Sci. USA 86 (1989) 9642-9646. Boer, P. and Steyn-Parve, E.P.: Isolation and purification of an acid phosphatase from baker's yeast. Biochim. Biophys. Acta 128 (1966) 400-402. Cregg, J.M., Barringer, K.J., Hessler, A.Y. and Madden, K.R.: Pichia pastoris as a host system for transformations. Mol. Cell. Biol. 5 (1985) 3376 3385. Dibenedetto, G.: Acid phosphatase in Schizosaccharomyces pombe, I. Regulation and preliminary characterization. Biochim. Biophys. Acta 286 (1972) 363-374. Feng, D.-F. and Doolittle, R.F.: Progressive sequence alignment as a prerequisite to correct phylogenetic trees. J. Mol. Evol. 25 (1987) 351-360. Field, C. and Schekman, R.: Localized secretion of acid phosphatase reflects the pattern of cell surface growth in Saccharomyces cerevisiae. J. Cell Biol. 86 (1980) 123-128. Hansche, P.E. and Beres, V.: Gene duplication in Saccharomyces cerevisiae. Genetics 88 (1978) 673-687. Harlow, E. and Lane, D.: Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988. Henikoff, S.: Unidirectional digestion with exonuclease III in DNA sequence analysis. Methods Enzymol. 155 (1987) 156-165. Himeno, M., Fujita, H., Noguchi, Y., Kono, A. and Kato, K.: Isolation and sequencing of a complementary DNA clone encoding acid phosphatase in rat liver lysosomes. Biochem. Biophys. Res. Commun. 162 (1989) 1044-1053. Hoffman, C.S. and Winston, F.: A ten-minute DNA preparation from yeast efficiently releases autonomous plasmids for transformation of Escherichia coli. Gene 57 (1987) 267-272.

Johnston, M. and Carlson, M.: Regulation of carbon and phosphate utilization. In: Jones, E.W., Pringle, J.R. and Broach, J.R. (Eds.), The Molecular and Cellular Biology of the Yeast Saccharomyces: Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1992, pp. 193-281. Kaiser, C.A., Michaelis, S. and Mitchell, A.: Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1994. Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (1970) 680-685. Liu, H., Veenhuis, M., McCollum, D. and Cregg, J.M.: An efficient screen for peroxisome-deficient mutants of Pichia pastoris. J. Bacteriol. 174 (1992) 4943-4951. Novick, P., Field, C. and Schekman, R.:Identification of 23 complemenration groups required for post-translational events in the yeast secretory pathway. Cell 21 (1980) 205-215. Odds, F.C. and Hierholzer, J.C.: Purification and properties of a glycoprotein acid phosphatase from Candida albicans. J. Bacteriol. 144 (1973) 257-266. Ogrydziak, D.M., Cheng, S.C. and Scharf, J.: Characterization of Saccharomycopsis lipolytica mutants producing lowered levels of alkaline extracellular protease. J. Gen. Microbiol. 128 (1982) 2271-2280. Ostanin, K., Harms, E.H., Stevis, P.E., Kuciel, R., Zhou, M.-M. and Van Etten, R.L.: Overexpression, site-directed mutagenesis, and mechanism of Escherichia coli acid phosphatase. J. Biol. Chem. 267 (1992) 22830-22836. Payne, G.S. and Schekman, R.: A test of clathrin function in protein secretion and cell growth. Science 230 (1985) 1009-1014. Perlman, D. and Halvorson, H.O.: A putative signal peptidase recognition site and sequence in eukaryotic and prokaryotic signal peptides. J. Mol. Biol. 167 (1983) 391-409. Pohlmann, R., Krentler, C., Schmidt, B., Schroeder, W., Lorkowski, G., Culley, J., Mersmann, G., Geier, C. and Waheed, A.: Human lysosomal acid phosphatase cloning expression and chromosomal assignment. EMBO J. 7 (1988) 2343-2350. Preuss, D., Mulholland, J., Franzusoff, A., Segev, N. and Botstein, D.: Characterization of the Saccharomyces golgi complex through the cell cycle by immunoelectron microscopy. Mol. Biol. Cell 3 (1992) 789 803. Preuss, D., Mulholland, J., Kaiser, C.A., Orlean, P., Albright, C., Rose, M.D., Robbins, P.W. and Botstein, D.: Structure of the yeast endoplasmic reticulum: localization of ER proteins using immunofluorescence and immunoelectron microscopy. Yeast 7 (1991) 891-911. Rickey, C.: Efficient electroporation of yeast. Mol. Biol. Reports (BioRad) April (1990) 2-3. Rubin, G.M.: Three forms of the 5.8-S ribosomal RNA species in Saccharomyces cerevisiae. Eur. J. Biochem. 41 (1974) 197-202. Sanger, F., Nicklen, S. and Coulson, A.R.: DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74 (1977) 5463-5467. Schmidt, G., Bartsch, G., Lamont, M.C., Herman, T. and Liss, M.: Acid phosphatase of baker's yeast: An enzyme of the external cell surface. Biochemistry 2 (1963) 126-131. Sikorski, R.S. and Hieter, P.: A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122 (1989) 19-27. Suomalainen, H., Linko, M. and Oura, E.: Changes in the phosphatase activity of baker's yeast during the growth phase and location of the phosphatases in the yeast cell. Biochim. Biophys. Acta 37 (1960) 482-490. Touati, E. and Danchin, A.: The structure of the promoter and N-terminal region of pH 2.5 acid phosphatase structural gene (appA) of E. coli: a negative control of transcription mediated by cyclic AMP. Biochimie 69 (1987) 215-221. Trimble, R.B., Maley, F. and Watorek, W.: Subunit structure and carbohydrate composition of the extracellular acid phosphatase of Rhodotorula glutinis. J. Biol. Chem. 256 (1981) 10037 10043.