Yeast Secretion Research Articles

The brefeldin A resistance protein Bfr1p is a component of polyribosome-associated mRNP complexes in yeast.

The yeast gene BFR1 was originally isolated from a genetic screen for high-copy suppressors of brefeldin A-induced lethality in Saccharomyces cerevisiae. While this result suggested a possible role for the encoded protein, Bfr1p, in the secretory pathway, subsequent data have not fully supported this conclusion. Alternatively, Bfr1p has also been found by yeast two-hybrid analysis to interact with Bbp1p, a component of the spindle pole body. Finally, we have reported that Bfr1p associates with cytoplasmic mRNP complexes containing Scp160p, raising the possibility that Bfr1p may function in mRNA metabolism. Here, we have explored this possibility further. We report that Bfr1p associates with yeast polyribosomes and mRNP complexes even in the absence of Scp160p, and that its interaction with Scp160p-containing mRNP complexes is RNA-dependent. Furthermore, we have determined by fluorescence microscopy and subcellular fractionation that Bfr1p and Scp160p demonstrate similar cytoplasmic localization with enrichment around the nuclear envelope/endoplasmic reticulum. Finally, we report that loss of Bfr1p disrupts the interaction of Scp160p with polyribosomes, thereby demonstrating that the relationship between these two proteins is functional as well as physical. Considered together, these data raise the intriguing possibility that Bfr1p may provide a link between mRNA metabolism, the chromosomal segregation machinery and perhaps secretion in yeast.

Identification of essential active-site residues in the cyanogenic β-glucosidase (linamarase) from cassava (Manihot esculenta Crantz) by site-directed mutagenesis

The coding sequence of the mature cyanogenic β-glucosidase (β-glucoside glucohydrolase, EC 3.2.1.21; linamarase) was cloned into the vector pYX243 modified to contain the SUC2 yeast secretion signal sequence and expressed in Saccharomyces cerevisiae. The recombinant enzyme is active, glycosylated and showed similar stability to the plant protein. Michaelis constants for hydrolysis of the natural substrate, linamarin (Km = 1.06mM) and the synthetic p-nitrophenyl β-D-glucopyranoside (PNP-Glc; Km = 0.36mM), as well as apparent pKa values of the free enzyme and the enzymeŐsubstrate complexes (pKE1 = 4.4Ő4.8, pKE2 = 6.7Ő7.2, pKES1 = 3.9Ő4.4, pKES2 = 8.3) were very similar to those of the plant enzyme. Site-directed mutagenesis was carried out to study the function of active-site residues based on a homology model generated for the enzyme using the MODELLER program. Changing Glu-413 to Gly destroyed enzyme activity, consistent with it being the catalytic nucleophile. The Gln-339Glu mutation also abolished activity, confirming a function in positioning the catalytic diad. The Ala-201Val mutation shifted the pKa of the acid/base catalyst Glu-198 from 7.22 to 7.44, reflecting a change in its hydrophobic environment. A Phe-269Asn change increased Km for linamarin hydrolysis 16-fold (16.1mM) and that for PNP-Glc only 2.5-fold (0.84mM), demonstrating that Phe-269 contributes to the cyanogenic specificity of the cassava β-glucosidase.

Genetic transformation and biotechnological application of the yeast Arxula adeninivorans.

The relatively unknown, non-pathogenic, dimorphic, haploid, ascomycetous yeast Arxula adeninivorans exhibits some unusual properties which are of biotechnological interest. The yeast is able to assimilate and ferment many compounds as sole source of carbon and/or nitrogen, it utilises n-alkanes and degrades starch efficiently. A. adeninivorans features such as thermo- and haloresistance as well as the yeast's uncommon growth and secretion behaviour should be especially emphasised. In media containing up to 20% NaCl, A. adeninivorans is able to grow at cultivation temperatures up to 48 degrees C. Additionally, the dimorphism of the yeast is unusual. Arxula grows at up temperatures of up to 42 degrees C as budding cells, which turn into mycelia at higher temperatures. This environmentally conditioned dimorphism is reversible and budding is reestablished when the cultivation temperature is decreased below 42 degrees C. Alteration of morphology correlates with changes in secretion behaviour. Mycelium cultures accumulate two-fold higher protein concentrations and contain two- to five-fold higher glucoamylase and invertase activities in the medium than budding cells. Based on these unusual properties, Arxula adeninivorans is used for heterologous gene expression and as a gene donor to construct more suitable yeasts for biotechnology. For example the Arxula glucoamylase gene was successfully expressed in Saccharomyces cerevisiae and Kluyveromyces lactis. Both transformed yeasts are able to assimilate and ferment starch as carbon source. A transformation system is used for heterologous gene expression which is based on integration of linearised DNA fragments in two to ten copies, e.g. into the 25S rDNA of A. adeninivorans by homologous recombination. The obtained transformants are mitotically stable. The expression of the lacZ gene from E. coli as well as the XylE gene from Pseudomonas putida indicates the suitability of A. adeninivorans as host for heterologous gene expression.

The p24 family member p23 is required for early embryonic development.

The p24 family of type I integral-membrane proteins, which are localised in the endoplasmic reticulum (ER), the intermediate compartment and the Golgi apparatus, are thought to function as receptors for cargo exit from the ER and in transport vesicle formation. Members of the p24 family have been found in a molecular complex and are enriched in COPI-coated vesicles, which are involved in membrane traffic between the ER and Golgi complex. Although expressed abundantly, simultaneous deletion of several family members does not appear to affect cell viability and protein secretion in yeast. In order to gain more insights into the physiological roles of different p24 proteins, we generated mice deficient in the expression of one family member, p23 (also called 24delta1, see for alternative nomenclature). In contrast to yeast genetics, in mice disruption of both p23 alleles resulted in early embryonic lethality. Inactivation of one allele led not only to reduced levels of p23 itself but also to reduced levels of other family members. The reduction in steady-state protein levels also induced structural changes in the Golgi apparatus, such as the formation of dilated saccules. The generation of mice deficient in p23 expression has revealed an essential and non-redundant role for p23 in the earliest stages of mammalian development. It has also provided genetic evidence for the participation of p24 family members in oligomeric complexes and indicates a structural role for these proteins in maintaining the integrity of the early secretory pathway.

Tansley Review No. 108: Molecular events of vesicle trafficking and control by SNARE proteins in plants.

Eukaryotic cells share a set of secretory pathways for the flux of membrane and protein material. In 1993, ideas about the functioning of three major proteins of the neurosecretory complex were consolidated in the SNARE hypothesis, which proposed that the interaction of these proteins provides both the specificity for vesicle targeting and the molecular machinery for fusion between vesicle and target membranes. Subsequetly, the organization, molecular mechanics and control of vesicle trafficking have become topics of intense research, and the hypothesis has evolved to accommodate new discoveries from the analysis of secretion in yeast and mammals. It is likely to be challenged again as more information comes to light about secretory processes in plants. New tools for measuring and manipulating vesicle traffic and secretion are now being used, drawing on in vivo fluorescence and capacitance recording as well as genetic engineering. These new technologies have already begun to yield details wholly unexpected from past studies. Here we focus on recent findings relating to the mechanisms of vesicle trafficking and the background to these developments, highlighting both current understanding of the molecular events of secretion and the gaps therein, as well as discussing emerging themes from work with plants. contents Summary 389 I. introduction 389 II. 1. The SNARE hypothesis 393 III. vesicle trafficking in plants 402 IV. regulation of vesicle trafficking in plant cells 406 V. conclusion 410 Acknowledgements 411 References 411.

SEM1, a homologue of the split hand/split foot malformation candidate gene Dss1, regulates exocytosis and pseudohyphal differentiation in yeast.

The exocyst is an essential multiprotein complex mediating polarized secretion in yeast. Here we describe a gene, SEM1, that can multicopy-suppress exocyst mutants sec3-2, sec8-9, sec10-2, and sec15-1. SEM1 is highly conserved among eukaryotic species. Its human homologue, DSS1, has been suggested as a candidate gene for the split hand/split foot malformation disorder. SEM1 is not an essential gene. However, its deletion rescued growth of the temperature-sensitive exocyst mutants sec3-2, sec8-9, sec10-1, and sec15-1 at the restrictive temperature. Cell fractionation showed that Sem1p is mainly cytosolic but also associates with the microsomal fraction. In linear sucrose gradients, Sem1p cosedimented with the exocyst component Sec8p. In diploid cells that normally do not form pseudohyphae (S288C background), deletion of SEM1 triggered pseudohyphal growth. This phenotype was abolished after reintroduction of either SEM1 or the mouse homologue Dss1 into the cells. In diploids that have normal capacity for pseudohyphal growth (Sigma1278b background), deletion of SEM1 enhanced filamentous growth. The functionality of both SEM1 and Dss1 in a differentiation process in yeast suggests that Dss1 indeed could be the gene affected in the split hand/split foot malformation disorder. These results characterize SEM1 as a regulator of both exocyst function and pseudohyphal differentiation and suggest a unique link between these two cellular functions in yeast.

Dominant negative alleles of SEC10 reveal distinct domains involved in secretion and morphogenesis in yeast.

The accurate targeting of secretory vesicles to distinct sites on the plasma membrane is necessary to achieve polarized growth and to establish specialized domains at the surface of eukaryotic cells. Members of a protein complex required for exocytosis, the exocyst, have been localized to regions of active secretion in the budding yeast Saccharomyces cerevisiae where they may function to specify sites on the plasma membrane for vesicle docking and fusion. In this study we have addressed the function of one member of the exocyst complex, Sec10p. We have identified two functional domains of Sec10p that act in a dominant-negative manner to inhibit cell growth upon overexpression. Phenotypic and biochemical analysis of the dominant-negative mutants points to a bifunctional role for Sec10p. One domain, consisting of the amino-terminal two-thirds of Sec10p directly interacts with Sec15p, another exocyst component. Overexpression of this domain displaces the full-length Sec10 from the exocyst complex, resulting in a block in exocytosis and an accumulation of secretory vesicles. The carboxy-terminal domain of Sec10p does not interact with other members of the exocyst complex and expression of this domain does not cause a secretory defect. Rather, this mutant results in the formation of elongated cells, suggesting that the second domain of Sec10p is required for morphogenesis, perhaps regulating the reorientation of the secretory pathway from the tip of the emerging daughter cell toward the mother-daughter connection during cell cycle progression.

A Role for Phospholipase D (Pld1p) in Growth, Secretion, and Regulation of Membrane Lipid Synthesis in Yeast

The SEC14 gene encodes a phosphatidylinositol/phosphatidylcholine transfer protein essential for secretion and growth in yeast (1). Mutations (cki1, cct1, and cpt1) in the CDP-choline pathway for phosphatidylcholine synthesis suppress the sec14 growth defect (2), permitting sec14(ts) cki1, sec14(ts) cct1, and sec14(ts) cpt1 strains to grow at the sec14(ts) restrictive temperature. Previously, we reported that these double mutant strains also excrete the phospholipid metabolites, choline and inositol (3). We now report that these choline and inositol excretion phenotypes are eliminated when the SPO14 (PLD1) gene encoding phospholipase D1 is deleted. In contrast to sec14(ts) cki1 strains, sec14(ts) cki1 pld1 strains are not viable at the sec14(ts) restrictive temperature and exhibit a pattern of invertase secretion comparable with sec14(ts) strains. Thus, the PLD1 gene product appears to play an essential role in the suppression of the sec14(ts) defect by CDP-choline pathway mutations, indicating a role for phospholipase D1 in growth and secretion. Furthermore, sec14(ts) strains exhibit elevated Ca2+-independent, phophatidylinositol 4,5-bisphosphate-stimulated phospholipase D activity. We also propose that phospholipase D1-mediated phosphatidylcholine turnover generates a signal that activates transcription of INO1, the structural gene for inositol 1-phosphate synthase.

Effects of signal sequences on the secretion of hen lysozyme by yeast: construction of four secretion cassette vectors.

Recombinant protein expression is important in many fields— medicine, biochemistry, molecular biology, etc.—as well as in industry. In general, bacterial, fungal, animal and plant cells have been used for this purpose. In the case of the bacterium Escherichia coli, recombinant proteins often accumulate as inclusion bodies, although expression level is high (Imoto et al., 1987). Therefore, the inclusion body must be renatured into active forms. Moreover, in many cases, methionine is not removed from the N-terminal end of the protein, which may affect the function or structure of the protein (Mine et al., 1997). On the other hand, as is the case for animal and plant cells, the level of expression is generally low (Haynes and Weissmann, 1983; Kaufman et al., 1987; Miyaji et al., 1990). Fungi, especially the yeast Saccharomyces cerevisiae, can secrete foreign protein in an active form and the secretion level is higher relative to that of animal or plant cells. Glycosylation occurs both in yeast as well as in animal cells (Ballou, 1990). Thus, expression in yeast is one of the best ways to acquire and purify large quantities of protein in an active form. Expression and secretion of various mammalian proteins in yeast have been reported: interferon α (Hitzeman et al., 1981), interferon γ (Derynck et al., 1983), interleukin 2 (Shaw et al., 1985) and epidermal growth factor (Brake et al., 1984), Fab (Horwitz et al., 1988). However, recombinant proteins are not always secreted from yeast, which is dependent on the target protein, host strain and secretion–signal sequence. The choice of a suitable secretion–signal sequence for a given protein is an important factor in the final yield of secreted protein. The addition of a secretion–signal sequence onto the mature protein is essential to acquire an active protein. One way to select the signal sequence is to use the original signal sequence of the desired protein gene (Jigami et al., 1986; Nakamura et al., 1986). However, it is limited only to secretory proteins. Alternatively, a yeast signal sequence may be used. In this case, it can be applied not only to secretory proteins but also to non-secretory proteins. Before a foreign protein is expressed in yeast, some trials are required to find out which signal sequence is most suitable. We constructed four ‘secretion cassette vectors’ containing four different yeast secretion signals: αF (Brake, 1990; Hitzeman et al., 1990), KILM1 (Skipper et al., 1985), PHO1 (Laroche et al., 1994) and SUC2 (Hitzeman et al., 1990).

Simple approach to reducing proteolysis during secretory production of human parathyroid hormone in Saccharomyces cerevisiae.

A gene coding for human parathyroid hormone (hPTH) was synthesized and cloned into a yeast expression and secretion vector containing the mating factor alpha pre-pro leader sequence and the galactose-inducible promoter, GAL10. The intact hPTH(1-84) was found to be secreted into the culture medium. As observed in the previous reports on the secretory production of hPTH in yeast, however, the proteolytic cleavage occurred as the culture proceeded, resulting in a significant loss of the intact hPTH. Attempts were therefore made to reduce the extent of proteolysis by simply controlling the culture conditions. The proteolytic cleavage was significantly reduced by the addition of an excess amount of l-arginine (>/=0.2M) to the culture medium, which resulted in a marked improvement in the yield of intact hPTH. To examine whether l-arginine affects the activities of intracellular proteases such as KEX2 endoproteinase or extracellular proteases, the proteolysis experiments were performed by incubating the commercial intact hPTH in a yeast host culture supernatant. The results demonstrated that l-arginine at high concentrations reduced the rate of hPTH proteolysis by inhibiting extracellular proteases.

Construction of a yeast expression system with positive selection for gene insertion in the absence of a specific phenotype

A positive-selection system of cloned inserts in Escherichia coli has been devised using a streptomycin-resistant (Sm r) gene and streptomycin-dependent (Sm d) E. coli. A vector, pHA394, based on the yeast expression vector pAM82 has the Sm r gene, which inactivates streptomycin (Sm) and invalidates the streptomycin dependence, resulting in a very low transformation efficiency. Replacement of the Sm r gene by the recombinant vector allows high-frequency transformation. This system was applied to the lysozyme gene. After the yeast secretion signal was fused to the lysozyme gene using an intermediate vector, pHA474, the Sm r gene of pHA394 was replaced by the fusion gene, followed by transformation of Sm d E. coli. Analysis of the transformants showed that the plasmid gene contained 100% of the lysozyme gene.

Subunit structure of the mammalian exocyst complex.

The exocyst is a protein complex required for the late stages of secretion in yeast. Unlike the SNAREs (SNAP receptors), important secretory proteins that are broadly distributed on the target membrane, the exocyst is specifically located at sites of vesicle fusion. We have isolated cDNAs encoding the rexo70, rsec5, and rsec15 subunits of the mammalian complex. The amino acid sequences encoded by these genes are between 21% and 24% identical to their yeast homologs. All three genes are broadly expressed and multiple transcripts are observed for rexo70 and rsec15. Characterization of cDNAs encoding the 84-kDa subunit of the mammalian complex revealed a novel protein. mAbs were generated to the mammalian rsec6 subunit of the exocyst complex. rsec6 immunoreactivity is found in a punctate distribution at terminals of PC12 cell processes at or near sites of granule exocytosis.

Accumulation of misfolded protein aggregates leads to the formation of russell body-like dilated endoplasmic reticulum in yeast.

RNAP-1, an aspartic proteinase from a filamentous fungus Rhizopus niveus, is secreted very efficiently in Saccharomyces cerevisiae. It is synthesized first as a precursor form with signal sequence and prosequence in its amino-terminus. Our previous study indicated that the prosequence of RNAP-I had important roles in its correct folding and secretion in yeast, and that a prosequence-deleted derivative of RNAP-I, delta pro, was not secreted but was retained and degraded in the yeast endoplasmic reticulum (ER). In the present study, we show that the accumulation of delta pro in the yeast ER caused elevated synthesis of ER resident chaperones, indicating that delta pro is recognized as an unfolded protein species in the ER. Our biochemical data demonstrated that delta pro formed aggregates which contained BiP, but not protein disulfide isomerase (PDI), in the ER. Immunoelectron microscopical analysis revealed that the delta pro aggregates were indeed visible as electron-dense regions in the ER and nuclear envelope. Such 'chaperone-associated misfolded protein bodies' were observed for the first time in yeast. Morphologies of the ER and nucleus were drastically altered by the accumulation of the delta pro aggregates. The ER lost its flat cisternal shape; the ER lumen extended aberrantly and the ER membrane irregularly proliferated. The misfolded delta pro proteins are probably sorted from the ordinary ER lumen to form the aggregates so that the ER function would not be grossly impaired, and the dilated ER may represent an ER subcompartment where the delta pro aggregates are degraded.

Sec2p mediates nucleotide exchange on Sec4p and is involved in polarized delivery of post-Golgi vesicles.

The small GTPase Sec4p is required for vesicular transport at the post-Golgi stage of yeast secretion. Here we present evidence that mutations in SEC2, itself an essential gene that acts at the same stage of the secretory pathway, cause Sec4p to mislocalize as a result of a random rather than a polarized accumulation of vesicles. Sec2p and Sec4p interact directly, with the nucleotide-free conformation of Sec4p being the preferred state for interaction with Sec2p. Sec2p functions as an exchange protein, catalyzing the dissociation of GDP from Sec4 and promoting the binding of GTP. We propose that Sec2p functions to couple the activation of Sec4p to the polarized delivery of vesicles to the site of exocytosis.

Characterization of a cDNA encoding a subunit of the rat brain rsec6/8 complex

Many proteins required for neurotransmission are homologous to proteins involved in the Golgi-to-plasma membrane stage of the yeast secretory pathway. A novel 17S complex composed of eight proteins including rsec6 and rsec8, the rat homologues of the yeast secretory proteins, Sec6p and Sec8p, has been identified in rat brain cytosol. Sec6p and Sec8p are components of a complex of at least seven proteins which are essential for secretion in yeast. While the complementary DNAs (cDNA) encoding rsec6 and rsec8 have been cloned [Ting et al. (1995) Proc. Natl. Acad. Sci. USA 92, 9613–9617], the other six components of the 17S complex remain undescribed. Using the peptide sequence obtained from p71, one of the subunits of the rat brain 17S complex, we isolated a full-length cDNA from a rat brain library. This cDNA is predicted to encode a hydrophilic protein of 82 kDa, similar in size to that observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for the endogenous rat brain rsec6/8 complex 71 kDa component. p71 contains domains of peptide sequence which display significant homology to regions of the tail domain of yeast type II myosin. Northern blot analysis of rat tissues indicates that messenger RNA transcripts of 3.1 and 4.4 kb encoding this protein are expressed broadly across several rat tissues in a pattern similar to that of rsec6 and rsec8 mRNA expression. A possible role for p71 as a point of interaction for proteins of the cytoskeleton and proteins involved in secretion is discussed.

Expression of a lipocalin in Pichia pastoris: secretion, purification and binding activity of a recombinant mouse major urinary protein

The proteins of the mouse major urinary protein complex (MUP), members of the lipocalin family, bind volatile pheromones and interact with the vomeronasal neuroepithelium of the olfactory system. We report the expression of a MUP protein using its native signal sequence for secretion in the methylotrophic yeast, Pichia pastoris. Mature recombinant MUP (rMUP) is secreted at a concentration of 270 mg/l in minimal medium and it is isolated from the culture supernatant by one step ion-exchange chromatography in a nearly pure form. Binding activity, tested with an odorant molecule which displays high affinity for native MUP, indicates that rMUP has a behavior similar to the native one. This finding suggests that the protein, and in particular its hydrophobic binding pocket, is properly folded.

Folding and Stability of the Z and Siiyama Genetic Variants of Human α1-Antitrypsin

Z (Glu342 --> Lys) and S(iiyama) (Ser53 --> Phe) genetic variations of human alpha1-antitrypsin (alpha1-AT) cause a secretion blockage in the hepatocytes, leading to alpha1-AT deficiency in the plasma. Using in vitro folding analysis, we have shown previously that these mutations interfere with the proper folding of polypeptides. To understand the fundamental cause for the secretion defect of the Z and S(iiyama) variants of alpha1-AT, we investigated in vivo folding and stability of these variant alpha1-AT using the secretion system of yeast Saccharomyces cerevisiae. Various thermostable mutations suppressing the folding block of the Z variant in vitro corrected the secretion defect as well as the intracellular degradation in the yeast secretion system. Significantly, the extent of suppression in the secretion defect of Z protein was proportional to the extent of suppression in the folding defect, assuring that the in vivo defect associated with the Z variant is primarily derived from the folding block. In contrast, the folding and secretion efficiency of S(iiyama) was not much improved by the same mutations. In addition, none of the rarely secreted S(iiyama) alpha1-AT carrying the stabilizing mutations for the wild type and Z variant were active. It appears that the major defect in S(iiyama) variant is the loss of stability in contrast to the kinetic block of folding in the Z variant.

Reduction of BiP Levels Decreases Heterologous Protein Secretion in Saccharomyces cerevisiae

Increased levels of the endoplasmic reticulum-resident protein folding chaperone BiP would be expected to either increase protein secretory capacity by improved solubilization of folding precursors or decrease secretory capacity by binding and retaining misfolded proteins. To address this question, the relationship between BiP levels and heterologous secretion in yeast was determined. A yeast strain was constructed in which BiP expression is tunable from 5 to 250% of wild-type levels, and this strain was used to explore the effect of varying BiP level on overall secretion of three heterologous proteins: human granulocyte colony-stimulating factor, Schizosaccharomyces pombe acid phosphatase, and bovine pancreatic trypsin inhibitor. For all three proteins examined, reduction in BiP expression below wild-type level diminished overall secretion, whereas 5-fold BiP overexpression from a constitutive glycolytic promoter did not substantially increase or decrease secretion titers. These results are consistent with a positive role for BiP in promoting membrane translocation and solubilization of folding precursors but are inconsistent with a negative role in proofreading and improper retention of heterologous secreted proteins.

An integrating vector for tunable, high copy, stable integration into the dispersed Ty delta sites of Saccharomyces cerevisiae.

We have constructed a yeast integration vector targeted to chromosomal Ty delta sequences and used it to create Saccharomyces cerevisiae strains with stable tandem integrations ranging from 1 to 30 vector copies. The vector carries the bacterial NEO gene, allowing copy number to be tuned by varying G418 resistance, which generally increases with copy number as determined by quantitative Southern blot. Tandem integration into a single site is most commonly observed, but single-copy and two-site integration is also observed. Bovine pancreatic trypsin inhibitor was constitutively expressed and secreted using the NEO-based delta vector, and secretion levels were 2-10-fold improved relative to commonly used 2 mu multicopy yeast plasmids. The NEO-based Ty delta vector is a powerful tool for stable heterologous protein expression and secretion in yeast.

Mutagenesis of endopolygalacturonase from Fusarium moniliforme: histidine residue 234 is critical for enzymatic and macerating activities and not for binding to polygalacturonase-inhibiting protein (PGIP).

The sequence encoding the endopolygalacturonase (PG) of Fusarium moniliforme was cloned into the E. coli/yeast shuttle vector Yepsec1 for secretion in yeast. The recombinant plasmid (pCC6) was used to transform Saccharomyces cerevisiae strain S150-2B; transformed yeast cells were able to secrete PG activity into the culture medium. The enzyme (wtY-PG) was purified, characterized, and shown to possess biochemical properties similar to those of the PG purified from F. moniliforme. The wtY-PG was able to macerate potato medullary tissue disks and was inhibited by the polygalacturonase-inhibiting protein (PGIP) purified from Phaseolus vulgaris. The sequence encoding PG in pCC6 was subjected to site-directed mutagenesis. Three residues in a region highly conserved in all the sequences known to encode PGs were separately mutated: His 234 was mutated into Lys (H 234-->K), and Ser 237 and Ser 240 into Gly (S 237-->G and S 240-->G). Each of the mutated sequences was used to transform S. cerevisiae and the mutated enzymes were purified and characterized. Replacement of His 234 with Lys abolished the enzymatic activity, confirming the biochemical evidence that a His residue is critical for enzyme activity. Replacement of either Ser 237 or Ser 240 with Gly reduced the enzymatic activity to 48% and 6%, respectively, of the wtY-PG. When applied to potato medullary tissue, F. moniliforme PG and wtY-PG caused comparable maceration, while the variant PGs exhibited a limited (S 234-->G and S 240-->G) or null (H 234-->K) macerating activity. The interaction between the variant enzymes and the P. vulgaris PGIP was investigated using a biosensor based on surface plasmon resonance (BIAlite). The three variant enzymes were still able to interact and bind to PGIP with association constants comparable to that of the wild type enzyme.