Appl Microbiol Biotechnol (1990) 33:307-312

Applied Microbiology Biotechnology © Springer-Verlag 1990

Regulated overproduction and secretion of yeast carboxypeptidase Y Trine L. Nielsen*, Steen Holmberg*, and Jens G. L. Petersen** Department of Yeast Genetics, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Copenhagen Valby, Denmark Received 13 November 1989/Accepted 30 January 1990

Summary. Carboxypeptidase Y (CPY) is a gly~osylated yeast vacuolar protease used commercially for synthesis of peptides. To increase the production of CPY i n Saccharomyces cerevisiae we have placed its coding region (PRC1) under control of the strongly regulated yeast G A L l promoter on multicopy plasmids and introduced the constructs into vpll mutant strains. Such mutants are known to secrete CPY. High levels of CPY production were obtained by induction of the G A L l promoter when the cells had left the exponential phase, resulting in a growth-phase-dependent CPY production similar to that of cells with PRC1 under the control of its own promoter. Introduction of a high copy number 2Ix-URA3-LEU2d plasmid with G A L l p - P R C I fusion in a vpll strain resulted in a 200-fold increase of secreted CPY (about 40 mg/1) as compared to a vpll mutant carrying a single copy of the wild-type PRC1 gene. The overproduced, secreted CPY was active and had the normal N-terminal sequence. Sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed two forms of active CPY, probably due to different levels of glycosylation.

Introduction Carboxypeptidase Y (CPY), encoded by the PRC1 gene (Wolf and Weiser 1977), is a 61000-dalton glycoprotein located in the vacuole of the yeast Saccharomyces cerevisiae. CPY is synthesized as a 67000-dalton inactive precursor (proCPY), which is processed into the mature form upon or just before delivery to the vacuole by cleavage of a part of the N-terminal region (Hasilik and Tanner 1978; Stevens et al. 1982). Mutant yeast cells (vpl or vpt) that secrete up to 90% of immunoreactive * Present address: Institute of Genetics, University of Copenhagen, Oster Farimagsgade 2A, DK-1353 Copenhagen K, Denmark ** Present address: Novo-Nordisk A/S, Novo All~, DK-2880 Bagsv~erd, Denmark Offprint requests to: T. L. Nielsen

CPY have been isolated (Bankaitis et al. 1986; Rothman and Stevens 1986). In the periplasm, the secreted proCPY is slowly converted into the mature, active form by some unknown mechanism (Rothman and Stevens 1986). CPY belongs to the serine carboxypeptidases. In addition to peptidase activity, CPY has the ability to catalyse aminolysis of C-terminal peptide esters in vitro, resulting in elongation of the peptide chain (Widmer and Johansen 1979). The commercial application of CPY for synthesis of small peptides prompted us to construct S. cerevisiae strains which overproduce and secrete CPY. The production of CPY is known to increase only eightfold when PRC1 is placed on a 2 tx based plasmid either behind its own promoter or the yeast promoters of A D H 1 or P H 0 5 (Stevens et al. 1986). We have placed PRC1 under control of the strongly regulated G A L l promoter from S. cerevisiae (St. John and Davis 1981). The G A L l promoter has previously been used for regulated production of heterologous proteins in yeast (see, for example, G o f f et al. 1984; Schultz et al. 1987). We show that PRC1 under control of the G A L l promoter placed on a high copy number plasmid in a vpll mutant results in a 200-fold overproduction of secreted, active CPY as compared to a vpll strain with one chromosomal copy of PRC1.

Materials and methods Plasmids and strains. Plasmid pTSY3 (Fig. la) consists of the yeast-bacterial shuttle vector YEp24 with a 3.2-kb yeast DNA insert containing the PRC1 gene (Stevens et al. 1986). Plasmid pCGS109 (Fig. lb) contains an 850-bp fragment of the S. cerevisiae GALl promoter (Goff et al. 1984). The 2 ~t based plasmids pGPR7 and p72UG containing PRC1 under control of the GALl promoter were constructed as shown in Fig. 1. All S. eerevisiae strains used in this study were isogenic vpll mutants, which have identical growth rates on glucose and/or galactose before transformation by GALl promoter constructs. Strain TSY10-7D (MATa, Aprel::LEU2 vpll-1 leu2-3 1eu2-112 ura3-52) was obtained froil~ 1. ~tc~cn~, Institute of Molecular Biology, University of Oregon, USA. In strain TSY10-7D the LEU2 insertion in the PRC1 locus was replaced by the wild-type allele of

308 A

HB

B Bg

s "~

! sa~[ Pvu~

[

H~

Klenow

~

%.~Lg -

k~

~--41111~

_~P ~

.TSV~

I~

_

E

~ "~

~

/ /*~

-I /

/~ I

L.

~ .CGS~O9 ~}

~~

~ ,

~ . mH ~N I ~,~ow~

B~p ~

/

"'

'

/I ~

~

/~

/E

~

H (B/S)

~

iIvvu" Klenow

~

~ B

k~. ~~@'/

'

.J

~l

/ /~

/g

Hindl ~ ~,igation

B~

/ ~_

~

~s "

Sm

I

ligation (B/A) B

Partial I

~.

I

Al~::~

~

~

~%

V

/~'

"

H Sm H i u..~I I

~ ~,A p72 11~

"~-

I A~cI

.i.../,~> ~ %~ e ~ Iigation

~ / ~~

~

~/

~"~

,

(~)

B

'~s k

-H (B/S)

~. , I t t t l ' ~ ~tt ~ ~ . .

~.~ B

~ ~

pGPR7 9.9 kb ~

x

~

~S I

~/

~

I /

N~ ..

[

~B

~3.0kb

/

~

H~ ~ ~

~ ,

~,c,

, coding region PRCI

.............

GALplromoter

I Klenow

. ~

S~a~

(E) {B/A)B

'..........~

~ _U

Bg

,4i ligation

~ ~+_ ~_

@~

(Sm/E)

(B/A)B

..........~ ~

Y '~"~

~

d

0i

Fig. 1. Construction of 2 ~ based plasmids containing PRC1 under control of the GALl promoter. From plasmid pTSY3 (a) the 1.9-kb AecI-PvulI fragment containing the coding region of PRC1 (Valls et al. 1987) was inserted into plasmid pCGS109 (b) resulting in plasmid pGPR7 (c). Secondly, the 3.2-kb SalI-PvuII PRC1 fragment of pTSY3 was inserted into the BamHI site of the LEU2d plasmid pMP78 (Erhart and Hollenberg 1983) (d) giving p72 (e). Plasmid p72U (tO is p72 with the yeast URA3 gene inserted into the HindIII site located close to PRC1. Plasmid p72U was further used to place PRC1 under GALlp control by replacing the 3.0-kb SmaI-BglII fragment with the 2.1-kb EcoRI-BglII fragment from pGPR7 (e), giving p72UG (g). A, AeeI; B, BamHI; Bg, BglII; E, EcoRI; H, HindIII; P, PvuII; S, SalI; Sin, SmaI; Klenow, Klenow fragment of DNA polymerase I and dNTP's were added to fill out sticky ends. Amp, ampicillin-resistance gene; ORL pBR322 origin of replication; 2#, fragment of the yeast 2 Ix plasmid containing the replication origin

PRC1, resulting in strain T2641 (MATa vpll-1 leu2-3 leu2-112 ura3-52), or by a deletion allele of PRC1, resulting in strain W2579 (MA Ta Aprcl vpll-1 leu2-3 leu2-112 ura3-52), using the two-step gene replacement technique described by Winston et al. (1983). Strain T2648 (MATa Aprel::LEU2 vpH-1 leu2-3 leu2-112 ura3-52 chal::GALlp-PRC1) has PRC1 under control of the GALl promoter integrated in one copy into the CHA1 gene (Petersen et al. 1988). This strain was constructed from TSY10-7D by two-step gene replacement (Winston et al. 1983) using the integration plasmid pICG9 constructed as shown in Fig. 2. The Escheriehia coli strain used was DH5 (Hanahan 1985).

Transformation and plasmid stability of yeast. Yeast transformation was carried out as described by Ito et al. (1983) using lithium acetate. The stability of plasmids in stationary-phase yeast cells was determined by plating culture samples on YPD medium (1% yeast extract [Difco], 2% peptone [Difco] and 2% glucose) and subsequent replica-plating of about 100 colonies to SC medium lacking uracil.

Media and growth of yeast. S. cerevisiae strains were grown at 30° C in synthetic complete (SC) medium (0.67% Yeast Nitrogen

Bacteria and DNA techniques. Growth and transformation of E. coli by treatment with calcium chloride and isolation of plasmid

Base (Difco, Detroit, Mich, USA), buffered with 5 g NaOH and 10 g citric acid/1 (pH 5.8) and supplemented with nutrients as described by Petersen et al. 1983). The carbon source was glucose and/or galactose as indicated.

309 (B/A) B

the specific activity of 130 units (U)/mg of a CPY standard (Sigma). For measurement of intracellular activity, cells were prepared and crushed by mixing with glass beads (212-300 ~tm, Sigma) on a whirli-mixer. After centrifugation the CPY activity of the supernatant was measured. This was done in the presence of 1.3 M guanidine hydrochloride (Fluka Chemic AG, Buchs, Switzerland) in order to prevent binding of the cytoplasmic inhibitor of CPY. Extracellular activity was measured directly on culture supernatants after 10 rain centrifugation at 5000 rpm.

.GP.,

J EcoR I Sal I

l e ow ~

// (H/P)~ ~

(E)

(B/A) B

~ ligation (Bg/~) f (B/A)

~

~ m m u ~

~

/~

~

B

B BIs)

,

' .........

Preparation of culture supernatants for sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE). The supernarants from 20-ml samples of cell cultures were made up to 1 raM. in EDTA and, after dialysis and lyophilization, dissolved in 0.5 ml H20. After centrifugation the supernatants were dried, the residues dissolved in 50 Ixl H20 and 25 Ixl analysed by SDS-PAGE.

SDS-PAGE. This was performed in 6%-10% gradient gels accord-

PRCl

ing to Laemmli (1970). The proteins were visualized by staining with Coomassie Brillant Blue R250 (Sigma).

,~ coding region P R C I

............ ~

.................

~o~ot~,

~B 10.9kb~B/Bg(S

CPYpurification. CPY was purified by affinity chromatography by binding to the CPY specific inhibitor para-aminobenzylsuccinic acid according to Johansen et al. (1976).

plCG9

F~g. ~, Construction of an integration plasmid containing PCRI under control of the GALl promoter. A 2.5-kb HindIII-ClaI fragment of the CHA1 gene (Petersen et al. 1988) was cloned into YIp5 giving plasmid YIp5-CHA1 (b). YIp5-CHA1 was linearized with B~llI, and the EcoRI-SalI GALlp-PRC1 fragment from pGPR7 (a) was inse~ed, resulting in plasmid pICG9 (c). C, ClaI; N, NruI. Other abbreviations are explained in the legend to Fig.

Endoglycosidase H (Endo H) treatment. This was performed as recommended by the supplier (Boehringer) after boiling the samples in 0.025% SDS for 3.5 min. Amino acid sequencing. N-Terminal amino acid sequence determination was performed for four cycles with a 470A gas-phase sequenator (Applied Biosystems, Foster City, Calif, USA) in combination with an on-line High Pressure Liquid Chromatographer, model 120A (Applied Biosystems) according to the manufacturers instructions.

1

Protein determination. Total protein was measured with the BioDNA followed standard procedures (Maniatis et al. 1982). Restriction enzymes, the Klenow fragment of DNA polymerase I and T4 DNA ligase were used according to the manufacturer (Boehringer, Mannheim, FRG).

Rad (Richmond, Calif, USA) protein assay after boiling the samples in 0.5 M NaOH for 5 min. Bovine serum albumin was used as standard. Results

Measurement of CPY activity. The activity of CPY was measured from the decrease in absorbance at 339 nm of 0.2 mM FA-PhePhe (N-(3-(2-furyl)acryloyl)-L-phenylalanyl-L-phenylalanine(Sigma, St. Louis, Mo, USA)) in 50 mM 2-(N-morpholino)-ethanesulphonic acid (MES, Sigma) and 1 mM ethylenediaminetetraacetic acid (EDTA) (Merck, Darmstadt, FRG), adjusted to pH 6.5 with NaOH. The concentration of CPY in mg/1 was calculated from

C P Y production f r o m the native PRC1 9ene T h e PRC1 gene has b e e n c l o n e d b y Stevens et al. (1986) o n the 2 Ix b a s e d p l a s m i d YEp24, r e s u l t i n g i n p l a s m i d p T S Y 3 (Fig. la). T h e p r e s e n c e o f p l a s m i d pTSY3 i n

Table 1. Carboxypeptidase Y (CPY) production from wild-type PRC1 and PRC1 under GALl promoter control in vpll strains grown to stationary phase in synthetic complete (SC) media Yeast strain

T2641 TSYI0-7D[pTSY3] T2648 TSY10-7D[pGPR7] W2579[p72UG]

State of PRCI

PRC1 2 ~t-PRC1 GALlp-PRC1 2tx-GALlp-PRCI 2 p~-LEU2d-GALlp-PRC1

Growth rate (generation/h)

Plasmid stability (%)

glua gal

glu + gal glu

0.40 0.37 0.40 0.37 0.26

0.40 0.35 0.40 0.35 0.26

0.40 0.35 0.28 0.19 0.00

Intracellular Extracellular CPY activity (mg/1) CPY activity (rag/l)

gal glu + gal glu

ND b NDND

75 73 75 ND N D N D 80 15 51 95 ND 83

0.00 0.18 0.00 0.00 0.00

gal

glu + gal glu

gal

0.00 0.15 0.00 0.00 ND

0.00 0.16 0.18 0.30 0.30

0.15 0.2 3.20 3.5 0.35 3.5 0.21 13.2 ND 42.2

0.20 3.50 0.00 0.00 0.00

glu + gal

Plasmid-bearing strains were grown without uracil and W2579[p72UG] was grown with only 6 mg leucine/1 medium. The values are the means of three independent experiments ~ The carbon source was either 2% glucose (glu), 2% galactose (gal) or 2.5% glucose plus 2.5% galactose (glu+gal) b ND = not determined

310

yeast has been shown to result in an eightfold elevation in the vacuolar level of immunoreactive CPY (Stevens et al. 1986). Yeast strains containing the vpH mutation secrete about 80% of immunoreactive CPY (Rothman and Stevens 1986). To obtain secretion of overproduced CPY, we transformed the vpll strain TSY10-7D with plasmid pTSY3. When grown on SC medium lacking uracil to maintain the plasmid, TSY10-7D[pTSY3] produced 3.5 mg/1 enzymatically active extracellular CPY and only 0.2 mg/l active intracellular CPY (Table 1). Thus the increased gene dosage of PRC1 resulted in a 17-fold overproduction of active, extracellular CPY, as compared to a single copy PRC1 vpll strain T2641 (Table 1).

PRC1 during exponential growth, we used mixed glucose and galactose medium, thereby obtaining repression of the GALl promoter as long as glucose was present in the medium (Adams 1972; St. John and Davis 1981). Strain TSY10-7D with the GALlp-PRC1 multicopy plasmid pGPR7 grown on a medium with 2.5% glucose and 2.5% galactose resulted in a significant increase in CPY production (extracellular activity 13.5 mg/1, Table 1). Similarly, strain T2648 with one integrated copy of GALlp-PRC1 secreted 3.5 mg/l active CPY when grown on mixed glucose/galactose medium, a tenfold increase as compared to growth on galactose medium (Table 1). Growth rates and plasmid stabilities of the TSY10-7D transformants were similar on glucose and mixed glucose/galactose (Table 1).

Galactose-inducible PR C1 expression The GALl gene of S. cerevisiae is strongly induced by galactose and repressed in the presence of glucose (St. John and Davis 1981). To increase the expression of PRC1, we constructed the 2 ix based plasmid pGPR7 (Fig. lc) with PRC1 under control of the GALl promoter (GALlp). Furthermore, we constructed a vpll strain with GALlp-PRC1 integrated in one copy into the genome as described in Materials and methods. The resulting strain T2648 and the vpll strain TSY10-7D transformed with plasmid pGPR7 were grown on galactose medium to induce the GALl promoter. In both cases only very low levels of active extracellular CPY were measured (0.35 mg/1 and 0.21 mg/1, respectively, Table 1). Growth rates and stability of plasmid pGPR7 were reduced as compared to the same strain grown on glucose medium. This may well have been caused by the expression of PRC1. In yeast cells harbouring the wild-type PRC1 gene the steady-state level of CPY mRNA is increased about tenfold in the early stationary phase (Distel et al. 1983). With strain TSY10-7D[pTSY3] we found a similar growth phase dependency of extracellular CPY activity, which reached the maximum level of 3.5 rag/1 in the early stationary phase (Fig. 3a). In order to imitate the growth-phase-dependent regulation and avoid high-level expression of GALlp~•

100 a

• ~ '~ ~

10.

~ ~

~"

~ ~

~ N

b &.~-4,~.~4, ./oO--O--O~o

0.0~- ~ H~

SDS-PAGE of culture supernatants and purified CPY

j&IA~A

[

~4

4'8 Hrs

While some commonly used yeast 2 ix based plasmids have a copy number of about eight per cell, plasmids containing the poorly expressed LEU2d marker can be brought to a copy number of about 35 per cell (Erhart and Hollenberg 1983). We constructed plasmid p72UG (Fig. lg) containing GALlp-PRC1, URA3 and LEU2d. The vpll leu2 yeast strain W2579, transformed with plasmid p72UG, grown on SC medium with a mixed carbon source and lacking uracil, produced 13.4 mg/1 of active extracellular CPY and grew with a rate similar to that of TSY10-7D transformants (data not shown). To select for increased copy number of p72UG by leucine depletion, only 6 mg/l of leucine was added to the medium (normal concentration in SC is 20 mg/1), resuiting in production of 42 mg/l secreted active CPY (Table 1). This is 200-fold more than produced extracellularly by the vpH strain T2641 with one copy of PRC1 and corresponds to about 2% of the total yeast protein or 99% of the total active CPY. As was found for strain TSY10-7D[pTSY3] (Fig. 3a), strain W2579[p72UG] shows a growth-phase-dependent CPY production with the maximum reached in the early stationary phase (Fig. 3b). Strain W2579, transformed with a LEU2dcontaining plasmid (Fig. ld, e, f, or g) and grown under leucine depletion, always resulted in a low growth rate (Table 1 and data not shown).

gig t ° ~ ° "'---• • S ~

//

GALl promoter directed CPY overproduction from a LEU2d high copy number plasmid

24

4'8

~2

Fig. 3a, b. Growth and CPY production, a Strain TSY107D[pTSY3] grown on synthetic complete (SC) medium with 2% glucose and lacking uracil, b Strain W2579[p72UG] grown on SC medium containing 2.5% glucose, 2.5% galactose, 6 rag/1 leucine and no uracil

Cultures of vpll yeast cells grown to stationary phase on mixed glucose/galactose were centrifuged and equal volumes of supernatant were analysed by SDS-PAGE (Fig. 4). In culture fluids of TSY10-7D containing plasmid pTSY3 (lane f) or plasmid pGPR7 (lane c) and in that of strain W2579 with plasmid p72UG (lane b) increasing amounts of a protein with the mobility of mature CPY were observed. This corresponds well to the ratios of the measured enzyme levels of these strains (Table 1). The gel pattern of W2579[p72UG] suggests

311

Fig. 4. Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE) of supernatants from stationary phase cultures grown in SC medium or SC medium without uracil for plasmid-bearing strains. Unless otherwise indicated, the carbon source was 2.5% glucose and 2.5% galactose. Lane a, molecular weight markers (Pharmacia LKB Biotechnology, Uppsala, Sweden; catalogue no. 17-0446-1) in daltons× 10-3; lane b, W2579[p72UG] grown with 6mg/1 leucine; lane c, TSY107D[pGPRT]; lane d, CPY standard (41xg); lane e, TSY107D[pGR7] grown on 2% galactose; lane f, TSY10-7D[pTSY3];lane g, T2641; lane h, W2579 that more than 90% of the extracellular protein is CPY. When TSY10-7D[pGPR7] was grown on galactose medium (Fig. 4, lane e), no CPY band was seen. This agrees with the measured level o f only 0.21 m g / l extracellular CPY (Table 1). Further analysis by SDS-PAGE of culture supernatant o f W2579[p72UG] (Fig. 5, lane c) resolved a band

Fig. 5. SDS-PAGE of purified and endoglycosidase H (Endo H)treated CPY. Lane a, molecular weight markers (Pharmacia, catalogue no. 17-0446-01) in daltons × 10-3; lane b, CPY standard; lane c, supernatant from a culture of W2579[p72UG] grown on SC medium with 2.5% glucose, 2.5% galactose, 6 rag/1 leucine and no uracil; lane d, same as c, after purification by affinity chromatography; lane e, Endo H-treated CPY standard; lane f, same as d, Endo H treated

with mobility similar to that of mature, active CPY (61000 daltons, lane b), and in addition high amounts of a protein of approximately 56000 daltons. SDSP A G E after purification of active CPY by affinity chromatography showed the same 56000 and 61000 dalton protein bands (Fig. 5, lane d), suggesting two active forms of CPY. Deglycosylated, mature CPY has a size of 51000 daltons (Stevens et al. 1982). Treatment of the purified CPY from W2579[p72UG] with Endo H, which removes carbohydrate moities from glycoproteins, resuited in a single protein band of approximately 51000 daltons (Fig. 5, lane f) similar to an Endo H-treated CPY control (lane e). Mature CPY has the N-terminal amino acid sequence Lys-Ile-Lys-Asp-Pro-Lys (Martin et al. 1982). Amino acid sequencing of the CPY purified by affinity chromatography gave the N-terminal sequence Lys-IleLys-Asp. This showed that the two species have identical and correct N-terminals. In addition, the specific activity of the purified CPY was 126 U / r a g which is close to the value of 130 U / m g of the CPY standard,

Discussion

High expression of P R C 1 during the exponential growth phase due to strong yeast promoters ( A D H 1 and P H 0 5 promoters, Stevens et al. 1986; A D H 1 and T P I promoters, our unpublished observations) has not resuited in levels of CPY significantly higher than were obtained with the P R C 1 promoter. Similarly, only low levels of CPY were produced by strains containing P R C 1 under control of the G A L l promoter induced during exponential growth (Table 1). By repression of the G A L l promoter ,during exponential growth and induction when the growth rate decreases, we have achieved growth-phase-dependent overproduction of secreted CPY in a vpH strain (Fig. 3b). In an almost stationary phase, a vpll strain containing a 2 ~t-LEU2dG A L l p - P R C 1 plasmid reached a level of 42 mg/1 of active secreted CPY. This level represents a 200-fold overproduction as compared to a vpll strain with a single copy of the P C R 1 gene. Only small amounts of intracellular active CPY were measured. SDS-PAGE revealed no extracellular proCPY, indicating high capacity for extracellular processing of the proenzyme. This is somewhat in contrast to the slow processing of CPY secreted from vpl strains found by Rothman and Stevens (1986). Presumably, this difference reflects the fact that Rothman and Stevens (1986) used bovine serum albumin as carrier protein in the medium, thus inhibiting the protease-catalysed maturation of proCPY. High level production of CPY by a vpll strain resuited in two molecular weight forms of active extracellular CPY (Fig. 5, lane d) probably with different carbohydrate contents (lane f). The low molecular weight CPY species could be due to insufficient capacity o f the glycosylation system to process the large amounts of CPY traversing the secretory pathway in CPY over-

312 p r o d u c i n g y e a s t as p r o p o s e d f o r a h e t e r o l o g o u s p r o t e i n b y G r e e n et al. (1986). T h e y i e l d o f C P Y m i g h t b e f u r t h e r i n c r e a s e d . As d e s c r i b e d f o r p r o d u c t i o n o f a h e t e r o l o g o u s p r o t e i n in y e a s t b y S c h u l t z et al. (1987), i n c r e a s e d e x p r e s s i o n o f GAL4, e n c o d i n g a p o s i t i v e r e g u l a t o r o f G A L g e n e s , m i g h t r e s u l t in i n c r e a s e d e x p r e s s i o n o f P R C 1 u n d e r GALl promoter control. Acknowledgments. We are grateful to T. H. Stevens for stimulating discussions and for providing us with plasmid pTSY3 and yeast strain TSY10-7D, and to D. T. Moir, Collaborative Research, Inc., MA, USA, and C. P. Flollenberg, Institut far Mikrobiologie, Universit~it Dfisseldorf, FRG, for providing us with plasmids pCGS109 and pMP78, respectively. We thank I. Svendsen for performing the amino acid sequencing and K. Breddam for instruction in affinity chromatography purification. We are grateful to M. C. Kielland-Brandt and T. Nilsson-Tillgren for advice, fruitful discussions and critical reading of the manuscript. J. R. Winther is thanked for advice and stimulating discussions. Carlbiotech (Carlsberg Biotechnology), Copenhagen, and Center for Mikrobiologi, Lyngby, Denmark, are acknowledged for financial support.

References

Adams BG (1972) Induction of galactokinase in Saccharomyces cerevisiae: kinetics of induction and glucose effects. J Bacteriol 111:308-315 Bankaitis VA, Johnson LM, Emr SD (1986) Isolation of yeast mutants defective in protein targeting to the vacuole. Proc Natl Acad Sci USA 83:9075-9079 Distel B, A1 EJM, Tabak HF, Jones EW (1983) Synthesis and maturation of the yeast vacuolar enzymes carboxypeptidase Y and aminopeptidase U. Biochim Biophys Acta 741:128-135 Erhart E, Hollenberg CP (1983) The presence of a defective LEU2 gene on 2 Ix DNA recombinant plasmids of Saccharomyces cerevisiae is responsible for curing and high copy number. J Bacteriol 156:625-635 Goff CG, Moir DT, Kohno T, Gravious TC, Smith RA, Yamasaki E, Taunton-Rigby A (1984) Expression of calf prochymosin in Saccharomyces cerevisiae. Gene 27:35-46 Green R, Schaber MD, Shields D, Kramer R (1986) Secretion of somatostatin by Saccharomyces cerevisiae. Correct processing in an a-factor-somatostatin hybrid. J Biol Chem 261:75587565 Hanahan D (1985) Techniques for transformation of E. coli. In: Glover DM (ed) DNA cloning I. A practical approach. IRL Press, Qxford, pp 109-114 Hasilik A, Tanner W (1978) Biosynthesis of the vacuolar yeast gly-

coprotein carboxypeptidase Y. Conversion of the precursor into the enzyme. Eur J Biochem 85:599-608 Ito H, Fukuda Y, Murata K, Kimura A (1983) Transformation of intact yeast cells treated with alkali cations. J Bacteriol 153:153-168 Johansen JT, Breddam K, Ottesen M (1976) Isolation of carboxypeptidase Y by affinity chromatography. Carlsberg Res Commun 41:1-14 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York Martin BM, Svendsen I, Viswanatha T, Johansen JT (1982) Amino acid sequence of carboxypeptidase Y. I. Peptides from cleavage with cyanogen bromide. Carlsberg Res Commun 47:1-13 Petersen JGL, Kielland-Brandt MC, Holmberg S, NilssonTillgren T (1983) Mutational analysis of isoleucine-valine biosynthesis in Saceharomyces eerevisiae. Mapping of ilv2 and ilv5. Carlsberg Res Commun 48:21-34 Petersen JGL, Kielland-Brandt MC, Nilsson-Tillgren T, Born~es C, Holmberg S (1988) Molecular genetics of serine and threonine catabolism in Saccharomyees eerevisiae. Genetics 119: 527534 Rothman JI-I, Stevens TH (1986) Protein sorting in yeast: mutants defective in vacuole biogenesis mislocate vacuolar proteins into the late secretory pathway. Cell 47:1041-1051 Schultz LD, Hofman KJ, Mylin LM, Montgomery DL, Ellis RW, Hopper JE (1987) Regulated overproduction of the GAL4 gene product greatly increases expression from galactose-inducible promoters on multicopy expression vectors in yeast. Gene 61 : 123-133 Stevens T, Esmon B, Schekman R (1982) Early stages in the yeast secretory pathway are required for transport of carboxypeptidase Y to the vacuole. Cell 30:439-448 Stevens TH, Rothman JH, Payne GS, Schekman R (1986) Gene dosage-dependent secretion of yeast vacuolar carboxypeptidase Y. J Cell Biol 102:1551-1557 St. John TP, Davis RW (1981) The organization and transcription of the galactose gene cluster of Saccharomyces. J Mol Biol 152:285-315 Vails LA, Hunter CP, Rothman JH, Stevens TH (1987) Protein sorting in yeast: the localization determinant of yeast vacuolar carboxypeptidase Y resides in the propeptide. Cell 48:887897 Widmer F, Johansen JT (1979) Enzymatic peptide synthesis. Carboxypeptidase Y catalyzed formation of peptide bonds. Carlsberg Res Commun 44:37-46 Winston F, Chumley F, Fink GR (1983) Eviction and transplacemerit of mutant genes in yeast. Methods Enzymol 101:211228 Wolf DH, Weiser U (1977) Studies on a carboxypeptidase Y mutant of yeast and evidence for a second carboxypeptidase activity. Eur J Biochem 73: 553-556