Copyright 0 1990 by the Genetics Society of America

Isolation and Characterization of PEPS, a Gene Essential for Vacuolar Biogenesis in Saccharomyces cerevisiae

Carol A. Woolford,* Colleen K. Dixon,* Morris F. Manolson,* Robin Wright? and Elizabeth W. Jones*

*Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, and ?Department ofMolecular and Cellular Biology, University of Calgornia, Berkeley, Calgornia 94720

Manuscript received May 3, 1989 Accepted for publication April 30, 1990

ABSTRACT pep5 mutants of Saccharomyces cerevisiae accumulate inactive precursors to the vacuolar hydrolases.

The PEP5 gene was isolated from a genomic DNA library by complementation of the pep5-8 mutation. Deletion analysis localized the complementing activity to a 3.3-kb DNA fragment. DNA sequence analysis of the PEP5 gene revealed an open reading frame of 1029 codons with a calculated molecular mass for the encoded protein of 117,403 D. Deletion/disruption of the PEP5 gene did not kill the cells. The resulting strains grow very slowly at 37 O . The disruption mutant showed greatly decreased activities of all vacuolar hydrolases examined, including PrA, PrB, CpY, and the repressible alkaline phosphatase. Apparently normal precursor forms of the proteases accumulated in pep5 mutants, as did novel forms of PrB antigen. Antibodies raised to a fusion protein that contained almost half of the PEP5 open reading frame allowed detection by immunoblot of a protein of relative molecular mass 107 kD in extracts prepared from wild-type cells. Cell fractionation showed the PEP5 gene product is enriched in the vacuolar fraction and appears to be a peripheral vacuolar membrane protein.

T HE vacuole of the yeast Saccharomyces cerevisiae contains a number of the major hydrolases of

the cell, including protease A (PrA), protease B (PrB), carboxypeptidase Y (CpY), the repressible alkaline phosphatase, the large aminopeptidase and at least one RNase (WIEMKEN, SCHELLENBERG and URECH 1979). Most, if not all, of these hydrolases are synthe- sized as inactive precursors [see JONES (1984) for review]. Like secreted proteins, the precursors to vac- uolar hydrolases pass through the endoplasmic retic- ulum and Golgi complex. They are then sorted from secretory proteins and directed to the vacuole (STE- VENS, ESMON and SCHEKMAN 1982). The precursors are thought to be processed to their active mature forms in the vacuole (WOOLFORD et al. 1986; AM- MERER et al. 1986).

Many of the mutants initially isolated as being de- ficient in CpY activity proved to contain pleiotropic mutations that affected several vacuolar hydrolases (JONES 1977). A total of 16 genes were identified in this screen. The pep5 mutation resulted in reduced levels of PrA, PrB and CpY and in a number of other phenotypic changes as well (JONES 1977;JoN~S 1983).

We describe here the isolation and analysis of the PEP5 gene and its product. The DNA sequence en- codes an open reading frame of 1029 codons. Anti- bodies raised to a P E P 5 fusion protein detect a poly- peptide of 107 kD in yeast extracts. Although normal vacuolar contents are greatly altered and the vacuole

Genetics 125: 739-752 (August, 1990)

morphology is significantly different in a pep5-8 mu- tant, disruption of the P E P 5 gene is not a lethal event. Cell fractionation shows that the PEP5 gene product is enriched in vacuolar membranes.

Recently D U L I ~ and RIEZMAN (1 989) have reported the cloning and characterization of the E N D l gene of S. cerevisiae. DNA sequence comparison has shown that PEP5 and E N D l are the same gene.

MATERIALS AND METHODS

Materials: Exonuclease I11 and mung bean nuclease were purchased from New England Biolabs. Restriction enzymes, T4 DNA ligase, and the Klenow enzyme were purchased from New England Biolabs, Bethesda Research Laborato- ries, or Boehringer Mannheim Biochemicals. Protease A, DNase I , 3P-indoleacrylic acid and lyticase L8137 were purchased from Sigma Chemical Co.; [a-"P]dCTP and Hy- bond-map (for purifying poly(A)+ RNA) were purchased from Amersham; ["S]dATP and Random Primer Extension Kit were purchased from New England Nuclear; nitrocel- lulose type HAHY (0.45 pm) was obtained from Millipore; Nytran (0.45 pm), DEAE-nitrocellulose (NA45) and nitro- cellulose BA85 were purchased from Schleicher and Schuell; Sequenase DNA Sequencing Kit and GeneScribe-Z vectors were purchased from United States Biochemicals. A Vectastain ABC kit was obtained from Vector Laboratories, and a goat anti-rabbit IgG-horseradish peroxidase conjugate was obtained from Bio-Rad.

Media: YEPD (JONES, ZUBENKO and PARKER 1982) and synthetic media (ZUBENKO, PARK and JONES 1982) were prepared for yeast cultures; LB and M9CA media (MANIA- TIS, FRITSCH and SAMBROOK 1982) were prepared for bac- terial cultures.

740 C. A. Woolford et al.

Strains: All yeast strains in our laboratory were derived from strain X2180-1B (MATa gal2 SUCZ) or from crosses between the strains in our isogenic series and strains con- genic to strain X2180-1B obtained from D. BOTSTEIN, ex- cept for the recipient strain used to generate an insertion of the TKPl gene into the PEP5 gene. BJ4075 (MATa his3- A200 trpl-Alol) and BJ4112 (MATa trpl-A101 leu2-AI), derivatives of X2 180 obtained from P. HIETER, were mated and a diploid was selected as the parent strain for this disruption. The relevant genotypes of other strains are as follows: BJ926 MATalMATa trpl/+ hisll+ prcl-l26/prcl- 126 pep4-3/pep4-3 prbl-l122/prbI-l122 canllcanl gal21 gal2; BJ2760 MATa pep5-8 trpl leu2 ura3-52; BJ3607 MATa pep5-8 leu2 ura3-52; 85922 and BJ1088 MATa trpl pep5-8 (different sources of the same isolate); BJ492 and BJ1983 MATa trpl (different sources of the same isolate); BJ4334 MATaIMATa his3-A200/+ trpl-Al0l~trpI-Al01 +lleu2-AI; BJ 1984 MATa trpl pep4-3; BJ 1824 MATa trpl leu2 ura3-52 pep4-3; BJ3044 MATa lys2-801 ura3-52 can1 prblAl.6R; and BJ3131 MATaIMATa ura3-52/ura3-52 &PI/+ leu21 leu2 +lade6 +/hisl. Strains BJ4339-BJ4350 and BJ4393- BJ4396 are tetrads resulting from the sporulation of BJ4334 after a single copy of the PEP5 locus was disrupted with the TKPl gene. Strains BJ5323-BJ5330 and BJ5335-BJ5346 are tetrads resulting from the sporulation of BJ3131 after a single copy of the PEP5 locus was deleted and URA3 was inserted at that site. Strain BJ4021 is strain BJ3607 trans- formed with pSS5 (see Plasmids below). Strain BJ4019 is strain BJ3607 transformed with YEp24.

Bacterial strains HBlOl (MANIATIS, FRITSCH and SAM- BROOK 1982), RR1 (MANIATIS, FRITSCH and SAMBROOK 1982) and JM 101 (YANISCH-PERRON, VIEIRA and MESSING 1985) were used to propagate plasmids.

Plasmids: The parental plasmids used were YEp24 (BOT- STEIN et al. 1979), YCp50 (Kuo and CAMPBELL 1983) and Yip5 (STRUHL et al. 1979). The original complementing plasmid pCW5A1 is a derivative of YCp50. Two deletion derivatives of pCW5A1 were constructed: pANr and pAEc. These plasmids were made by cleavage of pCW5A1 with NruI or EcoRI, respectively, followed by ligation. The BglII- NruI fragment of pCW5A1 was subcloned into the BamHI and NruI sites in YCp50 to give pCN5. The SphI fragment of pCW5A1 was inserted into the Sphl site of YEp24 to yield pSS5. Yi5A1 was constructed by subcloning the ClaI- EagI fragment of pCW5Al (the entire insert) into the ClaI and EagI sites of YIp5. Clal and EagI cut in the vector sequences of pCW5Al on either side of the BamHI site of insertion. The disruption plasmid ppep5::TRPI was con- structed by first subcloning the SphI fragment from pCW5A1 into pBR322, and then introducing the EcoRI fragment bearing the TRPl gene from pCEN3-TRPI (FUTCHER and CARBON 1986) into the EcoRI site of the PEP5 bearing fragment. The disruption plasmid ppep5A::URA3 was constructed by oligonucleotide directed mutagenesis as described by BRIZUELA, DRAETTA and BEACH (1 987). A single stranded template carrying the SphI-NruI fragment encoding PEP5 was hybridized to a 40-mer DNA primer. The primer sequence was that of the PEP5 gene from nucleotides 34-53 and 2898-2917 and created a HindIII site where the two parts of the primer joined. After the deletion plasmid was isolated, a DNA fragment carrying the UKA3 gene was inserted into the new HindIII site. This plasmid carries a deletion of the PEPS gene corresponding to amino acids 18-966.

Nucleic acid preparation: Bacterial plasmid DNA was purified from Brij-deoxycholate treated spheroplasts by ce- sium chloride-ethidium bromide density gradient centrifu- gation (PETES et al. 1978). DNA minipreparations were

made by the alkaline lysis method of BIRNBOIM and DOLY (1 979). Yeast genomic DNA was prepared from spheroplasts (DAVIS et al. 1980) either as described by LAST, STAVEN- HAGEN and WOOLFORD (1984) or by the method of HOLM et al. (1986). Yeast RNA was prepared from cells harvested after growth at 30' in YEPD medium to an Asoo of 1-2, as described previously (HEREFORD and ROSBASH 1977; KIRBY 1965; LARKIN 1985). Poly(A)+ RNA was isolated using Hybond-map from Amersham as directed by the manufac- turer. DNA was labeled in vitro to >I X 10' cpmlpg with [a-3'P]dCTP using the Random Primer Extension Kit ac- cording to the manufacturer's instructions. The techniques used for preparation and analysis of DNA fragments on agarose gels and most of the general procedures used have been described previously (MANIATIS, FRITSCH and SAM- BROOK 1982). DNA to be used as hybridization probes was isolated from agarose gels using NA45 DEAE membrane according to the directions of the manufacturer. Single- stranded DNA templates were generated using GeneScribe- Z vectors according to the manufacturer's protocol.

Electrophoresis, transfer, and hybridization of nucleic acids: Electrophoresis of DNA on 0.8% agarose gels and of RNA on 1.2% agarose-formaldehyde gels, transfer to nitro- cellulose or Nytran filters, and hybridization to in vitro labeled DNA probes were performed as described elsewhere (WOOLFORD et al. 1986).

DNA sequencing: Subclones of the PEP5 gene region were constructed and single-stranded templates were gen- erated. Sequencing reactions were performed by the di- deoxy chain-termination method (SANGER, NICKLEN and COULSON 1977) using a Sequenase Kit as directed by the manufacturer. Areas with G-C compressions were resolved using dITP (MILLS and KRAMER 1979). The open reading frame region was sequenced completely on both strands. NBRF-PIR, SWISS-PROT, and the translated GenBank data bases were searched using the FASTA program of PEARSON and LIPMAN (1988). Hydropathy predictions were determined by the method of KYTE and DOOLITTLE (1 982). Protein secondary structure predictions were determined by the method of CHOU and FASMAN (1 974) using a program written by WILLIAM E. BROWN, Carnegie Mellon University.

Preparation of yeast extracts: Extracts were prepared by a Braun homogenizer as described previously (JONES, ZU- BENKO and PARKER 1982) except that 1 X 10" cells were pelleted and resuspended in 2 ml of 0.1 M Tris-HC1 buffer, pH 7.6, and the extracts were clarified by centrifugation at 27,000 X g. Alternatively, extracts were prepared by using detergents. 3 X lo8 cells were harvested and washed once with 2 ml of PBS extract buffer (20 mM sodium phosphate, pH 7.4, 0.15 M NaCI). T o the cell pellets 0.4 g glass beads (0.44-0.46 mm diameter) and 50 pl of 1 % SDS were added. The protein was extracted by 90 sec of vortexing followed by 3 min of boiling. The extracts were briefly chilled in an ice bath and 450 pl of 1% Triton X-100 was added. The cell debris was removed by centrifugation at 27,000 X g for 20 min.

Induction and purification of fusion proteins: Plasmid constructions were maintained in Escherichia coli RR1. The EcoRI-XbaI fragment of pAEc was inserted into the pATH3 vector (T. KOERNER, unpublished communication) in order to fuse the TrpE protein to the open reading frame of the PEPS-encoded protein. Induction with 3P-indoleacrylic acid (IAA), harvesting, and initial purification of TrpE fusion protein was by the method of T. J. KOERNER and A. M. MYERS (personal communication) and as described in MOEHLE, DIXON and JONES (1989). Insoluble protein was solubilized by boiling in SDS and then size-fractionated on preparative SDS-polyacrylamide gels (LAEMMLI 1970). The

pCW5A 1

S. cereuisiae PEP5 Gene 74 1

R e G L G D R S T R D X N H TG 8 s N n n

+ G A A H

I OW

pANr N L I

na pA Ec

N h + FIGURE 1 .-Restriction maps and

conlplementation results for plasmid R I - ~ C W S A I and its derivatives. Kestric-

tion sites are indicated (A, Aval; G ,

S Xbal). Thin lines denote genomic se-

S quences; wide bars denote vector se- + auences. T h e extent of the PEP^

ppep5::TRPI s S open reading frame is indicated by an open arrow. See MATERIALS AND METHODS for details o f construction.

- I

ppep5A::URAd S N

, 1 K b , gels were briefly stained in Coomassie brilliant blue R and then briefly destained in methano1:water:acetic acid 2:7: 1 . The fusion protein was excised from the gel; the gel slice was equilibrated against 24 mM Tris, 192 mM glycine, 0.1 % SDS; and the protein was electroeluted using a Schleicher and Schuell Elutrap.

Antibody production and purification: The eluted fu- sion protein was used to immunize rabbits under contract with Bethyl Laboratories (Montgomery, Texas). All injec- tions were subcutaneous with Freund's complete adjuvant, and each injection contained approximately 75 pg of pro- tein. Rabbit serum that reacted with PEP5-encoded protein from yeast extracts was pooled and (NH&S04 was added to a final 50% saturation. The 50% (NH4)2S04 pellet was resuspended in and was dialyzed extensively against 20 mM Tris pH 7.2. After dialysis, insoluble material was removed by centrifugation at 12,000 X g for 20 min. A small piece of nitrocellulose (0.5 X 1.5 cm) was incubated in 250 pl of electroeluted fusion protein at approximately 300 pg/ml in a microfuge tube for 6 hr at 4" with shaking. The strip was transferred to 1 ml of serum and incubated overnight at 4" on a rocker. After washing the filter four times in 20 ml of 20 mM Tris pH 7.2, it was transferred to a 1.5-ml microfuge tube and bound antibodies were eluted using 500 pl of 0.2 M glycine pH 2.3 for 1 hr at 4". The solution was neutralized by addition of 15% v/v 1 M Tris. The purified antibody was used at a dilution of 1:lOO for immunoblots. Antibody to CpY was from T. STEVENS (STEVENS, ESMON and SCHEKMAN 1982). Antibody to PrA was obtained by immunizing rabbits with PrA that had been treated with endoglycosidase H.

The serum was affinity purified against a column containing PrA linked to CNBr-activated Sepharose 4B (WILCHEK et al. 1971). Antibody to PrB was obtained from C. MOEHLE (MOEHLE, DIXON and JONES 1989).

Immunoblots: Yeast cell extract proteins were separated by subjecting them to polyacrylamide gel electrophoresis (PAGE) (LAEMMLI 1970). Typically, 7.5% acrylamide gels were used for assessing CpY and PEPS-encoded protein, and 10% gels were used for PrA and PrB. Immunoblots were made as described (BURNETTE 1981). The proteins were transferred to nitrocellulose in 24 mM Tris, 192 mM glycine, 20% methanol. The extent of protein transfer to nitrocellulose was assessed by staining with Ponceau S and destaining with water. Immune complexes were visualized by staining using a Vectastain ABC kit according to the manufacturer's instructions, except that the secondary an- tibody was diluted by an additional factor of 10, or using a goat anti-rabbit IgG-horseradish peroxidase conjugate man- ufactured by Bio-Rad.

Purification of vacuolar membranes: Vacuolar mem- branes were prepared by osmotic lysis of spheroplasts fol- lowed by flotation of intact vacuoles on Ficoll gradients. The method was essentially as described by UCHIDA, OHS- UMI and ANRAKU ( 1 985) with the modifications of YOSHIH- ISA, OHSUMI and ANRAKU ( 1 988) and with additional slight variations. Cells were harvested at an Asao of 5-6 and washed with 50 mM glycine-NaOH pH 10, 2 mM dithiothreitol prior to spheroplasting with lyticase (4000 units/lOX cells) for 2 hr in 1.2 M sorbitol, 10 mM Tris-HCI pH 7.5, 2 mM dithio- threitol. Spheroplasts were washed twice with 1.2 M sorbitol

742 C. A. Woolford et al.

PrA .. , -

1

1 2 3 4 1 2 3 4

I : ~ c : r x ~ . : L).-\lature size 1'1-12 ; I I ~ CpY antigens are restored bv l)(:\V3:\ 1. (:ell rstr;tcts from stwins 13J 1983 (lanes 1 , Pep'); Bj2760 t w m f b l m w l w i t 1 1 p(;\V.5/\ 1 (hnrs 2, p e p 5 - 8 Pep' tr;lnsfornl;lnt); l%,JSiCiO (laws 3 , p ~ p 5 - 8 ) ; I I ~ QJ I824 (lanes 4, p r p 4 - 3 ) were prc- pal'cd I)! t l l c . tlctcrgrnt I l l r l l l o d (see \ ~ A ' I ' E R I A I S A N D METHODS) and w r c s~~l!jrc-tc*tl t o SDS I'A(;E (30 pg protein/hne). Proteins were t r a d i w w l t o nirrorc~llulosc. and prol)cd with P r h o r CpY antihod- ics ; I I I ~ thr imnllll~e rornplcxes \yere detected using :I \'ectastain / \ I%(: K i t .

and resuspended i n 10 volumes of 12% Ficoll, 10 mM MES- Tris pH 6.9, 0.1 I ~ M MgCI? (buffer A), homogenized 10 times with a loose-fitting Dounce homogenizer, and sub- jected to a low speed spin at 4500 X g for 10 min to remove intact cells, mitochondria and nuclei. The low speed spin supernatant was recentrifuged at 70,000 X g for 50 min and vacuoles were collected from the top of the tube. Vacuoles were floated up through a Ficoll gradient by resuspending i n buffer A, overlaying with 2 volumes of 7% Ficoll, 10 mM MES-Tris pH 6.9. 0.5 mM MgCl? and then centrifuging at 70,000 X g for 50 min. To release vacuolar content, the vacuolar float was homogenized 10 times with a tight fitting Dounce homogenizer in 10% sucrose, 10 mM MES-Tris pH 6.9, 5 mM MgCI,, 2 mM phenylmethylsulfonyl fluoride (PMSF) (30 ml/mg protein) and centrifuged at 100,000 X g for 45 min, pelleting the vacuolar membranes.

Purity of the vacuolar membranes was determined by the sensitivity of ATPase activity to 1 nmol/mg protein bafilo- rnycin (vacuolar ATPase) (BOWMAN, SIERERS and ALTEN- DORF 1988). 1 mM azide (mitochondrial ATPase) and 100 mM vanadate (plasma membrane ATPase) (reviewed in BOWMAN and BOWMAN 1986). ATPase assays were per- formed as described by UCHIDA, OHSUMI and ANRAKU ( 1 985) with released inorganic phosphate determined by the method of AMES (1 966). The vacuolar membranes were in general essentiallv free of mitochondria with less than 10% plasma membrane contamination (data not shown).

Sodium carbonate extraction of vacuolar proteins: Frac- tionation ofperipherally bound proteins from integral mem- brane proteins was done by the method of FUJIKI et al. ( 1 982). Approximately 4 mg of vacuolar membranes were resuspended in 12.5 ml ice-cold I00 mM Na2C03 pH 1 1, 2 ITIM I'MSF, 1 n m EDTA and homogenized 10 times with a tight-fitting Dounce homogenizer. Following a 30-min in- cubation on ice, membranes were spun down at 100,000 x g for 45 min. The supernatant containing the peripherally bound proteins was brought to 14% trichloroacetic acid (TCA), incubated on ice for 5 min and spun at 12,000 X g for 10 min to collect the precipitated proteins. TCA was removed from the pellet by two washes in ice cold ethyl ether followed by lyophilization to remove the organic sol- vent. Approximately .io% of the vacuolar membrane pro- tein as extracted from the membrane by the Na2C03 treatment.

Protein content of cell fractions was determined after solubilizing the samples i n SDS-PAGE denaturation buffer and assaying by a modified Lowry procedure as described in BENNETT ( 1 982). The vacuolar membranes and sodium

carbonate insoluble samples were solubilized in denatura- tion buffer on ice overnight (SARAFIAN and POOLE 1989). All other samples were denatured by boiling for 4 min.

Transformation: Bacteria were transformed by using the CaCI? protocol (MANIATIS, FRITSCH and SAMBROOK 1982). Yeast cells were transformed by the CaCI2 method of BRUS- CHI, COMER and HOWE (1987) or by a modification of the lithium acetate method of ITO et al. (1983). Cells were harvested and washed in 100 mM lithium acetate in T E (0.01 M Tris, 0.001 M EDTA, pH 7.5). Prior to DNA addition, the cells were incubated at 30" for 30 min in lithium acetate/TE. After DNA addition cells were incu- bated 1 hr at 30" and then 30 min at 37". Cells were pelleted and resuspended in SOS (1 M sorbitol/33% YEPD/ 0.0 125 M CaCI2), before plating on selective plates.

Genetic methods: The procedures used for routine spor- ulation, dissection, and scoring of nutritional markers have been described previously (HAWTHORNE and MORTIMER 1960). The pep5 marker was scored on the basis of an inability to catalyze cleavage of acetylphenylalanine B- naphthyl ester (APE) in agar overlays (JONES 1977) and the inability to catalyze cleavage of benzoyltyrosine-p-nitroani- lide (WOOLFORD et al. 1986). Growth at 37" was tested in three ways: 1) replica plating of cells from a master, 2) spotting a given number of cells, and 3) streaking for isolated colonies. Plates were made in duplicate. One set was placed in a plastic Petri dish bag that was then closed but not made air tight.

Native gel electrophoresis: For the visualization of re- pressible alkaline phosphatase activity, samples of extracts were subjected to electrophoresis in polyacrylamide gels, incubated in the substrate a-naphthylphosphate, and stained using fast black K diazonium salt, as described in JONES, ZURENKO and PARKER ( 1 982).

Electron microscopy: Aliquots of 50 ml YEPD liquid medium were inoculated to an AGO" of 0.4 and grown at 30" for 2.5 hr. The culture was then rapidly mixed with 10 ml 5 x fixative (20% glutaraldehyde, 0.5 M sodium cacodylate, 10 mM CaCI2, 50 mM MgC12, pH 6.9). After incubation at room temperature for 5 min, the cells were pelleted by centrifugation (3000 rpm for 5 min, Sorval GSA rotor) and resuspended in 5 ml 1 X fixative. Fixation was then allowed to proceed for 1 hr at 4". The fixed cells were washed six times with buffer alone ( 1 00 mM sodium cacodylate, 2 mM CaCI2, 5 mM MgCI?, pH 6.9) and stored in buffer overnight at 4". The fixed cells were then pelleted, resuspended in 2 ml buffer, and mixed with 2 ml of freshly prepared 4% aqueous potassium permanganate. After 5 min at room temperature, the cells were gently pelleted by centrifugation and incubated as a pellet for 1 hr at room temperature to complete fixation. The cells were washed repeatedly with distilled water, dehydrated in a graded series of ethanol, and embedded in SPURR'S (1969) resin as described in WRIGHT and RINE ( 1 989). Silver-to-gold interference color sections were cut using a diamond knife. Sections were mounted on grids with formvar coated bars, stained for 30 sec in 1:lO Reynolds' lead citrate:0.1 M NaOH (REYNOLDS 1963), and observed using a Philips 300 electron microscope operated at 80 kV. Micrographs were taken on Kodak SO- 163 film, developed in HC-110 developer (Eastman Kodak Company).

RESULTS

Cloning the PEP5 gene: Plasmids capable of com- plementing the pep5-8 mutation were recovered from the YCp.50 bank (a gift from M. ROSE, J. THOMAS and

S. cerevisiae PEP5 Gene 743

TABLE 1

Yi5Al integrates at the PEP5 locus

No. of the following tetrad types"

Parental NOnpdrentdl Cross Genotypes Ura+:Ura- ditype ditype Tetratype

1) MATa pep5-8 ura3-52 Pep+Ura+ transformant X MATa pep54 ura3-52 2:2 32 0 0 2) MATa pep5-8 ura3-52 Pep+Ura+ X MATa PEP5 ura3-52 2:2 33 0 0

For cross 1 the tetrad types were as follows: parental ditype, 2 Ura+Pep+:2 Ura-Pep-; nonparental ditype, 2 Ura+Pep-:2 Ura-Pep+;

For cross 2 the tetrad types were as follows: parental ditype, 4 Pep+:O Pep-; nonparental ditype, 2 Pep+:2 Pep-; tetratype, 3 Pep+:l Pep-. tetratype, 1 Ura+Pep-: 1 Ura-Pep-: 1 Ura+Pep+: 1 Ura-Pep+.

TABLE 2

pep5 is linked to rnal

No. of the following tetrad types

Marker pair ditype ditype Tetratype cM Parental Nonparental

mal-pep5 48 0 6 5.6 mal-ade4 11 3 40 54

ade4-pep5 10 5 39 64

P. NOVICK). After transformation of BJ2760 by the CaCI2 procedure (BRUSCHI, COMER and HOWE 1987), approximately 32,000 Ura+ transformants were rep- lica plated onto YEPD and tested for CpY activity as a test for complementation of the pep5 mutation (JONES 1977). Six different transformants were con- sidered to be Pep+ because of their ability to catalyze cleavage of acetylphenylalanine &naphthyl ester and benzoyl tyrosine-p-nitroanilide (WOOLFORD et al. 1986). Plasmids capable of effecting complementation of the pep5-8 mutation, after being passaged through E. coli, were recovered from all six of the transform- ants. Restriction digest analysis indicated that the six plasmids contained the same genomic DNA, and only pCW5A1 has been further investigated. The restric- tion map for the plasmid is shown in Figure 1.

Mutations in the PEP5 gene are pleiotropic and lead to reduced levels of the vacuolar proteases PrA, PrB and CpY (JONES 1977). As can be seen in lanes 3 in Figure 2, the forms that accumulate in such mutants appear to correspond to the fully glycosylated post- Golgi precursors of PrA and CpY. The pCW5A1 plasmid contains a sequence that complements the processing defect in the pep5-8 mutant and restores production of mature forms of the proteases (lanes 2 of Figure 2). Comparable results are seen for PrB (data not shown, but see Figure 9A).

Genetic data proving that the inserted fragment in pCW5A1 originated from the PEP5 region of the chromosome are shown in Table 1. The entire ge- nomic DNA insert was removed by digestion with ClaI and EagI, both of which cut only in the vector on either side of and close to the original BamHI site. The fragment was cloned into the ClaI and EagI sites

of the integrating plasmid YIp5 (STRUHL et al. 1979). This plasmid, Yi5A 1, was then linearized at the XbaI site in the putative PEP5 gene region and was used to transform BJ3607 to Ura+ (HINNEN, HICKS and FINK 1978). The transformants were Pep+. Plasmid loss and DNA blot analysis (SOUTHERN 1975) showed that the transformants stably maintained their Ura+Pep+ phenotype and that the plasmid had integrated into the chromosomal site from which the sequence origi- nated. One such transformant, Yi8 (BJ3 116), was crossed to a strain having the genotype pep5-8 ura3- 52 and to another strain having the genotype PEP5 ura3-52. An analysis of the tetrads from the first cross indicated that sequences responsible for the Ura+ and Pep+ phenotypes were integrated and tightly linked: all 32 tetrads segregated 2:2 for and were parental ditype with respect to the two markers. The results obtained in the cross to the PEP5 parent indicated that the sequence responsible for the Pep+ phenotype was closely linked to the PEP5 locus, since no Pep- segregants were recovered in 33 tetrads.

In order to determine which portion of the insert carried the PEP5 gene, a number of deletion deriva- tives or subclones were constructed and tested for retention of the ability to complement the peps-8 mutation. As shown in Figure 1, pANr, a derivative of pCW5Al that lacks insert DNA to the right of the NruI site, retains the ability to complement. Plasmid pGN5, which contains the BgZII-NruI fragment, also complements the pep5-8 mutation. If all sequences to the left of the SphI site are deleted, the resulting plasmid pSS5 still complements. However this con- struct has only been made using a high copy number vector (YEp24).

The pep5-8 mutation was localized to chromosome XZIZ by OFAGE mapping (P. HIETER kindly provided a nitrocellulose blot). pep5-8 shows linkage to rnal (Table 2). The order is centromere-pep5-rnal-ade4, as determined by a three factor cross.

Sequence analysis: The DNA sequence of the PEP5 gene region was determined from the SphI site to beyond the NruI site. The sequence of the open reading frame region was determined from both strands (Figure 3). The region contains an open read-

744 C. A

1 ATGTCCC GA CTCCTGGAGGCAAT CCAGCT CG GAATATTCCCA AAGAGATCCT

6 1 ~TT'FTG~AGEAG6TT;"ECC~ATEGTBCT~GCgCC~AA?AC€TT~CG~AG~TA~AA~AGaA

1 2 1 GATCgTCAAACTCTCA CA AGCTGT TT AAATATTATAAAAGTTGTC D Q T L T T A V q E N I I K V V Y T T

1 8 1 CAATCGCAAGTAATACATGAATTTCAATCTTTTCCTCATGATT~CAAATCACTTTCCTG

241 YGaCATCABCGEGG$"T'TFTCECGBAG~GT€AG~TG~TECATTG~CYC~TT~CCEA Q Q V I H E F Q S F P H D F Q I T F L

301 A AACAGTAT TAAACTAG CTGCCAAATAGAGAACAACTAT CCATTCACAACTC

361 G CTGAAAAACGGT T TACATACCCTA TTCAGTTGTCTCCATATCAAATGACCTT

4 2 1 TCCTGTATTGTGGTTGGATTCATTAATGGGAAAATCATCCTTATTAGAGGTGACATTTCA

481 AGAG TAGAGGATCTCAACAAAGGATTATATATGAAGATCCAAGTAAAGAGCCAATAACA

M S E S S W R Q 8 Q L T k N I P T R D P

T R V B K L W L P N R E Q L Q H S Q V

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S C I V V G F I N G K I I L I R G D I S

R 8 R G S Q Q R I I Y E D P S K E P I T

Woolford et al.

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LBO 541 GfT~ATATaCC~CqTqCG$CG~AA%GG~TT~TTATTFTG~GG~TA~AA%TT~AAgAA~CC~T 2oo

6 0 1 T € A T A T T F T A A ~ T G E T A ~ ~ T A g A G ~ C C ~ C C ~ A A ~ T T € G G ~ T T Z ~ T T ~ ~ T 2 2 0

6 6 1 GGCCTGGATCTAAATTGTGCATCTTTTAATCCGGCAACAAACCAATTTATATGTTGCTTA G L D L N C C S F N P A T N E F I C C L 240

721 AGCAACTTTATCGAATTTTTCACCTCTACTGGAAAAAAGCATCAATTCGCATTTGATCTA S N F I E F F S S S G K K H Q P A F D L 260

7 8 1 TCGCTGAGAAAGAGGATATFTGTGTAGATAAAGATCACATTTTGATTGTCACTGACCAA S L R K R I C V D K D H I L I V T E E 280

8 4 1 ACAGGTGTACCAACTACATCCATAAGTGTTAATGAACTGTCGCCCACAATAATTAACCGA T G V P T T S I S V N E L S P T I I N R 300

9 0 1 ATATTCATCATTCATGCTATAAAATCATTTCTTTCAATTTTGTTGTTTCTACTCCA I F I I D A K N K I I S L N F V V S S A 320

9 6 1 ATTATTGATATTTTTTCAACCTCTCAGAGCGGTAAAAACATCACTTATCTGCTAACTTCT I I D I F S T S Q S G K - Y L L T S 340

1021 GAAGGAGTAATGC TA GA AACTCCAAAATCTTTAG TCAAATTAACATCATCATC E G V M f i g T T P K S L M Q I N I I I 360

1081 CAAAAGGAACTGTACCCTT TGCTTTACAAT AGCGAAACACCATTCATTATCACCGCTA Q K E L Y P J A L Q Z A K Q H S L S P L 380

1141 GATGTTCAGGAAATTCATAAAAAATATGGTGATTATCTCTTTAAAAAGGGACTCAGGAAC 4oo D V Q E I H K K Y G D Y L F K K G L R K

1201 GAAGCAACAGACCAGT CA ACAATGTCTGGACGTTGTGGAAACTAGTCAAATCATTTCA E A T D Q e T Q C L D V V E T S E I I S 420

1261 AAATTTGGTGTCAAGGAGGTTCCTCACCCAGAGAGTATGAGGAACTTGGCTCACTATCTG K F G V K * E V P D P E S M R N L A D Y L 440

1 3 2 1 TGGTCTT GA CAAG TT TATTTCTCAACCCG TCATGTTACCCTTTTGTTAATTGTT W S E T K % g I S Q R $ H V T L L L I V 460

1381 TTAATCAAATTGAAGGATGTTGAAGGAATTGATACTTTTATCCACCATTTCGATAGAAAA L I K L K D V E G I D T F I Q H F D R K 480

1441 GGCATTTGGAATGAAGGTGTGTGATGGACCATATGGATGATGTGACGTTTTTTTATTCA G I W N E G V V M D D M D D V T P F Y S 500

1501 GATAACGATT TTTTGATT AGATTTAATCTTAGAGCTTATGAAACACTCACATTTTAAG D N D T F D E D L I L E L M K E S D F K 520

1561 C~TCECTEATBCAPC€TG~AAAKG~GT~TT~~GG~~5AT€AATTA'fTG~GG~TAp

1621 T T A T T G A A T C T A T T G C A T A A C C C T G T A A A A G C C A T A A A A T T

1681 G g T G ~ ~ C C E ~ ~ A T E C C ~ T G ~ G A % G T B C T ~ G ~ ~ A C E G G k G G k G T E A C g G ~ T

1 7 4 1 GAAACTAATGCTTTAC GATAGAAGTA ACAGGGAAG C CCAT CA ET N A L I E V T T G K q Y P 5 ?TGp 1801 GTGG TCTGG CC GAGAGATACGACGGGCG

V 6 L fi 8 R D T T G 6"EfiETGflTAp"fFF 1861 TACAGTTACAAAACATTCTTCAACTACATGAACTCAAATGGTACATCAGACGCAATGAGC

1921 GAGTCTTCAG GGCATCCCACGMCATG G GCCTA TT TC TC GC GAAGCCAT C

1 9 8 1 A'fTGBTTJCASTTCATTCGTTAC S F V T " B " c ~ ~ G ~ ~ G G ~ c ~ A G ~ G ~ c T ~ T

2041 TTGGCAT TT CCAGCAGT TG GGG G CG G TA GCAAG AA L A 8 4 Q Q 4 #. G T 6 $" fi 8 Q 5 mG"GTAtT

2101 T T A T A T G A C T T A T A T T T G A A T T T G G C G C ~ T G A T G T A C A C G A C T G G L Y D L Y L N L A Q N D V P E R I D D W

2161 CGTTCAAGAGCAACTGGCGTATTCCGTGAGAGC TAAATTGGTGTATTCTGCCGCAAGT R S R A T C V L R E S q K L V Y S A A S

2221 AATAATACTAGCAAAAGGGTGGATAACTCAATAATGCTGTTAATTTCCCATATGGATCAA N N T S K R V D N S I M L L I S H M D Q

2281 ACTAGTGCTTCACCAAAACATAAGACGAAAATTGACATAGCTTCATTTCCCAATGATAAC S S A S A K D K T K I D I A S F A N D N

2341 CCCGAGATGGATTTGCTGAGTACATTTAGGCCTATGACGTT~TCAAGAACCAAGTACT P E M D L L S T F R A M T L N E E P S T

2401 TGTCTCAAATTCCTAGATATGCCACAGAGGAACCCAACCTCT ACAAGTAGCATTG C L K F L E K Y C T E E P K L E Q V A L

2461 AGTTACTTTGTTTCTAATAAACTAATCT CAAGCAGATGGGCCGTAATGAAGTACTGAAA S Y P V S N K L I ' T F K E M G G N E V L K

2521 GAAAnAGTATTGAGGCCAATTATAGAGGGGGAAAGAATGCCACTGTTGGATATAATTAAA E K V L R P I I E G E R M P L L D I I K

2581 GCGCTATCCCGTACAAATGTAGCCCACTTTGGGCTGATACAAGACATCA~ATTGATCAT A L S R T N V A H F G L I Q D I I I D H

2641 GTCAAAACCCAAGATACAGAAATCMGGAACGWC AATTCAATCTTACCATAAA V K T E D T E I K R N E K T I E S Y D K

2701 GACTTAAAGGAGAAAAACAAGAAGTTG GAACACCATTAATTCAGATCAACCTCTCCAC E L K E K N K K L % N T I N S D Q P L H

2761 GTACCCCTGAAG TCAAACGTGTTTCATGTGTAGACTGACATTGCATATTCCTGTAGTT V P L K q a C F M C R L T L D I P V * V

2821 TTTTTTAAATGTGGTCACATTTACCACCAACATTGTCTAAATGAGGAAGAAGATACTCTA F F K C G H I Y H Q H C L N E E E D T L

2881 CP$CG$GAgAA@GCTCTTThAATGTCCC TGCTTGGTGCACTTAGAAACCTCCAAC L F K C P Y C L V D L E T S N

L L N L L H N P V K A I K Y I K S L P I

Y S Y K T F F N Y H N S N G T S D A ~ S

E S S f i A S H E H F k P % B f i g S K P E

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980 2941 AAACTTTTTCAAGCTCAACACGAACTACTTGAAAAGAATGATCTTTTCAATTTTGCATTA

3001 AACAGTCAAGAAGGTAGTACAGACCGTTFCApGTCATAACAC GTTTTTAGGTAGAGGT

3061 GCCATCAGTTATTCTGACATCACTATTTAAtgatggatcataacgatctattgtcgccgc

K L F E A Q H E V V E K N D L L N F A L l O O O

N S E E C S R D R V I T k F L G R G 1 0 2 0

E S E Y S D I T I 1029

FIGURE 3.-Sequence of the PEP5 gene and its flanking regions. The DNA sequence is shown together with the predicted amino acid sequence of the PEPS-encoded protein. Underlined amino acids identify the positions of potential signals for asparagine-linked glycosylation (N-X-T or N-X-S) (STRUCK and LENNARZ 1980). Asterisks at nucleotides 1336 and 2878 identify the EcoRI and XbaI sites used in constructing the fusion polypeptide used for generating antibodies.

ing frame of 3087 bp that encodes a 1029 amino acid polypeptide. The calculated molecular mass of this polypeptide is 1 17,403 D. Extreme codon bias toward major isoacceptor tRNA species has been observed in several highly expressed genes of S . cerevisiae (SHARP, TUOHY and MOSURSKI 1986). An examination of co- don utilization for the PEP5 gene shows excellent agreement with the codon bias of lowly expressed yeast genes. There are no significant sequence simi- larities between that predicted for the PEPS-encoded protein and any other sequence in the NBRF-PIR, SWISS-PROT and translated GenBank data bases. However, the sequence is nearly identical to that recently reported for the END1 gene (DULI~ and RIEZMAN 1989).

There are nine potential Asn-linked glycosylation sites, two of the sequence N-X-S and seven of the sequence N-X-T (STRUCK and LENNARZ 1980). The overall net charge from 281 charged amino acids is - 1. Computer-generated predictions of the hydropa- thy (KYTE and DOOLITTLE 1982) reveal no obvious transmembrane or signal sequences. Secondary struc-

ture predictions (CHOU and FASMAN 1974) show short regions of a-helix and @-sheet throughout the open reading frame.

Transcript analysis: RNA blot analysis was used to determine the size of the transcript homologous to the PEP5 gene. Poly(A)+ RNA isolated from strains BJ1983 (PEP5) and BJ4342 (pep5::TRPl) were frac- tionated on an agarose-formaldehyde gel and trans- ferred to nitrocellulose. The blot was probed with the radioactively labeled, gel-purified 1.5-kb EcoRI-XbaI fragment from plasmid pAEc. Figure 4 shows the results of the experiment. The size of the transcript encoded by PEP5 is 3200 nucleotides, in good agree- ment with a gene having a 3087 nucleotide long open reading frame.

Antibodies to the PEPB-encoded protein: The EcoRI-XbaI fragment from the PEP5 region was fused to the TrpE gene in the plasmid pATH3. Expression of an 89-kD fusion protein was induced by 30-indole- acrylic acid. This polypeptide was gel purified and injected into rabbits. Antiserum from these rabbits was affinity purified against the original TrpE fusion

S. cerevisiae PEP5 Gene 745

1 2 3 4 k D

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FIGL~RF. 4.-RNA blot of polv(A)' RNA isolated from strains 13.119X3 ( P E P S ) and BJ4342 (p tp5 : :TXPl ) . Total RNA \vas pre- pared from log;Irithnlirally grnwn cells and poIy(A)+ R N A \+';IS then isol;lred ( I pg/Iane). The Mot was prol,ed rvith the in vitro I;lbeletl I:'roRI-ShnI fragt~~ent t h a t is internal to PI7'5.

protein, and then used for immunoblot experiments to visualize the PEPS-encoded protein in yeast ex- tracts. Initially we only detected the probable PEPS- encoded antigen in cell-free extracts prepared from strain B.J402 1, which carries the PEP5 gene on a high copy number plasmid. By greatly increasing the amount of extract loaded on the gels we were able to detect the protein in wild type extracts. Figure 5 shows an immunoblot in which lanes 2 (PEPS) and 3 (pep5::TRPl) were loaded with 175 pg of protein and lanes 1 and 4 (PEP5 gene borne by YEp24) were loaded with 100 pg of protein.

A protein of relative molecular mass 107 k D was detected in wild-type extracts (lane 2), was present in large amounts in extracts from the strain bearing the PEPS gene in high copy (lanes 1 and 4); and was not detected in extracts from the strain bearing a pep5::TRPl insertion mutation (lane 3). In addition, more of this polypeptide was seen when extracts were prepared from plasmid-bearing cells grown under conditions selective for retention of the plasmid (lane 4) rather than nonselectively (lane 1). The 107-kD polypeptide was detected in extracts from log-phase cells and was never seen in extracts from stationary phase cells. It could be detected in extracts prepared in the Braun homogenizer but not in those prepared by an SDS extraction protocol.

- 116 4- - 93 - 66

IIG(!RE 5.-Identification of the PEP5 gene product by immu- nohlot. Yeast cell extracts were m;de from strains B.1400 I ( p r p 5 - 8 tr;unsfnrntctl with 21 high ropy number plasmid carrying P E P S . lanes I and 3 . I O 0 pg/Iane), I31 19XJ ( P E P 5 , lane 2, 17.i pg/I;me), and BJ4342 ( p ~ p 5 ; ; T R P l . lane 5 , 175 pg/Iane). <:ells were grow1 in either selertivr n~ctliurn (lane 4) or rich nlcdium (lanes I -?) , har- vested a t :I tlrtlsity ;IrountI I O ' ceIIs/mI, and protein was estracted \vithout the use of detergents. After transfer to nitrocellulose. the I h t s \+.ere itlculxlted with affinity purified antibodies generated against the TrpE-PIP5 fusion plvpt*ptitle (see MATERIALS A N D

MKI'HOIX). In~mutw con~plrses ~ w r c detected using a \'ectastain A 1 3 C K i t .

Localization of Pep5p: The PEPS gene product was sought in subcellular fractions obtained from a protease deficient strain (BJ926). Cells were converted to spheroplasts, lysed, and the sample centrifuged through two Ficoll gradients. The PEPS-encoded pro- tein in each fraction was visualized by an immunoblot experiment (Figure 6). Samples of the initial homog- enate, the low speed spin pellet, the pellet from the first Ficoll gradient (which would include plasma membrane, lysed vacuoles and any endoplasmic retic- ulum, nuclei, mitochondria, or Golgi complex carried over from the low speed spin pellet), and the vacuolar float of the second Ficoll gradient are shown in Lanes 1, 2, 3 and 6. The purity of the vacuolar fraction had been previously determined by the sensitivity of the ATPase activity present to various inhibitors (see Ma- terials and Methods). Lane 5 contains yeast cell extract from a strain carrying the high copy number PEP5 plasmid, and Lane 4 is a strain carrying the plasmid vector YEp24. There is an enrichment for the 107,000-D species in the vacuolar fraction. T o deter- mine if the PEP5 gene product was soluble in the vacuole or membrane associated, the vacuolar con- tents were released by homogenization and the vacu- olar membranes were pelleted. The PEP5 gene prod- uct is membrane associated (lane 7). Sodium carbonate extraction of the vacuolar proteins was done to deter- mine if the PEP5 gene product was peripherally as- sociated with (lane 8) or was an integral component (lane 9) of the vacuolar membrane. Since the protein is solubilized by sodium carbonate treatment, it is most likely not an integral membrane protein, but rather is peripherally bound to the membrane. We do not believe that the band of higher molecular weight (see

746 C . A. Woolford et al.

FIGURE (~.-Enrichnwnt of the P I P 5 genc product i l l \ucuoI;w nwnhrxnes. Strain BJ926 \<IS grown, converted t o sphcropl;lsts, and osmotically lysed. I'urifiecl vacuolar n1enlbranes were isolated and tllcl1 tlutetl with sodiunl carbonate t o solubilize peripheral n w I n I ) 1 . ; I I w proteins. 1:r;lctions were probed with affini:y purified ;unril)otlics t o the Pf:'P5 gene product. h c h lane contains 'LO pg of protcin, tlrtcrnlinctl a f ~ e r soluhilimtion o f t h e sample in denatura- tion l)uffer. I.;lne I : hornogcnate of lysed cells; lane 2: low speed spin pellct; lane 3: pellet from the first I;icoll gradient; lane 4: cell estract ol'a str;lin with ;I single copy of P I Y 5 (BJ4019); lane 5: cell estract of ;I strain carrying PEP5 o n ;I high C O ~ V number plasmid (13,1402l); h n c 6: K I C I I O I ~ ~ Iloat of the second I;icoll gradient; lane 7: vacuolar nIcInl~I.;~ncs; lane X: sodium carbonate soluble proteins; ; ~ n d lane 9: sodium c;lrl,on;lte-insoluble proteins.

lanes 1-6) represents a protein related to the PEP5 gene product because it is present at the same amount whether PEP5 is in single copy or is overexpressed by a multicopy plasmid (compare lanes 4 and 5).

Electron microscopy: Electron microscopy shows the vacuole of a Pep+ strain as consisting of a number of densely stained bodies (Figure 7B). pep5 mutant cells do contain densely staining material, but its mor- phology is quite aberrant compared to wild type vac- uoles (Figure 7A). We are not certain if these very small densely staining bodies in pep5 cells are vestigial vacuoles or are, in fact, a new structure that has arisen.

Disruption of the PEP5 gene: In order to deter- mine if PEP5 function is essential for growth, we constructed a disruption mutation by inserting a 1.5 kb fragment carrying the T R P l gene into the EcoRI site of the coding region of PEP5. This plasmid, ppep5::TRPI (see Figure 1) was digested with SphI and was then used to transform the diploid BJ4334 to Trp+. A Trp+ diploid was sporulated and dissected. Each ascus gave four surviving spores. All of the 18 asci showed segregations of 2 Pep+Trp-:2 Pep-Trp+. Further proof that we had constructed the insertion is given in Figure 8. Genomic DNA of the parent diploid and four haploid spore clones derived from one tetrad was digested with XbaI, blotted to nytran, and probed with the 6.3-kb SphI fragment. The wild type haploids and diploid possess an approximately 13 kb fragment and a 5 kb fragment. The pep5::TRPI disrupted spore clones lack the 13-kb fragment but possess a smaller 1 1.3-kb fragment and an additional 1.7-kb fragment, due to the XbaI site in the TRPZ gene. These results indicate that we had indeed dis-

FIGUKF. 7.--Elcctrotl micrographs of yc;lst cells stained for vac- uoles. Yeast cells were fixed. thin-sectioned and the mounts were stained with Ie;ltl citrate to visualize the wcuoles a s dense staining 1)otlies. A, p ~ p 5 - 8 . (BJ922); B, PI:'P5, (BJ492).

rupted one of the two copies of the PEP5 gene present in the diploid.

A deletion/disruption mutation, pep5A::URA3, in which a 1.1-kb fragment carrying the URA3 gene replaces deleted nucleotides 54-2897 of the open reading frame was constructed. The plasmid bearing the mutation, ppep5A::URA3 (see Figure I) , was di- gested with SphI and EcoRI and the DNA was used to transform the diploid BJ3 131 to Ura+. Two Ura+ diploids were sporulated and dissected. As with the previous disruption, each ascus gave four surviving spores. All 31 asci from one diploid and the 46 asci from the second diploid showed segregations of 2Pep+Ura-:2Pep-Ura+. Genomic DNA from the spores derived from two tetrads and the parental diploid was digested with SstI. Blotting of the DNA and hybridization to the 6.3-kb SphI fragment con- firmed the construction. The wild-type haploids and diploid possess a 5-kb fragment (encoding the PEP5 ORF) and a 2.5-kb fragment representing down- stream homology to the probe. The pep5A::URA3 disrupted spore clones lack the 5-kb fragment but possess a 3.2-kb fragment instead, in agreement with the length of DNA deleted and inserted at the PEP5 locus (data not shown). Because all segregants bearing the disruption or deletion/disruption mutation are viable and Pep-, we conclude that PEP5 function is not essential for growth.

Pleiotropic effects of the pep5 disruption muta- tion: The pep5::TRPI disruption mutation profoundly depresses production of mature species of PrA, PrB and CpY (Figure 9A). The three defects cosegregate in tetrads. A representative tetrad is shown in Figure 9A. For CpY, substantial levels of an antigen that corresponds in size to that of the normal, larger (p2) precursor is present (compare to lane 6 in which p2 that accumulates in a pep4 mutant is seen). For PrB, some antigen is present that is comparable in size to the normal, 40-kD intermediate (see lane 6) as is a small amount of antigen of mature size. Two addi- tional bands are present; both are larger than the 37 kD intermediate previously described (MOEHLE, DIXON and JONES 1989), being about 38 and 39 kD in size. Plate tests reveal little or no activity for either PrB or CpY. For PrA some mature sized antigen is

S. cerevisiae PEP5 Gene 747

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c k three ways. As stated above, pep5::TRPl segregants, m l i j pep5A::URA3 segregants, and the original pep5-8 mu-

2 3

with pep5 mutations when replica plates were incu- bated at 3 7 " , we tested for temperature sensitivity in tc

C C L tant grow at 37" when replica plated from a master. Growth is slower than wild-type strains, but definitely visible within 3 days. Next we tested for growth from low cell density spottings: 500-3000 cells from segregants from 15 tetrads, 10 segregating for pep5A::URA3, 5 for pepS::TRPl, were spotted onto YEPD medium and the plates incubated at 37" both in a plastic bag and unwrapped. Figure 10 shows the growth pattern of five tetrads incubated in these con- ditions for three days (cells from tetrad three proved not to have the correct genotype when later tested). First, the pep5A::URA3 segregants show tight temper- ature sensitivity for growth when the plates are bagged (lA,C; 2A,B; e.g.). For the other ten tetrads tested (five of them segregating the pep5::TRPl allele) the pep5 bearing segregants also show tight tempera- ture sensitivity for growth at 37" when grown on bagged YEPD plates. Second, tetrads on the un- wrapped plate show neither solely a 2:2 nor a 4:O segregation for growth at 37". For the 15 tetrads the

FIGURE 8.-DNI\ hlot of total yeast DNA from PEP5 and ppp5::TRP I strains. D N A was prepared from spheroplasts. digested with X h n l . fractionated on an ;~garose gel and transferred to nitro- cellulose. T h e hlot WIS probed with the 6.3-kh Sphl fragment. T h e strains represented are the parental diploid (Bj4334) and spore clones BJ4339-4342. T h e strains having TRP I inserted into the PEP5 gene possess an ;~dditional 1.7-kb Fragnlent.

present, as is a larger species. We are unsure whether this larger species corresponds to the normal PrA precursor, since we lack an appropriate control (the pep4-3 mutation eliminates this antigen, since it is a nonsense mutation in the PrA structural gene).

Production of activity for the repressible, vacuolar alkaline phosphatase (BAUER and SICARLAKIE 1975) is greatly depressed in the pep5::TRPl segregants (Fig- ure 9B). We assume that a modifier gene is segregat- ing in these tetrads [there are high high (3A, 4D, e.g.) and low high (3D, 4B) wild-type segregants; high low (3B, 4A) and low low (3C, 4C)pepS::TRPl segregants] that may correspond to the one that was previously described (JONES, ZURENKO and PARKER 1982).

Because vacuolar hydrolases share the early por- tions of the secretion pathway with secreted enzymes (STEVENS, ESMON and SCHEKMAN 1982), we tested the segregants for production of secreted invertase on a native activity gel (CARLSON, OSMOND and BOTSTEIN 1981). Derepression of invertase activity was normal in all segregants (data not shown).

Recently D U L I ~ and RIEZMAN (1 989) reported that end 1-1 and two disruption alleles of END1 cause temperature sensitivity for growth at 37 ". Because we had observed no temperature sensitivity associated

ratio was 4:9:2 for 4:0, 3:1, and 2:2 segregations of growth: no growth at 37". We infer that a second segregating gene influences the ability of pep5 segre- gants to grow at 37" if the plates are unbagged. And thirdly, there is an occasional pep5 mutant that can escape the tight temperature sensitivity for growth on bagged plates (see segregant 6A). A pep5-8 bearing strain behaved just as did the null mutants when plated in low density in this manner (it is spotted adjacent to the positive control on each plate in Figure 10). The third method used for testing for temperature sensi- tivity and the relationship to low cell density plating was to streak for isolated colonies. Segregants from the 15 tetrads tested above were streaked on YEPD plates and incubated at 37" in bags or unwrapped. Growth was checked after 3 and 6 days. After 3 days of open incubation, only 3 of the 30 pep5 segregants failed to show growth of individual colonies. Even for these 3, there was growth at the initial heavy inoculum streak. Eighteen of the 30 pep5 segregants showed growth only at the initial high cell density streak (no single colonies) when the plates were bagged. Yet after 6 days, only four of those 18 still showed no evidence of isolated colonies. Six of the 18 strains had only tiny isolated colonies. To test whether this slow growth was due to adaptation we retested selected strains for the ability to grow rapidly at 37" upon subculturing. Two of the four strains that had produced no isolated colonies, one of the six poor growers, as well as two better growers were restreaked, bagged, and incu- bated at 37". The strains all retained their respective growth phenotype on restreaking. Thus, the slow

748 C . A. Woolford et al.

I ~ L ~ R F . 9.-l'leiotropic effects o f p p p 5 nlutants. A, Precursor f o r m of v;~cuol;~r proteases accumuI;Ite in p ~ p 5 : : T R P l strains. Cell extr:~cts (50 pg protein/lane) fronl strains I314342 (lanes 1, ~ P ~ S : : T R I ' / ) . 13,14341 (I;tncs 2, PEP5) . t%14340 (lanes 3, ppp5::TRI'/) , €3J4:439 (lanes 4, / ' E / ' 5 ) . Ii.1 I O X X (lane\ 5. p p 5 - 8 ) . and RJ 1 9 X 4 (lanes 6 . ppp4-3) were prrp;~retl. Cells were grown i n rich Inetlium for 48 hr and ;qqm)xitmtely 2 X I O " cells ~verc I~;~rvcstetl and protein cxtractcd without the use o f detergents. Blotted proteins were prolwd w i t h PrA, PrR. or CpY alItil)odic<, ; ~ n d immune complexes ~crrc vismli7~d using an Ig(~-horscr;disl~ peroxidase conjugate m;tn~rfarturerI hv 13io-RxI. P and 5.1 refer I(] ~ I X ~ C I I I W ~ and n u t u r c si/cd antigens, rrspectively. R. Reprrssible alkaline pl1osphat;we activity i n meiotic segregants from a PPPS::TRPI/+ (liploid. I ~ . s I I . ; I c . [ \ of I ~ , j 4 ~ ~ ~ ~ ~ ; - ~ . 1 ~ : ~ ~ ~ : ~ (lanes Ia-ld) and I$j4:350-4339 (lanes 2;1-4rl) were lo;~tled. PEP5 genotypes for these tetrads (from left t o 1-igllt) ~ r c - + - +, - + + -, + - - +, - + - +. Relevant genotypes of control strains are: pep5-8 (l3,lIOSX. lane 5 ) ; p r h / - A / . h R (RJ3044, 1a11e 6 ) ; ; 1 1 1 ( 1 p p 4 - 3 (I%,] 1984. lane 5) . Cells were grown i n low phosphate nledium ;II 30" for 48 hr a s tlescribc4 in JONFS, ZURENKO and I ' A H K L K (19x2) except for the atltlition of 30-fold higher concentr;ttion of FeCI:,. Approxin1;ltely 1 X 10"' cells were harvested. and protein \ \ x est~.;~c.tctl \vithout the ~ I S C of detergcws (75 pg/lane). Vislt;llization of repressible alk;~line pl~osph;~t;~se x t i v i t y \cas performed :IS described i n JONES. ZI'BENKO and PARKER ( 1 9x2)).

F I G U R E I O.--l'ernper;~ture scmitivity of growth at low cell den- sity. Cclls were- spotted ;It l o w cell density (500-5000 cells) ; ~ n d

incub;Itctl ; ~ t 37" for three clays: I , Imggrd; I I . unhaggetl. Segregants spotted are Iron1 tetrads from the cross hetero7ygous for

(tctr;~ds 4-6) (tetrad 3 is exclucletl from RFSUI.TS)I. PI?5 genotypes for these tetr;~tls are: - + - +, - - + +, - + + -, - + + -, - - + +. Untlerneath the tetr;~ds are spotted :I wild type strain (+) a n d . t o its right. RJ 1088, dlich carries the p ~ p 5 - 8 allele (-).

j ~ ~ f ~ 5 . l : : l ' / t ~ 4 3 [1$15323-BJ5330 ( t e t ~ ~ t l ~ 1 ; t n d 2) ;III<I 13J5335-5346

growth is not due to long-term adaptation. When pep5 strains are grown at 30" on YEPD, they

grow rapidly, but cell densities at stationary phase are only %-Y2 of wild-type densities. If glycerol replaces

the glucose in YEPD, pep5 strains grow poorly at 30" and not at all at 37". Diploids homozygous for the pep5-8, the pep5::TRPI, or the pep5A::URA3 muta- tions do not sporulate.

DISCUSSION

The pep5 mutant, like other pep mutants, was orig- inally isolated as being unable to catalyze cleavage of acetylphenylalanine @-naphthyl ester, an indication of a decrease in CpY activity (JONES 1977). The biolog- ical effects of the pep5 mutation are far reaching and include sensitivity to amino acid analogs, low amino acid pools, inability to utilize glycerol as a carbon source at high temperatures, sporulation deficiency, lysis of cells at stationary phase (JONES 1983), instabil- i ty to storage (E. JONES, unpublished observations), and low frequency of transformation (C. WOOLFORD, unpublished observations).

Initially we attempted to localize the pep5-8 muta- tion by s p o l l mapping (KLAPHOLZ and ESPOSITO 1982). The data from this mapping procedure were not definitive, leaving open possible assignment of pep5-8 to one of three chromosomes. Three factor crosses with markers for each of the three chromo- somes ruled out pep5-8 linkage to any of these chro- mosomes. It was only when the P E P 5 gene was cloned, and we could use a DNA fragment for OFAGE map- ping, that we succeeded in identifying the chromo- somal residence of the P E P 5 gene as chromosome

S. cerevisiae PEP5 Gene 749

XZZZ. It is worth noting that spoll data had excluded a chromosome XZZZ location.

We have shown that disruption of the PEP5 gene results in greatly decreased activities of all vacuolar hydrolases examined, including PrA, PrB, CpY and the repressible alkaline phosphatase. Apparently nor- mal precursor forms of the proteases accumulate in the mutants, as do novel forms of PrB antigen. The pep5-8 mutant was recently reported to secrete the large (p2) CpY precursor (ROTHMAN, HOWALD and STEVENS 1989). We assume that these cells may also secrete precursors to the other absent hydrolases.

As a start towards determining the immediate func- tion of the PEP5 gene product, the gene was cloned and sequenced and antibodies were raised to a fusion protein that contains almost half of the open reading frame of the PEP5 gene (514/1029 codons). The PEP5 gene encodes a 1029 residue polypeptide with a calculated molecular mass of 117,403 D. The se- quence does not show similarity to any sequence in the NBRF-PIR, SWISS-PROT or translated GenBank data bases. No sequence elements that might have provided clues about intracellular location or function were found. No obvious signal sequence or membrane spanning elements were identified, nor were func- tional motifs detected. Although hydrophobic stretches of sequence were detected, these stretches were predicted to assume &sheet structures rather than a-helices. The estimated size of the protein de- tected on immunoblots was 107 kD, substantially less than the predicted size of 117,403 D. At present we cannot distinguish whether this difference is due to an anomalous mobility of the protein in SDS-PAGE or a consequence of posttranslational proteolytic proc- essing. The molecular mass of the PEP5 gene product was not altered when cells were grown in the presence of tunicamycin or by Endo H treatment of cell extracts (data not shown). This suggests that the protein is not glycosylated. We are investigating whether the protein undergoes any other posttranslational processing events.

The PEP5 sequence is nearly identical to that of the ENDl gene (DULIC and RIEZMAN 1989). There are differences between the two sequences however. Co- don 37 is GCT (coding for A) in PEP5 and CGT (coding for R) in ENDI. In the region of codons 61 8- 625, there is a two nucleotide insertion in the ENDl sequence that causes a frameshift with respect to PEP5 and this is followed by a third nucleotide insertion that brings the two genes back into the same reading frame. The resulting amino acids coded for in this region are, for PEPS, TVFYSYKT; for ENDI, TMFFTVTKH. And at codons 769-770, the PEP5 sequence is ACGAAA (coding for TK); the corre- sponding ENDl sequence is AAGCAA (coding for KQ). We are certain of our sequence in these regions.

If these differences are real they might indicate a polymorphism that does not affect PEPSIENDI en- coded protein function. In the 5’ nontranslated region we find nucleotides AAC at -75 to -73 compared with TTT for ENDI. And in the 3’ untranslated region of PEP5 we find a C at position 3187 and D U L I ~ and RIEZMAN find a T at the corresponding position. DULIC and RIEZMAN (1989) found a short sequence similarity between ENDl and the a-subunit of the E. coli H+-ATPase.

D U L I ~ and RIEZMAN (1 989) detected a 6-7-kb tran- script in poly(A)+ RNA when a 2.2-kb EcoRI fragment that extends 5’ of the ENDl ORF by approximately 600 nucleotides was used as a probe. They stated that this transcript was no longer present in their disrupted strains and suggested that the 6-kb transcript is an ENDl encoded run-on transcript. We never detect this transcript in poly(A)+ RNA when a completely internal probe is used (the EcoRI-XbaI fragment). However, we do detect a transcript approximately 6 kb in size when we use the upstream 2-kb BglII frag- ment as a probe. We suggest that this might be the transcript DULIC and RIEZMAN are detecting and that the ENDl disruption might affect a region required for encoding or regulating this upstream transcript. Since both the pep5A::URA3 disruption (which should eliminate the 6-kb transcript) and the pep5::TRPl disruption (which should not) appear phenotypically similar, this transcript is not germane to the PEP5 phenotype.

Codon usage analysis implies that PEP5 is not a highly expressed gene. The low levels of antigen detected in immunoblots of wild-type cell extracts is in agreement with the codon usage analysis. The level of PEPS-encoded antigen appears to be growth stage dependent. The antigen has been detected only in cell-free extracts of log phase cells and not in extracts of stationary phase cells. PEPS-encoded RNA can be isolated from log phase cells, but not from stationary phase cells (C. WOOLFORD, unpublished observations). Because the protein cannot be detected in stationary phase cells it is possible that the protein is not a stable component of cells and might turn over in a fashion coordinated with the cell cycle. We have not yet tested whether the PEPS-encoded protein shows cell cycle dependent expression or presence. Cell cycle depend- ent expression might well lead to an underestimate of the protein’s abundance in cells. An alternative pos- sibility is that the PEP5 encoded protein is susceptible to proteolysis. When the gene product is looked for in a protease deficient strain, it is easily detectable at a single copy level whereas in a protease proficient strain it may be undetectable (compare Figure 6, lanes 1 and 4). Since protease levels rise markedly as cells enter stationary phase (SAHEKI and HOLZER 1975), the protein might be extremely labile in stationary

750 C. A. Woolford et al.

phase cell extracts and detection of it might require the use of protease deficient mutants.

Complementation tests indicate that the p e p 5 , v p t l l , vp19, and endl mutations are members of the same complementation group (cited in ROBINSON et al. 1988; D U L I ~ and RIEZMAN 1989; ROTHMAN, HOWALD and STEVENS 1989) and that the cloned END1 gene complements vptll (DULI~ and RIEZMAN 1989). It seems likely that the spoT7 mutation is also a member of the set, since it, like p e p 5 , maps near rna l (TSUBOI 1983; TANAKA and TSUBOI 1985) and the restriction maps of the two genes are similar. We were unable to obtain the spoT7 mutant to test for complementation.

Two of the eight v p t l l alleles are reported to be temperature sensitive lethal mutations (ROBINSON et al. 1988), as is the single endl allele (CHVATCHKO, HOWALD and RIEZMAN 1986). The tests we performed indicate that pep5 causes temperature sensitivity for growth but by no means causes lethality at 37 O . The fact that a complete deletion of the PEP5 gene results in very slow growth at 37 O implies that the PEP5 gene product helps in extending the temperature range of growth but that there is not an absolute requirement for it.

The degree of temperature sensitivity manifested by the pep5 segregants is subject to modification by a second gene (Figure 10). That this is not the same modifier gene that affects the levels of repressible alkaline phosphatase (Figure 9) emerged from the analysis, since the same tetrads were examined for temperature sensitivity and phosphatase levels and the two phenotypes did not cosegregate.

Microscopic examination indicates that pep5 mu- tants lack phase bright vacuoles. Abnormally small, indeed tiny, vesicles are present and accumulate the fluorescent dyes lucifer yellow and dichlorocarboxy- fluorescein (R. PRESTON, personal communication). Electron microscopic examination of pep5-8 mutant cells suggests that no normal vacuole is present but rather small, amorphous, dense vacuole-like struc- tures are present. We have not yet determined whether the hydrolase antigens present in pep5 mu- tant cells are located within these aberrant structures or whether, in the absence of a normal vacuole, the antigens end up at other locations within the cells.

Cell fractionation has shown that the PEP5 gene product is localized to the vacuolar membrane. SO- dium carbonate treatment of vacuolar membranes releases the PEP5 gene product, an indication that the protein is a peripherally bound membrane protein. Since there is no obvious structure resembling a clas- sical signal sequence, and there is no evidence of asparagine-linked glycosyl addition to the protein de- spite potential acceptors, we suspect that the PEPS- encoded protein is bound to the cytoplasmic side of the vacuolar membrane. This location is consistent

with the pleiotropic phenotype caused by pep5 muta- tions and, in combination with the aberrant vacuolar morphology of the pep5-8 mutant, suggests that PEP5 encodes a protein whose function is required for bio- genesis and maintenance of the vacuole structure it- self.

Since the vacuolar hydrolases play a substantial role in protein turnover during sporulation (ZUBENKO and JONES 198 1) and might well participate in RNA turn- over during this process (HOPPER et al. 1974), the sporulation defect of pep5 homozygotes can be readily understood. Alterations in the amino acid pools usu- ally stored in the vacuole (WIEMKEN and DURR 1974; MESSENGUY, COLIN and TEN HAVE 1980), and even analog sensitivity are also understandable. It is less apparent how a vacuole-specific defect could account for other aspects of pep5 pleiotropy such as aberrant carbon source utilization and low transformability (C. WOOLFORD, unpublished observations).

We thank WILLIAM E. BROWN for the computer program for analyzing protein secondary structure, CHARLES MOEHLE for assist- ance in searching the protein sequence data bases, PATRICIA WAL- TERS for assistance in mapping the PEP5 locus, and ROBERT PRES- TON for passing on the “bagging” technique from theJOHN PRINGLE lab.

This research was supported by U.S. Public Health Service grants DK18090 and GM29713 from the National Institutes of Health (NIH) to E.W.J., by Damon Runyon-Walter Winchell Cancer Fund Postdoctoral Research Fellowship DRG 826 to C.A.W., by the Medical Research Council of Canada to M.F.M., by American Cancer Society fellowship PF2946 to R.W., by U.S. Public Health Service grant GM35827 to JASPAR RINE, and in part by a grant from the Pittsburgh Supercomputing Center through the NIH Division of Research Resources cooperative agreement U41 RR04154.

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Communicating editor: M. CARLSON