Overexpression of plant histidinol dehydrogenase using a baculovirus expression vector system.

A cDNA encoding cabbage histidinol dehydrogenase, including the chloroplast transit peptide sequence, was overexpressed using a baculovirus expression vector system. The maximum level of the expression of histidinol dehydrogenase was reached 5 days after infection of the insect cells. Two forms of recombinant histidinol dehydrogenase with molecular masses of 53 and 52 kDa, respectively, were obtained by a one-step purification from the cell homogenate. Compared with the 52-kDa form, the 53-kDa form contained 10 additional amino acids at the N-terminus derived from the transit peptide. By incubating the cell homogenate for 2 h at 30 degrees C, the 53-kDa form could be completely converted to the 52-kDa form. This conversion was blocked by leupeptin. Eighty percent of the converted 52-kDa form had Cys at position 31 at the N-terminal amino acid and the rest had Met 33. Kinetic properties of the recombinant enzyme were virtually identical to those of histidinol dehydrogenase isolated from cabbage plants. The overexpression of recombinant cabbage histidinol dehydrogenase in insect cells, the proteolytic processing of the preprotein next to the N-terminus (compared to the mature cabbage enzyme), and its easy purification allow the preparation of large amounts of the active enzyme for structural and functional studies.     

Human fur gene encodes a yeast KEX2-like endoprotease that cleaves pro- beta-NGF in vivo

Extracts from BSC-40 cells infected with vaccinia recombinants expressing either the yeast KEX2 prohormone endoprotease or a human structural homologue (fur gene product) contained an elevated level of a membrane-associated endoproteolytic activity that could cleave at pairs of basic amino acids (-LysArg- and -ArgArg-). The fur-directed activity (furin) shared many properties with Kex2p including activity at pH 7.3 and a requirement for calcium. By using antifurin antibodies, immunoblot analysis detected two furin translation products (90 and 96 kD), while immunofluorescence indicated localization to the Golgi apparatus. Coexpression of either Kex2p or furin with the mouse beta- nerve growth factor precursor (pro-beta-NGF) resulted in greatly enhanced conversion of the precursor to mature nerve growth factor. Thus, the sequence homology shared by furin and the yeast KEX2 prohormone processing enzyme is reflected by significant functional homology both in vitro and in vivo.     

Molecular and enzymatic properties of furin, a Kex2-like endoprotease involved in precursor cleavage at Arg-X-Lys/Arg-Arg sites.

We have recently shown that furin, a mammalian homologue of the yeast precursor-processing endoprotease Kex2, is involved in precursor cleavage at sites marked by the Arg-X-Lys/Arg-Arg motif within the constitutive secretory pathway. In this study, we analyzed molecular and enzymatic properties of furin expressed in Chinese hamster ovary cells using gene transfer techniques. COOH-terminal truncation analyses indicate that the polypeptide region significantly conserved among the Kex2 family members is required for the endoprotease activity of furin, while the COOH-terminal unconserved region containing the Cys-rich domain and the transmembrane domain is dispensable. A mutant of furin truncated up to the transmembrane domain from the COOH-terminus was secreted into the culture medium as an active form. The sequence requirements for precursor cleavage of this truncated furin determined in vitro were similar to those of wild-type furin determined by expression studies in cultured cells. It had a strong resemblance to the Kex2 protease in the inhibitor profile and pH dependency. These observations support the notion that furin is the endogenous endoprotease involved in precursor cleavage at Arg-X-Lys/Arg-Arg sites.     

Expression and processing of recombinant human terminal transferase in the baculovirus system

Overproduction of human terminal transferase protein has now been accomplished by cloning the coding sequence of human terminal transferase into a baculovirus, where the expression of terminal transferase is under the control of the polyhedrin protein promoter. Two constructs were made, one producing a protein containing the entire terminal transferase fused to 12 amino acids from the NH2 terminus of the polyhedrin protein, and the other producing 58-kDa human terminal transferase. The terminal transferase levels expressed in cells infected with either recombinant baculovirus are around 10,000 units/10(7) cells at 48 h postinfection, about 200-fold greater than levels expressed in thymus and cultured lymphoblastoid cells. The chimeric polyhedrin/human terminal transferase protein produced in the infected insect cells has a molecular weight of about 60,000 while the nonfused recombinant human terminal transferase is identical in molecular weight to that present in human lymphoblastoid cells. Both forms of recombinant terminal transferase show immunological and enzymatic activity. When infected cells are pulse-labeled with [35S] methionine at 42-45 h postinfection, about 10% of newly synthesized protein is terminal transferase. Both forms of terminal transferase are phosphorylated in recombinant virus-infected cells as demonstrated by pulse-labeling infected cells with 32P-inorganic phosphate and isolation of labeled terminal transferase peptides by immunoprecipitation.     

Mutation of a tyrosine localization signal in the cytosolic tail of yeast Kex2 protease disrupts Golgi retention and results in default transport to the vacuole.

Kex2 protease processes pro-alpha-factor in a late Golgi compartment in Saccharomyces cerevisiae. The first approximately 30 residues of the 115 amino acid CO2H-terminal cytosolic tail (C-tail) of the Kex2 protein (Kex2p) contain a Golgi retention signal that resembles coated-pit localization signals in mammalian cell surface receptors. Mutation of one (Tyr713) of two tyrosine residues in the C-tail or deletion of sequences adjacent to Tyr713 results in loss of normal Golgi localization. Surprisingly, loss of the Golgi retention signal resulted in transport of C-tail mutant Kex2p to the vacuole (yeast lysosome), as judged by kinetics of degradation and by indirect immunofluorescence. Analysis of the loss of Kex2 function in vivo after shutting off expression of wild-type or mutant forms proved that mutations that cause rapid vacuolar turnover do so by increasing the rate of exit of the enzyme from the pro-alpha-factor processing compartment. The most likely explanation for these results is that mutation of the Golgi retention signal in the C-tail results in transport of Kex2p to the vacuole by default. Wild-type Kex2p also was transported to the vacuole at an increased rate when overproduced, although apparently not due to saturation of a Golgi-retention mechanism. Instead, the wild-type and C-tail mutant forms of Kex2p may follow distinct paths to the vacuole.     

Inhibition of African swine fever virus binding and infectivity by purified recombinant virus attachment protein p12.

The African swine fever virus protein p12, involved in virus attachment to the host cell, has an apparent molecular mass of 17 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. We have also identified 12- and 10-kDa forms of the p12 protein in infected Vero cells and found that the mature 17-kDa protein is the only form present in virus particles. The p12 protein has been produced in large amounts in Spodoptera frugiperda insect cells infected with a recombinant baculovirus. A 17-kDa protein that possessed the biological properties of the viral protein was produced, since it bound to susceptible Vero cells and not to receptor-negative L cells, which do not support virus replication. The binding of the baculovirus-expressed protein p12 to Vero cells was specifically blocked by virus particles. In addition, the recombinant protein purified by immunoaffinity chromatography blocked the specific binding of virus particles to susceptible cells and prevented infection, demonstrating that the p12 protein mediates the attachment of virions to specific receptors and indicating that blocking the p12-mediated interaction between African swine fever virus and receptors in Vero cells can inhibit infection. However, although antibodies specific for protein p12 are induced in natural infections and in animals inoculated with inactivated virus or recombinant protein p12, these antisera did not inhibit virus binding to the host cell or neutralize virus infectivity.     

Site-directed mutagenesis and expression of PC2 in microinjected Xenopus oocytes.

The biosynthesis and post-translational maturation of PC2, a neuroendocrine-specific Kex2-like endoprotease, following expression in Xenopus oocytes is described. The initial translation product was a 75-kDa membrane-associated protein which was released from the oocytes as a glycosylated 71-kDa protein. During extended chase periods, the extracellular 71-kDa protein was converted to a mature 68-kDa product. A deletion mutant lacking a putative COOH-terminal amphipathic helix was still membrane-associated, suggesting that this domain was not essential for attachment of PC2 to membranes. Two putative proregion cleavage site mutants were also constructed. Conversion of the 75-kDa peptide to the 71-kDa peptide involved cleavage at the sequence Lys-Arg-Arg-Arg (amino acids 78-81), since mutation of this sequence to Lys-Val-Arg-Leu resulted in the secretion of the 75-kDa peptide. Extracellular conversion of the 71-kDa peptide to the 68-kDa peptide involved cleavage at the sequence Arg-Lys-Lys-Arg (amino acids 106-109), since deletion of this tetrabasic sequence resulted in secretion of the 71-kDa peptide without further conversion to the 68-kDa form. Finally, a mutation which changed a catalytically important Asp to Asn did not affect processing of proPC2. These results may be relevant to our understanding of mechanisms in the intracellular sorting and maturation of proPC2 in neuroendocrine cells.     

Reconstitution of human DNA polymerase delta using recombinant baculoviruses: the p12 subunit potentiates DNA polymerizing activity of the four-subunit enzyme.

Eukaryotic DNA polymerase delta is thought to consist of three (budding yeast) or four subunits (fission yeast, mammals). Four human genes encoding polypeptides p125, p50, p66, and p12 have been assigned as subunits of DNA polymerase delta. However, rigorous purification of human or bovine DNA polymerase delta from natural sources has usually yielded two-subunit preparations containing only p125 and p50 polypeptides. To reconstitute an intact DNA polymerase delta, we have constructed recombinant baculoviruses encoding the p125, p50, p66, and p12 subunits. From insect cells infected with four baculoviruses, protein preparations containing the four polypeptides of expected sizes were isolated. The four-subunit DNA polymerase delta displayed a specific activity comparable with that of the human, bovine, and fission yeast proteins isolated from natural sources. Recombinant DNA polymerase delta efficiently replicated singly primed M13 DNA in the presence of replication protein A, proliferating cell nuclear antigen, and replication factor C and was active in the SV40 DNA replication system. A three-subunit subcomplex consisting of the p125, p50, and p66 subunits, but lacking the p12 subunit, was also isolated. The p125, p50, and p66 polypeptides formed a stable complex that displayed DNA polymerizing activity 15-fold lower than that of the four-subunit polymerase. p12, expressed and purified individually, stimulated the activity of the three-subunit complex 4-fold on poly(dA)-oligo(dT) template-primer but had no effect on the activity of the four-subunit enzyme. Therefore, the p12 subunit is required to reconstitute fully active recombinant human DNA polymerase delta.     

High-level expression of a mitochondrial enzyme, ornithine transcarbamylase from rat liver, in a baculovirus expression system

The mitochondrial enzyme, ornithine transcarbamylase (OTC) from rat liver was expressed in Spodoptera frugiperda (Sf) insect cells using a baculovirus vector. When insect cells were infected with recombinant Autographica californica nuclear polyhedrosis virus (AcNPV) containing a cDNA encoding the precursor form of OTC (pOTC) inserted into the polyhedrin gene, they expressed catalytically active enzyme at levels of approximately 2.5 micrograms/10(6) cells. About 25% of the active enzyme was a novel, partially processed product of pOTC containing four extra amino acids at the amino terminus of OTC. The most abundant protein found in mitochondria from infected insect cells was the normal processing intermediate iOTC, which contains 8 extra amino acids at the amino terminus of OTC. Whereas this species, present at 20 micrograms/10(6) cells, was not active and did not bind the transition-state analog inhibitor of OTC, delta-PALO, the novel processing product did bind and was affinity-purified, along with mature OTC, on a PALO-affinity column. The OTC expressed in insect cells was located in the same compartment of the mitochondrion as in rat liver. The incomplete processing occurred in vitro in both noninfected and infected insect cells. The high level of expression of iOTC using the baculoviral expression system provides a means of overproducing an obligatory intermediate in the mitochondrial import process.     

大鼠20 α－羟类固醇脱氢酶在昆虫细胞中的表达

大鼠２０α羟类固醇脱氢酶（２０α－Ｈｙｄｒｏｘｙｓｔｅｒｏｉｄｄｅｈｙｄｒｏｇｅｎａｓｅ,２０αＨＳＤ）ｃＤＮＡ片段,被插入杆状病毒（ＢａｃｕＩｏｖｉｒｕｓ）的转移载体ｐＢｌｕｅＢａｃⅢ,经野生型病毒ＤＮＡ的共转染,从被转染的昆虫细胞中获得重组病毒。Ｎｏｒｔｈｅｒｎｂｌｏｔ分析,重组病毒感染细胞有２０αＨＳＤ基因表达。感染细胞裂解液的Ｗｅｓｔｅｒｎ印迹法分析,３７ｋＤ的蛋白带被２０αＨＳＤ抗体识别．体外酶活性测定发现,感染细胞裂解液中含有２０αＨＳＤ酶促活性以上结果提示,大鼠２０αＨＳＤ在杆状病毒昆虫表达系统成功地获得表达,为今后大量制备和纯化２０αＨＳＤ创造条件。     

Heterologous expression of peptide hormone precursors in the yeast Saccharomyces cerevisiae. Evidence for a novel prohormone endoprotease with specificity for monobasic amino acids.

The peptide somatostatin (SRIF) exists as two different molecular species. In addition to the most common form, which is a 14-residue peptide, there is also a 14-amino acid amino-terminally extended form of the tetradecapeptide, SRIF-28. Both peptides are synthesized as larger precursors containing paired basic and monobasic amino acids at their processing sites, which, upon cleavage, generate either SRIF-14 or -28, respectively. In mammals a single prepro-SRIF molecule undergoes tissue-specific processing to generate the mature hormone whereas in some species of fish separate genes encode two distinct but homologous precursors prepro-SRIF-I and -II that give rise to SRIF-14 and -28, respectively. To investigate the molecular basis for differential processing of the prohormones we introduce their cDNAs into yeast cells (Saccharomyces cerevisiae). The signal peptides of both precursors were poorly recognized by the yeast endoplasmic reticulum translocation apparatus, consequently only low levels of SRIF peptides were synthesized. To circumvent this problem a chimeric precursor consisting of the alpha-factor signal peptide plus 30 residues of the proregion was fused to pro-SRIF-II. This fusion protein was efficiently transported through the yeast secretory pathway and processed to SRIF-28 exclusively, which is identical to the processing of the native precursor in pancreatic islet D-cells. Most significantly, cleavage of the precursor to SRIF-28 was independent of the Kex 2 endoprotease since processing occurred efficiently in a kex 2 mutant strain. We conclude that in addition to the Kex 2 protease, yeast possess a distinct prohormone converting enzyme with specificity toward monobasic processing sites.     

Mechanism of Kex2p inhibition by its proregion.

Many proteases are produced as zymogens bearing an N-terminal proregion acting both as intramolecular chaperone and as enzyme inhibitor. We studied here the inhibition mechanism of the yeast proprotein convertase Kex2p by its proregion. A recombinant secreted and soluble form of Kex2p was produced in Pichia pastoris and its enzymatic properties toward a fluorogenic synthetic peptide were characterized. Recombinant Escherichia coli-produced Kex2p proregion specifically and potently inhibited the enzyme, with an IC(50) of 160 nM. Exploration of the inhibition mechanism revealed that the proregion behaved as a mixed inhibitor.     

The sequence NPFXD defines a new class of endocytosis signal in Saccharomyces cerevisiae

The yeast membrane protein Kex2p uses a tyrosine-containing motif within the cytoplasmic domain for localization to a late Golgi compartment. Because Golgi membrane proteins mislocalized to the plasma membrane in yeast can undergo endocytosis, we examined whether the Golgi localization sequence or other sequences in the Kex2p cytoplasmic domain mediate endocytosis. To assess endocytic function, the Kex2p cytoplasmic domain was fused to an endocytosis-defective form of the alpha-factor receptor. Ste2p. Like intact Ste2p, the chimeric protein, Stex22p, undergoes rapid endocytosis that is dependent on clathrin and End3p. Uptake of Stex22p does not require the Kex2p Golgi localization motif. Instead, the sequence NPFSD, located 37 amino acids from the COOH terminus, is essential for Stex22p endocytosis. Internalization was abolished when the N, P, or F residues were converted to alanine and severely impaired upon conversion of D to A. NPFSD restored uptake when added to the COOH terminus of an endocytosis-defective Ste2p chimera lacking lysine-based endocytosis signals present in wild-type Ste2p. An NPF sequence is present in the cytoplasmic domain of the a- factor receptor, Ste3p. Mutation of this sequence prevented pheromone- stimulated endocytosis of a truncated form of Ste3p. Our results identify NPFSD as a clathrin-dependent endocytosis signal that is distinct from the aromatic amino acid-containing Golgi localization motif and lysine-based, ubiquitin-dependent endocytosis signals in yeast.     

Identification of Two Mannoproteins Released from Cell Walls of a Saccharomyces cerevisiae mnn1 mnn9 Double Mutant by Reducing Agents

In this report, we present the identification of the main polypeptides that are extracted from purified cell walls of a Saccharomyces cerevisiae mnn1 mnn9 strain by reducing agents. Treatment of the purified cell walls of this strain with beta-mercaptoethanol releases several mannoproteins, of which three, with apparent sizes of 120, 45, and 40 kDa, are the most abundant. Analysis of the amino-terminal sequences revealed that the 120-kDa mannoprotein is Bar1p, the protease involved in the so-called barrier activity in yeast cells, and that the 45- and 40-kDa mannoproteins are the Kex2-unprocessed and Kex2-processed forms of the gene product of open reading frame (ORF) YJL158c, an ORF that belongs to the PIR (protein with internal repeats) family of genes, composed thus far of PIR1, PIR2/HSP150, and PIR3. Accordingly we have named this gene PIR4, and Pir4 denotes the 40-kDa Kex2-processed form of the mannoprotein. We have characterized Pir4 and have shown the feasibility of using it as a fusion partner for the targeting of recombinant proteins to the cell wall.     

Production of recombinant Rhizopus oryzae lipase by the yeast Yarrowia lipolytica results in increased enzymatic thermostability

The gene encoding Rhizopus oryzae lipase (ROL) was expressed in the non-conventional yeast Yarrowia lipolytica under the control of the strong inducible XPR2 gene promoter. The effects of three different preprosequence variants were examined: a preprosequence of the Y. lipolytica alkaline extracellular protease (AEP) encoded by XPR2, the native preprosequence of ROL, and a hybrid variant of the presequence of AEP and the prosequence of ROL. Lipase production was highest (7.6 U/mL) with the hybrid prepropeptide. The recombinant protein was purified by ion-exchange chromatography. The ROL included 28 amino acids of the C-terminal region of the prosequence, indicating that proteolytic cleavage occurred below the KR site through the activity of the Kex2-like endoprotease. The optimum temperature for recombinant lipase activity was between 30 and 40 °C, and the optimum pH was 7.5. The enzyme was shown not to be glycosylated. Furthermore, recombinant ROL exhibited greater thermostability than previously reported, with the enzyme retaining 64% of its hydrolytic activity after 30 min of incubation at 55 °C.     

Maturation of barley cysteine endopeptidase expressed in Trichoderma reesei is distorted by incomplete processing

Maturation of barley cysteine endopeptidase B (EPB) in Trichoderma reesei was studied with metabolic in hibitors, Western blotting, and immuno microscopy. The inactive 42-kDa recombinant EPB proprotein, first detected in apical cells, was sequentially processed in a time-dependent manner to a secreted polypeptide of 38.5 kDa, and thereafter, to polypeptides of 37.5, 35.5, and 32 kDa exhibiting enzyme activity both in the hyphae and culture medium. The sizes of the different forms of recombinant EPB were in accordance with molecular masses calculated from the deduced amino acid sequence, assuming cleavage at four putative Kex2p sites present in the 42-kDa proprotein. Both the liquid and the zymogram in-gel activity assays indicated that the 32-kDa enzyme produced in T. reesei in vivo was 2 kDa larger and four times less active than the endogenous EPB. Brefeldin A treatment prevented the last Kex2p processing step of EPB from a 35.5- to a 32-kDa protein. This coincided with a significant increase in the immuno-gold label for EPB and in modified Golgi-like bodies, which suggests that the processing step probably took place in medial Golgi. A 30.5-kDa EPB polypeptide was observed when glycosylation was inhibited by tunicamycin (TM) or when deglycosylation was carried out enzymatically. Deglycosylation increased the enzyme activity twofold, which was also indicated by an increased fluorescence by TM treatment in the zymogram in-gel activity assay. Simultaneous incubation with TM and monensin produced a peptide of 31.5 kDa. Therefore, monensin may inhibit the final processing step of an unglycosylated EPB by an unknown protease in the fungus. In any case, the final recombinant EPB product in Trichoderma differs from the mature endogenous 30-kDa enzyme produced in barley.     

The effects of clathrin inactivation on localization of Kex2 protease are independent of the TGN localization signal in the cytosolic tail of Kex2p.

Localization of Kex2 protease (Kex2p) to the yeast trans-Golgi network (TGN) requires a TGN localization signal (TLS) in the Kex2p C-terminal cytosolic tail. Mutation of the TLS accelerates transport of Kex2p to the vacuole by an intracellular (SEC1-independent) pathway. In contrast, inactivation of the clathrin heavy-chain gene CHC1 results in transport of Kex2p and other Golgi membrane proteins to the cell surface. Here, the relationship of the two localization defects was assessed by examining the effects of a temperature-sensitive CHC1 allele on trafficking of wild-type (WT) and TLS mutant forms of Kex2p. Inactivation of clathrin by shifting chc1-ts cells to 37 degrees C caused WT and TLS mutant forms of Kex2p to behave identically. All forms of Kex2p appeared at the plasma membrane within 30-60 min of the temperature shift. TLS mutant forms of Kex2p were stabilized, their half-lives increasing to that of wild-type Kex2p. After inactivation of clathrin heavy chain, vacuolar protease-dependent degradation of all forms of Kex2p was blocked by a sec1 mutation, which is required for secretory vesicle fusion to the plasma membrane, indicating that transport to the cell surface was required for degradation by vacuolar proteolysis. Finally, after clathrin inactivation, all forms of Kex2p were degraded in part by a vacuolar protease-independent pathway. After inactivation of both chc1-ts and sec1-ts, Kex2 was degraded exclusively by this pathway. We conclude that the effects of clathrin inactivation on Kex2p localization are independent of the Kex2p C-terminal cytosolic tail. Although these results neither prove nor rule out a direct interaction between the Kex2 TLS and a clathrin-dependent structure, they do imply that clathrin is required for the intracellular transport of Kex2p TLS mutants to the vacuole.     

An extracellular exo-beta-(1,3)-glucanase from Pichia pastoris: purification, characterization, molecular cloning, and functional expression

An extracellular exo-beta-(1,3)-glucanase (designated EXG1) was purified to apparent homogeneity from Pichia pastoris X-33 cultures by ammonium sulfate fractionation, ion-exchange chromatography, and gel filtration. The native enzyme is unglycosylated and monomeric with a molecular mass of approximately 47kDa. At its optimal pH of 6.0, the enzyme shows highest activity among physiological substrates toward laminarin (apparent Km, 3.5 mg/ml; Vmax, 192 micromole glucose produced/min/mg protein) but also hydrolyzes amygdalin and esculin, and the chromogenic substrates p-nitrophenyl-beta-D-glucopyranoside and p-nitrophenyl-beta-D-xylopyranoside. The P. pastoris EXG1 gene was cloned by a PCR-based strategy using genomic DNA as template. This intronless gene predicts an ORF that encodes a primary translation product of 414 amino acids. We believe that this preproprotein is processed sequentially by signal peptidase and a Kex2-like endoprotease to yield a mature protein of 392 amino acids (45,376 Da; pI, 4.46) that shares 36-64% amino acid identity with other yeast exo-beta-(1,3)-glucanases belonging to Glycoside Hydrolase Family 5. It also possesses the eight invariant residues and signature pattern [LIV]-[LIVMFYWGA](2)-[DNEQG]-[LIVMGST]-X-N-E-[PV]-[RHDNSTLIVFY] shown by all Family 5 members. Overexpression of the cloned EXG1 gene in Pichia cells, followed by Ni-CAM HC resin chromatography, yielded milligram quantities of homogeneous recombinant EXG1 in active form for further characterization studies.     

Characterization of the yeast KEX1 gene product: a carboxypeptidase involved in processing secreted precursor proteins.

We have identified and partially characterized the Saccharomyces cerevisiae KEX1 gene product, Kex1p, to assess its role in processing secreted protein precursors. Anti-Kex1p antibodies identified a 113-kilodalton protein that was absent in cells in which the KEX1 gene has been disrupted and that was more abundant in cells overexpressing the KEX1 gene. Kex1p was found to be a membrane-associated glycoprotein with N-linked carbohydrate. The N-linked oligosaccharide(s) was modified in a progressive manner after synthesis, causing the glycoprotein to slowly increase in mass to 115 kilodaltons. After a Kex2p-mediated cleavage event at specific pairs of basic amino acids, alpha-factor and K1 killer toxin precursors have COOH-terminal dibasic residue extensions and require a carboxypeptidase B-like enzyme to process the precursors to maturity. A carboxypeptidase activity, with apparent specificity for basic amino acids, was detected in KEX1 cells. Disruption of the KEX1 gene abolished this activity, while overexpression of KEX1 increased it. Our results provide biochemical evidence consistent with earlier genetic work, that KEX1 encodes a serine carboxypeptidase involved in the processing of precursors to secreted mature proteins.