CHARACfERIZATION OF THE BAR PROTEINASE, AN EXTRACELLULAR ENZYME FROM THE YEAST SACCHAROMYCES CEREVISIAE Vivian L. MacKay, Jacque Annstrong, Carli Yip, Susan Welch, Kathy Walker, Sherri Osborn, Paul Sheppard, and John Forstrom ZymoGenetics, Inc. 4225 Roosevelt Way NE Seattle, Washington 98105 INTRODUCTION Haploid S. cerevisiae cells of the a mating type constitutively secrete an extracellular proteinase that cleaves the peptide mating pheromone (a-factor) secreted by mating-type a cells. DNA sequence analysis of the BARI gene that encodes Bar proteinase demonstrated that the primary translation product of 587 amino acids has strong homology to two-domain aspartic proteinases such as pepsin, chymosin, and others, but contains a unique third domain that is not homologous to these enzymes. When produced by wild-type yeast cells, the Bar enzyme exists as a heterogeneous, heavily glycosylated protein with apparent molecular weight >200,000 Da. By producing the proteinase in mutant yeast strains that are defective in glycosylation, we have been able to purify and characterize a homogeneous species. In this paper, we will describe some of the enzyme's physical properties and substrate requirements, as well as present data indicating that the third domain is required for secretion of the proteinase to the culture medium. BIOLOGY OF BAR PROTEINASE In heterothallic strains of the yeast S. cerevisiae, haploid cells exist as a or a mating types. These can grow and divide stably as haploids or can fuse to form stable ala diploids. The conjugation or mating process, which has been studied extensively in the last twenty years, has been shown to require the secretion of and response to mating type-specific peptide pheromones. (For reviews, see Cross et al., 1988; Herskowitz, 1989). Thus, a cells constitutively secrete a-factor which acts on a cells, and a cells produce a-factor which affects a cells. The pheromones act through cell type-specific surface receptors that are coupled to heterotrimeric G proteins. Response to the pheromone of the opposite cell type prepares the cell for fusion via several changes: synthesis of mating type-specific surface agglutination factors, alteration of cell wall mannan synthesis, induction or enhancement of

StructllTt! and F IIIICtion of thl! Aspartic Pro~inases Edited by B.M. Dunn, Plenum Press. New York, 1991

161

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8

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III

159

1 91

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ii

c c

g

g

I

c

Figure 1. Schematic representation of the primary translation product of the S. cerevisiae BARI gene. SP = putative signal peptide; I, II, and III = three domains of the polypeptide as predicted by sequence homology with aspartyl proteinases; D = predicted active site aspartic acid; C = cysteine; 0 = potential asparagine-linked glycosylation sites. Amino acids in each domain are indicated above the diagram.

transcription of several mating-specific genes, and arrest of the responding cell in the Gl phase of the cell cycle. If cells do not mate, they can recover from G 1 arrest and resume growth and cell division by one of two mechanisms: an adaptation response to the pheromone or inhibition/inactivation of the pheromone. In the latter case, Mata cells were shown to produce an extracellular activity, called Barrier, that acts as an antagonist of a.-factor (Hicks & Herskowitz, 1976). As shown in a later section, Barrier activity is a proteinase that cleaves a.-factor. Recently, Mata. cells have also been shown to have such an activity that inactivates a-factor (Steden et al., 1989). Mutant cells which lack these enzymes are supersensitive to the pheromone of the opposite cell type (Sprague & Herskowitz, 1981; Chan & Otte, 1982; Steden et al., 1989). CHARACfERISTICS OF BAR PROTEINASE PREDICTED FROM DNA SEQUENCE ANALYSIS OF THE CLONED BAR] GENE Using a barJ mutant that lacks Barrier activity, we cloned the gene encoding Bar proteinase by in vivo complementation of the mutation (MacKay et al., 1988). The BAR] gene was shown to be the structural gene for the enzyme by the ability of Schizosaccharomyces pombe (an unrelated yeast) transformed with the BAR] gene to secrete the activity. DNA sequence analysis demonstrated that the primary translation product (587 amino acids) has significant homology to a variety of aspartyl proteinases and appears to be organized into several domains (Figure I). Thus, there is a predicted amino-terminal signal peptide of 24 amino acids (von Heinje, 1986), two domains (205 and 159 amino acids separated by an 8 amino acid linker) with homology to the domains of aspartyl proteinases, particularly around the active site residues (Figure 2), and a third domain of 191 amino acids with no significant homology to these enzymes or other proteins in databases. (Domain boundaries were predicted solely from sequence homology.) Sequence homology between Bar's first two domains and bovine chymosin, for which crystal structure coordinates are available, allows the generation of a hypothetical structure for Bar proteinase (Figure 3). As expected for an aspartyl proteinase, mutation of the active site Asp in the second domain to either Glu or Ala abolished detectable activity, although the protein was still secreted into the culture medium (MacKay et al., 1988). Unlike other aspartyl proteinases, however, the putative active site triad in the second domain is Asp-Ser-Gly, rather than Asp-Thr-Gly. There are nine cysteine residues in the primary translation product: one in the proposed signal peptide, two in the first domain, three in the second domain, and three in the third domain. At least some of these appear to be disulfide bonded, as boiling partially purified fractions with reducing agents destroys the activity (although boiling in the absence of reducing agents does not). The polypeptide contains nine potential asparagine-linked glycosylation sites, all (or nearly all) of which are utilized. Since wild-type yeast cells generally hyperglycosylate such sites, the protein is secreted to the culture medium as a

162

PDB4AP -> Endothiapepsin PDB2AP -> Penicillopepsin PDBICMS -> Chymosin BARI ItndgTGHLEFILQHEEEMYYATTLDIGTPSQSLTVLFDTGSADFWVMds (50) PDB4AP: STGSATTTPIDSLDDAYITPVQIGTPAQTLNLDFDTGSSDLWVF-- (41) PDB2AP: AASGVATNTPTA-NDEEYITPVTIGG--TTLNLNFDTGSADLWVF-- (42) lCMS GEVASVPLTNYLDSQYFGKIYLGTPPQEFTVLFDTGSSDFWVP-- (43)

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snpfclpnsntssysnatyngeevkpsidcrsMSTYNEHRSSTY-QYLEN -SSETT-------ASEVDG-------------QTIYTPSKSTTAKLLSGA -STELP-------ASQQSG-------------HSVYNP--SATGKELSGY -SIYCK--------SNA------------CKNHQRFDPRKSSTF-QNLGK

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(143) (117) (118) (119)

BAR1 PDB4AP: PDB2AP: 1CMS

SGVLGIGFPRRESVKGYegapneyypnfpQILKSEKiidvvaySLFLNSP DGLLGLAFSTLNTVSPT---------QQKTFFDNAKAS--LDSPVFTADL DGLLGLAFSSINTVQPQ---------SQTTFFDTVKSS--LAQPLFAVAL DGILGMAYPSLASEYSI------------PVFDNMMNRHLVAQDLFSVYM

(193) (155) (157) (157)

BAR1 PDB4AP: FDB2AP: 1CMS

D-SGTGSIVF-GAIDESKFSGDLFTFPMVNE--YPT-IVDAPATLAmtiq GYHAPGTYNF-GFIDTTAYTGSITYTAVSTKQGFWEWTSTGYAVGS---KHQQPGVYDF-GFIDSSKYTGSLTYTGVDNSQGFWSFNVDSYTAGS---DRNGQESMLTLGAIDPSYYTGSLHWVPVTV-QQYWQFTVDSVTI SGVVV-

(238) (201) (202) (202)

BAR1 PDB4AP: PDB2AP: 1CMS

glgaqnksscehETFTTTKYPVLLDSGTSLLNAPKVIadkmasf-vNASY ------------GTFKSTSIDGIADTGTTLLYLPATVVSAYWAQVSGAKS --------------QSGDGFSGIADTGTTLLLLDDSVVSQYYSQVSGAQQ --------ACE------GGCQAILDTGTSKLVGPSSDILNIQQ-AIGA-T

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SEEEGIYILDCPV--SVGDVEYNFDFGdlqISVPLSSLILSPETEGSY-C SSSVGGYVFPCSA--TLPSFTFGVGSAR--IVIPGDYIDFGPISTGSSSC DSNAGGYVFDCST--NLPDFSVSISGYT--ATVPGSLINYGPSGDGST-C QNQYGEFDIDCDNLSYMPTVVFEINGKM-YPLTPSAYT-----SQDQGFC

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GFAVQPTN--DSMVLGDVFLSSAYVVFDLDnykislaqan FGGIQSSAGIGINIFGDVALKAAFVVFNGATTPTLGFASK LGGIQSNSGIGFSIFGDIFLKSQYVVFDSDG-PQLGFAPQA TSGFQSENHSQKWILGDVFIREYYSVFDRANN--LVGLAKAI

*

(372) (326) (323) (323)

Figure 2. Sequence conservation between the BARl primary translation product and members of the aspartyl proteinase family. Capitalized letters in the BARl sequence indicate conservation with one or more of the other three enzymes. Active site aspartic acids are marked with asterisks.

163

Figure 3. Three-dimensional structure assignment of the Bar proteinase as predicted from sequence homology with bovine chymosin. See Figure 2. First domain is on the right of the active site cleft and second on the left.

164

Wild-type yeast hyperglycosylation

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M = mannose; GlcNAc = N-

heterogeneous, heavily glycosylated moiety >200 kDa. In addition, the third domain is quite serine/threonine-rich and may contain substantial a-linked glycosylation (see below). Sequence analysis of the 5' noncoding region suggested several sequences that might be involved in regulation of the gene, that is, its expression only in Mata cells, not in Mata or a/a cells. Kronstad et ai. (1987) demonstrated that BARl is transcribed only in a cells because of a 5' binding site for a repressor protein that is present in a and a/a cells. Moreover, BARl transcription and expression of the proteinase are enhanced in a cells incubated with a-factor; two sequences which flank the repressor binding site are responsible for this increase. These sequences are found in the 5' noncoding region of several genes whose transcription is enhanced by a-factor treatment (Kronstad et ai., 1987). CHARACfERIZATION OF THE BARRIER ACTIVITY AS A PROTEINASE Purification and characterization of the enzyme has been hampered because of the heterogeneous hyperglycosylation of the protein. However, based on the extensive work of C. E. Ballou and co-workers (1990), we have recently developed stable mutant mnnl mnn9 yeast strains which add only a homogeneous ManlOGlcNAc2 oligosaccharide (similar to mammalian high mannose type) to asparagine-linked glycosylation sites (MacKay et ai., 1990; see Figure 4). Expression of Bar proteinase was enhanced approximately 20-fold by replacing the BARl promoter with the strong constitutive promoter from the TPll gene encoding triose phosphate isomerase (Alber & Kawasaki, 1982) and ligating the expression unit into a mUlticopy number plasmid. Bar proteinase secreted by the mnnl mnn9 transformants migrated as a 92-95 kDa homogeneous protein (Figure 5). The predicted 165

molecular weight of the polypeptide (assuming cleavage of the putative signal peptide) is 61.6 kDa, approximately the size of the polypeptide expressed in E. coli (data not shown). Thus, even in the mutant strains, Bar proteinase is substantially modified post-translationally by Nglycosylation and probably also by O-glycosylation (see below). We have purified the protein approximately 2000-fold to >90% purity by ethanol precipitation, followed by chromatography on DEAE-Sepharose, mono-Q, and S-200 resins. During purification, we employed a semi-quantitative biological assay which relies on the ability of isolated Bar proteinase to overcome the supersensitivity of bar] mutants to a-factor (Manney, 1983). This assay can detect as little as approximately 1 ng of the enzyme. The natural substrate for Bar proteinase is the peptide pheromone a-factor (available from Sigma) with the following sequence: Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr. The proteinase cleaves a-factor between Leu6 and LYS7, as determined by sequencing of cleavage products isolated by HPLC (Figure 6B). Although this site may be considered unusual for cleavage by an aspartyl protease, it should be noted that pepsin can cleave a-factor at one of several sites, one of which yields cleavage products with the same retention times as those from Bar cleavage (Figure 6C). Using a-factor cleavage as detected by HPLC as an assay, we have determined reaction conditions for the purified proteinase. The enyzme has a pH optimum of approximately 5.0 - 5.3 but retains >50% activity from ca. pH 2.6 to 6.8, and slight activity (1 - 2%) can be detected at pH 1.1. It does not require Ca+ 2 , other divalent cations, or any other cofactors. A variety of natural and synthetic peptides have been assayed for cleavage by Bar proteinase, as shown in Table 1. Bar proteinase apparently has quite strict substrate specificity for a-factor (substrate 1) and only closely related peptides (although Arg can substitute for Lys at the scissile bond, substrate 10), as it does not cleave a similar sequence

92·95

kd-+

2

3

4

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Figure S. Immunoblot of secreted Bar proteinase from wild-type and mutant yeast strains. Lanes 1, 2, and 4, protein secreted by mnnl mnn9 mutant strains (see Figure 4); lane 3, protein secreted by a mnn9 mutant; lane 5, protein secreted by a wild-type strain. Bar proteinase was concentrated from cell-free culture supernatants by ethanol precipitation (MacKay et 01., 1988) and detected with a rabbit polyclonal antibody raised against Bar polypeptide produced in E. coli.

166

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Figure 6. Detection of a·factor cleavage by HPLC. Standard reaction conditions (100 ~l) are 50 mM sodium citrate, pH 5.0, 5 ~g a·factor (ca. 30 ~M), and :'>50 ng purified enzyme for 15 minutes at 30D C. Reactions are terminated by the addition of 1.5 ~l of concentrated sulfuric acid and 90 ~l was subjected to HPLC on a C18 column. Under the gradient conditions used, substrate eluted at approximately 14 minutes. With Bar proteinase, the C·terminal and N·terminal cleavage products eluted at approximately 9.4 and 15.3 l1)inutes, respectively. a) No enzyme; b) Bar proteinase; c) porcine pepsin.

in platelet-derived growth factor (peptide 11) or in a synthetic peptide (14) provided by Ben Dunn. It is likewise inactive on two pep tides generally cleaved by aspartyl proteinases (12 and 13). Like other aspartyl proteinases, Bar requires four amino acids (P4 - PI) on the amino terminal side of the scissile bond (peptides 2-5), but is inactive with even four residues (P I .'- P4 ') on the carboxyl-terminal side of the bond (peptides 6-9), unlike other enzymes that require only three (Dunn et al., 1987). We have been unable to find any reversible inhibitors of Bar proteinase. As expected, the enzyme is resistant to EDT A, e-amino caproic acid, and a number of serine protease inhibitors, including phenylmethyl sulfonylfluoride, aprotinin, leupeptin, tosyl arginyl methyl ester, and tosyUysyl methyl ester. However, it is also completely resistant to 167

Table 1. Substrate specificity of Bar proteinasea Peptide 1 2 3

4 5 6 7 8 9 10 11 12 13

14 15

Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr His- Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln Trp-Leu-Gln-Leu-Lys-Pro-Gly Trp-Leu-Gln-Leu-Lys-Pro Trp-His-Trp-Leu-Gln-Leu-Arg-Pro-Gly-Gln-Pro-Met-Tyr ... -Pro-Thr-Val-Gln-Leu-Arg-Pro-Val-Gln-V al-Arg-Lys-· .. b Lys-Pro-lle-Glu-Phe-Nph-Arg-LeuC Ala-Pro-Ala-Lys-Phe-Nph-Arg-Leu C Arg-Phe-Leu-Glu-Nph-Lys-Pro-Gly-Gln-Proc,d Lys-Pro-Glu-lle-Nph-Lys-Ser-Glu-Lys-llec,e

Cleavage

+ + +

+

aScissile bond indicated by italics. bInternal sequence of the B chain of platelet-derived growth factor, which was reduced and alkylated prior to incubation with Bar proteinase. cSynthetic peptides kindly provided by Dr. Ben Dunn. dSequence based on ex-factor. eSequence based on a proposed internal cleavage site within Bar proteinase.

pepstatin A, although pepsin cleavage of a-factor under identical conditions was inhibited by 1 mM pepstatin. None of the uncleaved peptides in Table 1 inhibit Bar cleavage of a-factor. From its strict substrate specificity, it seems unlikely that any of the general reversible protease inhibitors will inhibit Bar proteinase. (yt/e have not yet investigated irreversible inhibitors such as diazoacetyl norleucine [DAN]). In summary, from the data available, it is not certain if the Bar enzyme is an aspartyl proteinase. Its sequence homology with classical aspartyl proteinases, proposed structure, inactivation by mutation of a putative active site aspartic acid, and its acid pH optimum are all consistent with this classification. However, its unique and strict substrate specificity and resistance to pepstatin A argue that this enzyme could differ significantly from aspartyl proteinases, possibly in structure or in mechanism of action. ROLE OF THE THIRD DOMAIN IN PROMOTING EXPORT OF BAR PROTEINASE In addition to the two presumably catalytic domains that are homologous to typical aspartyl proteinases, the Bar enzyme has a third domain of approximately 191 amino acids with a unique sequence. The domain contains three cysteines, four potential asparaginelinked glycosylation sites (see Figure 1), and 33% serine + threonine. SDS gel electrophoresis of nonglycosylated Bar polypeptide expressed in E. coli vs. the mnnl mnn9 yeast protein ± deglycosylation with endoglycosidase H (which removes N-linked, but not fungal O-linked carbohydrate) indicates that the polypeptide contains N-linked glycosylation as well as other substantial post-translational modification (S. Welch, unpublished). Based on analysis of expressed fragments of the polypeptide, a large part of the additional modification appears to occur in the third domain and probably arises from O-glycosylation 168

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schematic representation of Bar polypeptide. Top figure, full length polypeptide; second figure, replacement of the Bar signal peptide with the preprosequence from the S. cerevisiae MFa] gene. See text for explanation of other figures.

of this serine/threonine-rich region, although other modifications cannot be ruled out at this time. It should be noted that many extracellular fungal enzymes contain O-glycosylated serine/threonine-rich domains (e.g., Salovuori et al., 1987; Tomme et al., 1988). C-terminal deletion analysi~; indicated that at least part of the third domain is responsible for export of Bar proteinase. With the full length protein, at least 95% of the polypeptide is found in the culture medium Ilnder steady-state conditions. As shown in Figure 7, deletion of up to 140 amino acids from the carboxyl terminus did not decrease either activity or expOlt, but removing an additional 25 amino acids eliminated nearly all exported activity, although significant cell-associated activity and protein could be detected. Removing 197 amino acids including six from the proposed C-terminus of the second domain led to loss of both activity and export. However, deletion of nearly all of the first two domains while retaining the third domain abolished proteinase activity but not export of the polypeptide fragment. These results suggest that the 25 amino acids between the endpoints of A140 and A165 may contain a specific sequence necessary for protein export or stability of the extracellular enzyme. Alternatively, perhaps a minimal size of the third domain (or amount of O-glycosylation?) is required for export and is relatively independent of the precise sequence. Bar expression plasmids containing internal deletions within the third domain are under construction to test these possibilities. The properties of the third domain have been exploited in the design and construction of a novel leader for the secretion of heterologous recombinant proteins (Figure 7). A DNA restriction fragment encoding the proposed Bar signal peptide and the first 10 amino acids of the first domain was joined with another fragment representing the last 6 (approximately) amino acids of the second domain plus most of the third domain and an oligonucleotide encoding five amino acids of a Kex2 cleavage site. (The yeast Kex2 proteinase is a calciumdependent serine protease that nOlmally processes the precursors for a-factor and for killer toxin [Fuller et al., 1989].) This leader has been compared to the one commonly used for the secretion of heterologous proteins (MacKay, 1986), that is, the preprosequence from the 169

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MFa} gene that encodes the precursor for a-factor pheromone (Kurjan & Herskowitz,

1982). The Bar leader has been at least as effective as the MFa} signal for the secretion and export of human insulin precursor. platelet-derived growth factor. epidermal growth factor, transforming growth factor-a, and porcine urokinase.

SPECULATION ABOUT ACTIVE FRAGMENTS OF BAR PROTEINASE As shown in Figure 5, Bar proteinase is secreted from mnn} mnn9 mutant cells as an apparent 92-95 kDa protein. However, during purification the protein undergoes extensive proteolysis with little or no loss of activity. S-200 gel filtration of pooled fractions from mono-Q chromatography resulted in several overlapping protein peaks with Bar proteinase activity in all fractions. Western blot analysis of these fractions demonstrated the existence of multiple, fairly discrete fragments, at least some of which appeared to retain activity (Figure 8). We were particularly intrigued by the small fragment (ca. 22-23 kDa; last 3-4 lanes), as this is the size predicted if a glycosylated fragment were released from the first domain by cleavage near the end of the domain. Perhaps such a monodomain fragment could form an active homodimer as shown for the HlV protease (Darke et al .• 1989; Meek et al., 1989). It has also recently been reported (Bianchi et al., 1990) that under controlled conditions pepsin can cleave itself near the end of its first domain, releasing a fragment that can be isolated as an active homodimer. a In any case, our results suggest that fragments of Bar proteinase retain proteolytic activity, although the precise sites and the enzyme(s) responsible for cleavages are unknown at this time. a It should be noted however that, although we consistently observe cleavage of the purified enzyme, the small 22-23 kDa fragment is not always detected among the cleavage products. Western blots with an antibody specific for the third domain showed that some of the cleavages remove at least part of this domain. Moreover, cleavage patterns in purified samples of the mutant enzyme Bar:D287A indicate that some but probably not all of the cleavage is due to a contaminating protease.

170

ACKNOWLEDGMENTS We wish to thank Teresa Gilbert and Peter Lockhart for last-minute DNA sequencing and oligonucleotide synthesis, respectively, and Ben Dunn for peptide substrates and useful discussion.

REFERENCES Alber, T., & Kawasaki, G., 1982, Nucleotide sequence of the triose phosphate isomerase gene of Saccharomyces cerevisiae,J. Mol. Appl. Genet., 1:419.

Ballou, C. E., 1990, Isolation, characterization, and properties of Saccharomyces cerevisiae mnn mutants with nonconditional protein glycosylation defects, Meth. Enzymol., 185:440. Bianchi, M., Boigegrain, R. A., Castro, B., & Coletti-Previero, M.-A., 1990, N-terminal domain of pepsin as a model for retroviral dimeric aspartyl protease, Biochem. Biophys. Res. Comm., 167:339. Chan, R. K., & OUe, C. A., 1982, Isolation and genetic analysis of Saccharomyces cerevisiae mutants supersensitive to Glll11\!st by a-factor and a-factor pheromones, Mol. Cell. BioI., 2:11. Cross, R., Hartwell, L. H., Jackson, C., & Konopka, J. B., 1988, Conjugation in Saccharomyces cerevisiae, Ann. Rev. Cell BioI., 4:429.

Darke, P. L., Leu, C.-T., Davis, L. J., Heimbach, J. C., Diehl, R. E., Hill, W. S., Dixon, R. A. F., & Sigal, I. S., 1989, Human immunodeficiency virus protease: Bacterial expression and characterization of the purified aspartic protease, J. BioI. Chem., 264:2307. Dunn, B. M., Jimenez, M., Weidner, J., Pennington, M., Carter, M., & Parten, B., 1987, Kinetic product analysis of aspartyl proteinases utilizing new synthetic substrates and reversed phase HPLC, in: "Proteins," J. J. L'Italien, ed., Plenum, New York. Fuller, R. S., Brake, A., & Thorner, J., 1989, Yeast prohormone processing enzyme (KEX2 gene product) is a Ca2+-dependent serine protease, Proc. Natl. Acad. Sci., U.S.A., 86:1434. Herskowitz, I., 1989, A regulatory hierarchy for cell specialization in yeast, Nature (London), 342:749. Hicks, J. B., & Herskowitz, I., 1976, Evidence for a new diffusible element of mating pheromones in yeast, Nature (London), 260:246.

Kurjan, J., & Herskowitz, I., 1982, Structure of a yeast pheromone gene: A putative a-factor precursor contains four tandem copies of mature a-factor, Cell, 30:933. Kronstad, J. W., Holly, J. A., & MacKay, V. L., 1987, A yeast operator overlaps an upstream activation site, Cell, 50:369. MacKay, V. L., 1986, Secretion of heterologous proteins in yeast, in: "The Biochemistry and Molecular Biology of Industrial Yeasts," G. G. Stewart, I. Russell, R. D. Klein, and R. R. Hiebsch, eds., CRC Press, Boca Raton, Florida. MacKay, V. L., Welch, S. K., Insley, M. I., Manney, T. R., Holly, J., Saari, G. C., & Parker, M. L., 1988, The Saccharomyces cerevisiae BARl gene encodes an exported protein with homology to pepsin, Proc. Natl. Acad. Sci., U.S.A., 85:55. MacKay, V. L., Yip, C., Welch, S., Gilbert, T., Seidel, P., Grant, F., & O'Hara, P., 1990, Glycosylation and export of heterologous proteins expressed in yeast, in: "Recombinant Systems in Protein Expression," K. K. AlitaIo, M.-L. Huhtala, J. Knowles, & A. Vaheri, eds., Elsevier, Amsterdam. Manney, T. R., 1983, Expression of the BARl gene in Saccharomyces cerevisiae: Induction by the a mating pheromone of an activity associated with a secreted protein, J. Bacteriol., 155:291. Meek, T. D., Dayton, B. D., Metcalf, B. W., Dreyer, G. B., Strickler, J. E., Gorniak, J. G., Rosenberg, M., Moore, M. L., Magaard, V. W., & Debouck, C., 1989, Human immunodeficiency virus 1 protease expressed in Escherichia coli behaves as a dimeric aspartic protease, Proc. Natl. Acad. Sci., U.S.A., 86:1841.

Salovuori, I., Makarow, M., Rauvala, H., Knowles, J., & Kaariainen, L., 1987, Low molecular weight high-mannose type glycans in a secreted protein of the filamentous fungus Trichoderma reesei, BioITechnology,5:152.

171

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