TY - JOUR
AU - Sasnauskas, Kestutis
AB - Abstract Screening for genes affecting super-secreting phenotype of the over-secreting mutant of Kluyveromyces lactis resulted in isolation of the gene named KlMNN10, sharing high homology with Saccharomyces cerevisiae MNN10. The disruption of the KlMNN10 in Kluyveromyces lactis, as well as of MNN10 and MNN11 in Saccharomyces cerevisiae, conferred the super-secreting phenotype. MNN10 isolated from Saccharomyces cerevisiae suppressed the super-secretion phenotype in Kluyveromyces lactis klmnn10, as did the homologous KlMNN10. The genes MNN10 and MNN11 of Saccharomyces cerevisiae encode mannosyltransferases responsible for the majority of the α-1,6-polymerizing activity of the mannosyltransferase complex. These data agree with the view that the structure of glycoproteins in a yeast cell wall strongly influences the release of homologous and heterologous proteins in the medium. The set of genes namely the suppressors of the over-secreting phenotype, could be attractive for further analysis of gene functions, over-secreting mechanisms and for construction of new strains optimized for heterologous protein secretion. KlMNN10 has EMBL accession no. AJ575132. Yeast, Kluyveromyces lactis, Saccharomyces cerevisiae, Super-secretion, KlMNN10, MNN10, MNN11 1 Introduction Kluyveromyces lactis is a biotechnologically significant yeast which has already been exploited as a host for the production of heterologous proteins due to its secretory performance. Production of a foreign protein via the eukaryotic secretory pathway ensures high fidelity of folding, assembly and modification processes required for biological activity. Although high-level transcription of genes is attainable in eukaryotic systems, secretion of corresponding protein products often does not increase proportionally. In many cases, retention within the endoplasmic reticulum (ER) or the point of exit from the ER lumen into Golgi appears to be responsible for this bottleneck [1,2]. Once within the ER lumen, a secretory protein must be correctly folded and in some cases modified, assembled into a functional oligomer and released through the secretory pathway. The major rate-limiting step in constitutive protein secretion in eukaryotic cells occurs at the point of exit from the ER lumen into Golgi, and it is at this point that quality control is exerted. Extensive cellular machinery responsible for efficient and accurate maintenance of these processes within endogenous cellular proteins exists, but if a cell is genetically modified to express a high level of a heterologous secretory protein, one or more of these processes may become rate- or yield-limiting. Several publications have provided the approach to overcome such problems in S. cerevisiae by modulating cellular levels of soluble ER components, namely protein disulphide isomerase which catalyses the formation of native disulphide bonds in secretory proteins, or BiP which appears to act, in part, as a molecular chaperone for secretory proteins passing through the ER. Over-expression of syntaxins Sso1 and Sso2, functioning at the targeting/fusion of the Golgi-derived secretory vesicles to the plasma membrane, polyubiquitin, PSE1 and YAL048c ORF or the disruption of protease Yps1p, Yap3p-encoding genes enhance heterologous protein secretion also [2–9]. Quality and quantity of a product might be improved in mutants with an altered glycosylation process to prevent over-glycosylation or with a reduced vacuolar protease content to prevent proteolytic degradation [10–12]. Recently it has been shown that an increased dosage of polyubiquitin and PDI1 genes enhanced human protein secretion in K. lactis[13]. An empirical approach might be fruitful in search for genes which increase the rate and yield of secretory proteins, if over- or under-expressed. First of all it was demonstrated for the prochymosin-producing strain with two recessive ssc1 and ssc2 mutations, in which the distribution of product between the vacuole and the medium was shifted towards an improved release [14]. Later the SSC1 gene was shown to be identical to the PMR1 gene encoding Ca2+-ATPase, involved in the secretory pathway [15]. Other mutations like rgl2 act on the transcriptional level [16]. Previously we have described the isolation of K. lactis mutants over-secreting heterologous proteins in the medium. One of the recessive over-secreting mutant strains was used for the isolation of genes conferring super-secreting phenotype [17]. In this paper we present molecular and functional characterization of one of the isolated genes, KlMNN10, which in a high copy number suppresses elevated secretion of heterologous proteins. 2 Materials and methods 2.1 Strains and media K. lactis strains MD2/1 (MATα, ura3, argA1, lysA1) and MW270-7B (MATa, metA1, ura3, leu2) were kindly provided by Dr. M. Bianchi and Dr. M. Wesolowski-Louvel. K. lactis MD2/1-9 super-secreting mutant has been described previously [17]. S. cerevisiae CTY182 (MATαhis3 lys2 ura3) was used for the disruption experiments of MNN10 and MNN11 genes. Plasmids were constructed in Escherichia coli strain XL1-Blue (F′proAB lacIqΔ(lacZ) M15 Tn10 (Tetr)/recA1 endA1 gyrA96 (NaIr) thi1 hsdR17(r−k m+k) supE44 relA1 lac). All yeast cultures were incubated at 30 °C. E. coli strains were grown in Luria–Bertani broth. Growth media YEPD (1% yeast extract, 2% peptone, 2% glucose, Difco) and YNB (0.67% yeast nitrogen base, 1% casamino acids, 2% glucose, Difco) were used. K. lactis haploid strains were mated on ME media (5% malt extract, 3% agar, Difco). Sporulation was performed on SP medium (1% potassium acetate, 0.1% yeast extract, 0.05% glucose). YPS (0.67% yeast nitrogen base, 0.2% casamino acids, 0.1% glucose, 0.2% starch and 2% agar) plates were used for halo assays of α-amylase activity. 2.2 Plasmids Most recombinant DNA experiments were performed as described [18]. All enzymes were obtained from Fermentas UAB (Vilnius, Lithuania) and used according to the manufacturer's recommendations. Plasmid pBoriAMY used for α-amylase secretion and pKDU-HGH used for investigation of secretion of human growth hormone (HGH) has been described earlier [17,19]. The 3.8 kb URA3 cassette with S. cerevisiae URA3 gene flanked by direct repeats of Salmonella hisG DNA [20] was used for the disruption experiments of both S. cerevisiae and K. lactis genes. 2.3 DNA sequencing Double-stranded DNA sequencing was performed on both strands using dideoxy termination on Exonuclease III-nested deletions. The CycleReaderTM Auto DNA Sequencing KIT with Cy5 labelled M13/pUC sequencing primer (Fermentas UAB, Vilnius, Lithuania) was used. 2.4 α-Amylase halo assay For halo assay-yeast cultures were diluted to equal OD and 50 μl of each culture were spotted onto YPS plates and incubated at 30 °C for 24 h. The halos surrounding transformants were detected by the absence of iodine staining (3 mg ml−1 J2, 15 mg ml−1 KJ) of the digested starch as earlier described [17]. 2.5 HGH secretion K. lactis and S. cerevisiae were grown in shaker flasks in YNB medium (0.67% yeast nitrogen base, 1% casein hydrolysate, 2% glucose). After growth for 24 h galactose 2% was added for induction and supernatants were analysed by ELISA after 48 h. The culture was diluted to OD A600 nm=1 with phosphate-buffered saline (PBS) and 1 ml of culture was centrifuged and used for HGH ELISA in supernatant and yeast lysates obtained after the disruption of pellets with glass beads. Determination of HGH by monoclonal antibody-based ELISA was carried out as earlier described [17]. 2.6 Invertase assays Invertase secretion was performed as previously described [17]. Native gel electrophoresis of invertase and 2,3,5-triphenyltetrazolium chloride gel staining was carried out as described by Ballou [21]. 2.7 Disruption of the KlMNN10 gene in K. lactis and the MNN10, MNN11 genes in S. cerevisiae The disruption of the gene was performed according to the one-step gene replacement method. For the disruption of KlMNN10, the BamHI fragment of 2158 bp encoding KlMNN10 and the surrounding sequences, inserted into pUC57, was used for the construction of the disruption cassette. The Eco130I DNA fragment of 587 bp from the KlMNN10-encoding sequence was removed and changed by the blunt-ended BamHI–BglII fragment, containing the hisG–URA3–hisG cassette from the plasmid pNKY51 [20]. The resultant plasmid was digested with the Ecl136II and XbaI restriction endonucleases to obtain the hisG–URA3–hisG cassette flanked by KlMNN10-surrounding sequences necessary for homologous recombination, and was used for transformation of K. lactis diploid strain MD2/1xMW270-7B. Colonies were picked up and the organization of the target gene and its neighboring regions was examined by PCR using specific primers for appropriate genes. A diploid strain with the deleted target gene was plated on sporulation medium. The sporulated culture was treated with zymolyase and Ura+ colonies were isolated. The URA3 gene was removed by selection of clones growing on medium containing 5-fluoro-orotic acid (5-FOA). The K. lactis klmnn10 strain thus obtained was transformed with plasmids pBoriAMY and pKDU-HGH [17] to test the influence of the KlMNN10 gene deletion on α-amylase and HGH secretion. For the disruption of the MNN10 gene in S. cerevisiae, a 2160-bp DNA fragment of S. cerevisiae CTY182 encoding the MNN10 sequence was amplified by using the pair of primers: Left primer: 5′-ACTCGAGGCATGGACCTGAAAAGATT-3′ Right primer: 5′-ACTCGAGGACATGCCCACGTAAGGTAT-3′ The amplified fragment was inserted in the bacterial vector pUC57T. The replacement of the XmaJI–BspTI DNA fragment of 617 bp from the MNN10-coding sequence by the XbaI –Ecl136II fragment, containing the hisG–URA3–hisG cassette from the plasmid p19NKY, was performed [20]. The resulting structure containing the hisG–URA3–hisG cassette flanked by the target gene-specific sequences was used for transformation of S. cerevisiae CTY182 (MATαhis3 lys2 ura3). Ura+ transformants were selected and the organization of the target gene and its neighbouring regions was examined by PCR. The URA3 gene was removed by selection of clones growing on a 5-FOA agar plate. For the disruption of MNN11 in S. cerevisiae, a 1152-bp DNA fragment of S. cerevisiae CTY182 encoding the MNN11 sequence was amplified by using the pair of primers: Left primer: 5′-GACTCGAGGGCAAAACGTACTCCTC-3′ Right primer: 5′-CACTCGAGGCACCGTTTCCAGACTC-3′ The amplified fragment was inserted in the bacterial vector pUC57T. The BclI DNA fragment of 318 bp from the MNN11-coding sequence was replaced by the BglII–BamHI fragment, containing the hisG–URA3–hisG cassette. The S. cerevisae mnn11 clones were selected as described above for the mnn10. The S. cerevisiae mnn10 mnn11 strain was constructed by serial disruption of MNN10 and MNN11 in S. cerevisiae CTY182 as described above. 2.8 Cytohelicase assay Cytohelicase assay was carried out according to [22]. Cells were grown to exponential phase and 5×108 cells were suspended in 4 ml of buffered sorbitol solution (1.2 M sorbitol; 20 mM Tris–HCl, pH 8.0; 10 mM DTT). After 10 min, 1 ml of the solution containing 0.5 mg of cytohelicase (from Helix pomatia, Sigma) was added and cells were incubated at 30 °C. Lysis in samples taken at 10 min intervals was determined by measurements of A600 after dilution 1:20 in water. 2.9 Resistance to hygromycine B, SDS and vanadate To assess the degree of resistance to hygromycine B, SDS and vanadate the cells (about 107 cells ml−1) of fresh culture were diluted to 10−1, 10−2 and 10−3 in sterile water and 5 μl of each dilution were spotted on YEPD plates containing 10 μg ml−1 hygromycine B (from Streptomyces hygroscopicus, Sigma), or 10 mM Na3VO4, or 0.03% SDS for K. lactis; and 50 μg ml−1 hygromycine B, or 7 mM Na3VO4, or 0.1% SDS for S. cerevisiae. 3 Results and discussion 3.1 Sequence analysis and characterization of cloned gene Previously we have described the isolation of K. lactis mutants conferring elevated secretion of heterologous proteins. Both dominant and recessive mutations were obtained. One of the recessive mutant strains, K. lactis MD2/1-9, which secreted the heterologous proteins in five-fold excess compared to the wild-type strain, was used for cloning of genes conferring super-secreting phenotype [17]. The set of genes conferring super-secretion phenotype in K. lactis MD2/1-9 has been isolated. Sequence analysis of one of the cloned genes allowed to identify K. lactis ORF, named KlMNN10 sharing a high similarity to the MNN10 gene of S. cerevisiae. The KlMNN10 gene of K. lactis in a high-copy number state suppressed super-secretion phenotype but had no noticeable influence on super-secretion in a low-copy number centromeric plasmid state (data not shown). The amino acid sequence alignments of predicted gene products of S. cerevisiae MNN10 and K. lactis KlMNN10 are presented in Fig. 1 (BLAST, http://www.ncbi.nlm.nih.gov/). Both predicted proteins share 65% aa identity. Mostly the N-end differed in both predicted proteins, and it was extended in Mnn10p of S. cerevisiae. A high similarity was observed from the 123 aa of S. cerevisiae Mnn10p, which corresponded to 61 aa of the predicted KlMnn10p sequence. The membrane-spanning domains were identified at the N-end of both proteins. However, the K. lactis KlMnn10p membrane domain was larger in comparison to S. cerevisiae Mnn10p and consisted of two joined hydrophobic aa stretches, which were separated by a hydrophilic aa stretch in S. cerevisiae Mnn10p (Fig. 1). S. cerevisiae Mnn10p protein is the type-II membrane protein and is characterized as a subunit of a Golgi mannosyltransferase complex. Mnn10p and its distantly-related homologue Mnn11p (21% identity) are responsible for the majority of the α-1,6-polymerizing activity of the Golgi mannosyltransferase complex, which comprises five proteins Anp1p, Mnn9p, Hoc1p, Mnn10p and Mnn11p [23–25]. Furthermore, MNN10 (BED1) is required for polarized growth and efficient bud emergence in S. cerevisiae[26]. Tetrad analysis of S. cerevisiae mnn10 mutants has revealed that MNN10 is essential for germination of spores [23]. 1 View largeDownload slide Amino acid sequence alignment of predicted proteins of the K. lactis KlMnn10p and S. cerevisiae Mnn10p (BLAST, http://ncbi.nlm.nih.gov/). Identity of sequences is indicated in bold. Membrane domains are indicated in italic and underlined. KlMNN10 has the EMBL accession no. AJ575132. 1 View largeDownload slide Amino acid sequence alignment of predicted proteins of the K. lactis KlMnn10p and S. cerevisiae Mnn10p (BLAST, http://ncbi.nlm.nih.gov/). Identity of sequences is indicated in bold. Membrane domains are indicated in italic and underlined. KlMNN10 has the EMBL accession no. AJ575132. A high similarity to the Mnn10p of S. cerevisiae allowed to predict the function of the KlMNN10 gene in K. lactis, namely mannosylation of internal and external cell proteins. To confirm this prediction and further investigate functions and characterize the null allele phenotype related to heterologous protein secretion, the disruption of the genes K. lactis KlMNN10, S. cerevisiae MNN10 and its distantly related MNN11 (21% identity) responsible for manifestation of a similar protein complex in S. cerevisiae, was carried out by conventional techniques. 3.2 Secretion of heterologous and homologous proteins in K. lactis klmnn10, S. cerevisiae mnn10 and mnn11 mutants Since the KlMNN10 gene in a high-copy number state negatively affected the secretion of proteins, we have tested the influence of the klmnn10 mutation on the secretion of heterologous and homologous proteins. First of all, secretion of α-amylase in the K. lactis klmnn10 mutant was tested by halo formation on starch agar plates using shuttle plasmid pBoriAMY [5]. The results of this analysis presented in Fig. 2 show that the disruption of KlMNN10 in K. lactis enhanced secretion of α-amylase significantly. Human growth hormone (HGH) secretion in supernatants was analysed by ELISA. The results presented in Fig. 3 indicate that the KlMNN10 deletion in K. lactis enhanced HGH secretion approximately 3- to 4-fold. Comparison of the K. lactis klmnn10 to a wild-type strain revealed no significant changes of the intracellular HGH level. Hence, the influence of the KlMNN10 gene on the secretion process has been verified in favor of super-secretory type of the corresponding mutation. 2 View largeDownload slide α-Amylase secretion in the K. lactis wild-type and K. lactis klmnn10 mutant strains. 2 View largeDownload slide α-Amylase secretion in the K. lactis wild-type and K. lactis klmnn10 mutant strains. 3 View largeDownload slide ELISA of intracellular and extracellular human growth hormone (HGH) level in the K. lactis MD2/1 (wt) and K. lactis klmnn10 cell lysates (A) and supernatants (B). The amount of HGH was calculated for yeast supernatants and cells obtained from 1 ml yeast culture diluted to OD A600= 1. 3 View largeDownload slide ELISA of intracellular and extracellular human growth hormone (HGH) level in the K. lactis MD2/1 (wt) and K. lactis klmnn10 cell lysates (A) and supernatants (B). The amount of HGH was calculated for yeast supernatants and cells obtained from 1 ml yeast culture diluted to OD A600= 1. 3.3 Resistance to hygromycin B, SDS, vanadate and cytohelicase Since S. cerevisiae mnn10 mutants with mannosylation defects have been reported to be hypersensitive to aminoglycosides and SDS but resistant to vanadates [22,23,27], we tested the resistance of K. lactis klmnn10, S. cerevisiae mnn10, S. cerevisiae mnn11 and S. cerevisiae mnn10 mnn11 mutants to hygromycine B, SDS and vanadate. According to our data in Fig. 4, K. lactis klmnn10 as well as S. cerevisiae mnn10, S. cerevisiae mnn11 and S. cerevisiae mnn10 mnn11 are sensitive to hygromycin B and SDS, but resistant to vanadate in comparison to the corresponding wild-type strain. 4 View largeDownload slide Sensitivity of K. lactis and S. cerevisiae wild type and various null mutants cells to hygromycine B, vanadate (Na3VO4) and SDS. Fresh cultures (about 107 cells ml−1) cells were diluted to 10−1, 10−2 and 10−3 by sterile water and 5 μl of each dilution were spotted on YEPD (control) and YEPD plates containing 10 μg ml−1 hygromycin B, or 10 mM Na3VO4 or 0.03% SDS for K. lactis and 50 μg ml−1 hygromycin B, or 7 mM Na3VO4 or 0.1% SDS for S. cerevisiae, and incubated at 30 °C for 2 days. 4 View largeDownload slide Sensitivity of K. lactis and S. cerevisiae wild type and various null mutants cells to hygromycine B, vanadate (Na3VO4) and SDS. Fresh cultures (about 107 cells ml−1) cells were diluted to 10−1, 10−2 and 10−3 by sterile water and 5 μl of each dilution were spotted on YEPD (control) and YEPD plates containing 10 μg ml−1 hygromycin B, or 10 mM Na3VO4 or 0.03% SDS for K. lactis and 50 μg ml−1 hygromycin B, or 7 mM Na3VO4 or 0.1% SDS for S. cerevisiae, and incubated at 30 °C for 2 days. Since S. cerevisiae mnn10 and other under-mannosylated or under-glycosylated mutants were characterized as endo-β-1,3-glucanase hypersensitive, we tested sensitivity of mutants to the cell wall-hydrolyzing enzyme cytohelicase. The K. lactis klmnn10 mutant as well as the corresponding S. cerevisiae mnn10, S. cerevisiae mnn11 and S. cerevisiae mnn10 mnn11 mutants were significantly more sensitive to cytohelicase in comparison to corresponding wild-type strains (data not shown). Glucans that determine the rigidity of yeast cell walls are protected from enzymatic lysis by the overlaying mannoproteins. Sensitivity of yeast cells to cytohelicase depends, therefore, on the ability of cytohelicase to penetrate the mannoprotein layer. The enhanced sensitivity to cytohelicase, SDS, hygromycin B and resistance to vanadate reflects the alteration of cell wall structure and glycoprotein composition in both yeast genera. However, the sensitivity of K. lactis klmnn10 transformants, containing the S. cerevisiae MNN10 gene in a high-copy number state, was not distinguished from that of the wt strain. Hence, the MNN10 gene of S. cerevisiae could suppress the klmnn10 mutation in K. lactis. 3.4 Invertase secretion To investigate the influence of KlMNN10 and MNN10 disruption on homologous protein secretion, we tested the activity of secreted invertase. Study of invertase secretion in the K. lactis and S. cerevisiae mutants and in corresponding wt strains revealed that the disruption of KlMNN10 in K. lactis enhanced invertase secretion approximately 50%. Disruption of MNN10 or MNN11 or both in the corresponding S. cerevisiae mutants enhanced invertase secretion approximately 80%, 50% and 120%, respectively (Fig. 5). The introduction of recombinant plasmids, containing K. lactis KlMNN10 or S. cerevisiae MNN10 in a high-copy number plasmid state decreased the secretion of invertase in both yeasts (data not shown). In the long range of invertase investigation worldwide we did not find data on a significant influence of glycosylation on the enzymatic activity of invertase. Therefore, we concluded that the disruption of KlMNN10 and MNN10 enhanced secretion of invertase, like that of HGH and α-amylase described above. 5 View largeDownload slide Invertase secretion in the K. lactis and S. cerevisiae wild type and various null mutants. Invertase activity is expressed in comparative units. 5 View largeDownload slide Invertase secretion in the K. lactis and S. cerevisiae wild type and various null mutants. Invertase activity is expressed in comparative units. 3.5 Native gel electrophoresis of internal invertase To directly confirm the influence of the klmnn10 mutation in K. lactis on protein glycosylation we investigated the mobility of invertase in a native gel. The rate of invertase migration in the native gel depends on a number and size of the N-linked oligosaccharide chains. Invertase from S. cerevisiae contains 14 potential glycosylation sites, and the secreted enzyme usually possesses 9–11 carbohydrate chains. K. lactis invertase contains even 24 potential glycosylation sites [21,23]. Data on crude invertase migration visualized by an activity stain using the chromophore 2,3,5-triphenyltetrazolium chloride as described by Ballou [21] are presented in Fig. 6. The external invertase migrates as diffused bands or spots on a native gel at room temperature because of instability of the dimers and the differences in glycosylation level [21]. These results clearly demonstrate that the spots of invertase from K. lactis klmnn10 and S. cerevisiae mnn10 lysates migrate faster in comparison to the corresponding wild-type enzymes. The mnn11 mutation did not affect significantly migration of invertase in S. cerevisiae lysates in comparison to wt. Native invertase gel-electrophoretic patterns point to a function of KlMNN10 similar to that defined for S. cerevisiae MNN10. These results demonstrate the alteration of the glycosylation level in mutants, reflected by an increase in invertase mobility. 6 View largeDownload slide Native invertase gel electrophoretic patterns. Invertase spots were detected with the activity stain as described by Ballou [21]. Cells were disrupted after induction for invertase secretion and samples obtained from equivalent amounts of cells were applied for electrophoresis on a nondenaturating 5% acrylamide gels. The K. lactis (A) and S. cerevisiae (B) invertase mobility patterns were obtained separately because of the difference in migration rate. The rate of S. cerevisiae invertase mobility was significantly higher in comparison to that of K. lactis, because of the differences in protein molecular mass and glycosylation level. 6 View largeDownload slide Native invertase gel electrophoretic patterns. Invertase spots were detected with the activity stain as described by Ballou [21]. Cells were disrupted after induction for invertase secretion and samples obtained from equivalent amounts of cells were applied for electrophoresis on a nondenaturating 5% acrylamide gels. The K. lactis (A) and S. cerevisiae (B) invertase mobility patterns were obtained separately because of the difference in migration rate. The rate of S. cerevisiae invertase mobility was significantly higher in comparison to that of K. lactis, because of the differences in protein molecular mass and glycosylation level. 3.6 Conclusions Since the early 1990s, non-Saccharomyces yeast species have gained more prominence in biotechnology. In this respect, Kluyveromyces has been shown to have significant advantages over traditional baker's yeast in production of certain proteins. High-level secretion of correctly folded and processed recombinant serum albumin, chymosine, interleukine-1β, and immunoglobin can be achieved by using the K. lactis expression systems [11,28–31]. In this paper we demonstrated that cell wall structure significantly influenced secretion of proteins in the media. The glycoproteins in the cell wall of S. cerevisiae are modified with both N-linked and O-linked glycans. The O-linked structures are chains of 4–5 mannoses attached to serines and threonines, whereas the N-linked glycans comprise the conventional core structure to which a long outer chain structure of up to 200 mannose residues can be attached [32]. This mannan modification is so extensive that the mannoproteins constitute up to 40% of the dry weight of the yeast cell wall [33]. Mannans provide an external layer to the cell wall, which is believed both to contribute to its structural integrity and to serve for the exclusion of hydrolytic enzymes and other compounds such as aminoglycosides and detergents. The external protein layer, in particular the N-linked side-chains of the mannoproteins, also determine the permeability of the cell wall for macromolecules [32,33]. Our results confirmed that KlMNN10, MNN10 and MNN11 influenced secretion of proteins in both yeasts and that the cell wall structure and mannan composition could significantly affect the last stages of protein secretion in the media in both yeasts. Probably, defects in protein mannosylation do not influence the protein secretory process directly, but may have a significant impact on the release of secretory proteins into the media at the final steps. Acknowledgements This work was supported by the Lithuanian State Science and Study Foundation and the National Programme: Molecular Basis of Biotechnology, Grant No. 219/2490/05. Abbreviations Abbreviations HGH human growth hormone SDS sodium dodecyl sulphate References [1] Shuster J.R. ( 1991) Gene expression in yeast: protein secretion. Curr. Opin. Biotechnol.  2, 685– 690. Google Scholar CrossRef Search ADS PubMed  [2] Robinson A.S. Hines V. Wittrup K.D. ( 1994) Protein disulfide isomerase overexpression increases secretion of foreign proteins in Saccharomyces cerevisiae. Biotechnology (NY)  12, 381– 384. Google Scholar CrossRef Search ADS   [3] Knittler M.R. Haas I.G. ( 1992) Interaction of BiP with newly synthesized immunoglobin light chain molecules: cycles of sequential binding and release. EMBO J.  11, 1573– 1581. Google Scholar PubMed  [4] Chow T.Y-K. Ash J.J. Dignard D. Thomas D.Y. ( 1992) Screening and identification of a gene, PSE-1, that affects protein secretion in Sacharomyces cerevisiae. J. Cell Sci.  101, 709– 719. Google Scholar PubMed  [5] Chen Y. Pioli D. Piper P.W. ( 1994) Overexpression of the gene for polyubiquitin in yeast confers increased secretion of a human leukocyte protease inhibitor. Biotechnology (New York)  12, 819– 823. [6] Ruohonen L. Toikkanen J. Tieaho V. Outola M. Soderlund H. Keranen S. ( 1997) Enhancement of protein secretion in Saccharomyces cerevisiae by overproduction of Sso protein, a late-acting component of the secretory machinery. Yeast  13, 337– 351. Google Scholar CrossRef Search ADS PubMed  [7] Kerry-Williams S.M. Gilbert S.C. Evans L.R. Balance D.J. ( 1998) Disruption of the Saccharomyces cerevisiae YAP3 gene reduces the proteolytic degradation of secreted recombinant human albumin. Yeast  14, 161– 169. Google Scholar CrossRef Search ADS PubMed  [8] Wolf A.M. Litske-Petersen J.G. Nilsson-Tillgren T. Din N. ( 1999) The open reading frame YAL048c affects the secretion of proteinase A in S. cerevisiae. Yeast  15, 427– 434. Google Scholar CrossRef Search ADS PubMed  [9] Egel-Mitani M. Andersen A.S. Diers I. Hach M. Thim L. Hastrup S. Vad K. ( 2000) Yield improvement of heterologous peptides expressed in ysp1-disrupted Saccharomyces cerevisiae strains. Enzyme Microb. Technol.  26, 671– 677. Google Scholar CrossRef Search ADS PubMed  [10] Hinnen A. Buxton F. Chaudhuri B. Heim J. Hottiger T. Meyhack B. Pohlig G. ( 1994) Gene expression in recombinant yeast. In: Gene expression in recombinant microorganisms  ( Smith A. Ed.), pp. 121– 193 Marcel Dekker, New York. [11] Gellissen G. Hollenberg C.P. ( 1997) Application of yeast in gene expression studies: a comparison of Saccharomyces cerevisiae, Hansenula polymorpha and Kluyveromyces lactis– a review. Gene  190, 87– 97. Google Scholar CrossRef Search ADS PubMed  [12] Sagt C.M.J. Kleizen B. Verwall R. De Jong M.D.M. Müller W.H. Smits A. Visser C. Boonstra J. Verkleij A.J. Verrips C.T. ( 2000) Introduction of an N-glycosylation site increases secretion of heterologous proteins in yeasts. Appl. Environ. Microbiol.  66, 4940– 4944. Google Scholar CrossRef Search ADS PubMed  [13] Bao W. Fukuhara H. ( 2001) Secretion of human proteins from yeast: stimulation by duplication of polyubiquitin and protein disulfide isomerase in Kluyveromyces lactis. Gene  272, 103– 110. Google Scholar CrossRef Search ADS PubMed  [14] Smith R.A. Duncan M.J. Moir D.T. ( 1985) Heterologous protein secretion from yeast. Science  229, 1219– 1224. Google Scholar CrossRef Search ADS PubMed  [15] Rudolph H.K. Antebi A. Fink G.R. Buckley C.M. Dorman T.E. LeVitre J. Davidow L.S. Mao J. Moir D.T. ( 1989) The yeast secretory pathway is perturbed by mutations in PMR1, a member of a Ca2+ ATPase family. Cell  58, 133– 145. Google Scholar CrossRef Search ADS PubMed  [16] Sakai A. Shimizzu Y. Hisshinuma F. ( 1988) Isolation and characterization of mutants which show an oversecretion phenotype in Saccharomyces cerevisiae. Genetics  119, 499– 506. Google Scholar PubMed  [17] Bartkevic̆iūt≐ D. Sasnauskas K. ( 2003) Studies of yeast Kluyveromyces lactis mutations conferring super-secretion of recombinant proteins. Yeast  20, 1– 11. Google Scholar CrossRef Search ADS PubMed  [18] Sambrook J. Russell D.W. ( 2001) Molecular cloning. A laboratory manual , third ed. CSHL Press, Cold Spring Harbor, New York. [19] Bartkevic̆iūt≐ D. Šiekštele R. Sasnauskas K. ( 2000) Heterologous expression of the Kluyveromyces marxianus endopolygalacturonase gene (EPG1) using versatile autonomously replicating vector for a wide range of host. Enzyme Microb. Technol.  26, 653– 656. Google Scholar CrossRef Search ADS PubMed  [20] Alani E. Cao L. Kleckner N. ( 1987) A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains. Genetics  116, 541– 545. Google Scholar CrossRef Search ADS PubMed  [21] Balou C.L. Isolation, characterization, and properties of Saccharomyces cerevisiae mnn mutants with nonconditional protein glycosylation defects. Methods in enzymology. vol. 185  ( 1990) Academic Press, New York 440– 470. [22] Uccelletti D. Pacelli V. Mancini P. Palleschi C. ( 2000) vga mutants of Kluyveromyces lactis show cell integrity defects. Yeast  16, 1161– 1171. Google Scholar CrossRef Search ADS PubMed  [23] Dean N. Poster J.B. ( 1996) Molecular and phenotypic analysis of the S. cerevisiae MNN10 gene identified a family of related glycosyltransferases. Glycobiology  6, 73– 81. Google Scholar CrossRef Search ADS PubMed  [24] Jungman J. Rayner J.C. Munro S. ( 1999) The Saccharomyces cerevisiae protein Mnn10p/Bed1p is a subunit of a Golgi mannosyltransferase complex. J. Biol. Chem.  274, 6579– 6585. Google Scholar CrossRef Search ADS PubMed  [25] Kojima H. Hashimoto H. Yoda K. ( 1999) Interaction among the subunits of Golgi membrane mannosyltransferase complexes of the yeast Saccharomyces cerevisiae. Biosci. Biotechnol. Biochem.  63, 1970– 1976. Google Scholar CrossRef Search ADS PubMed  [26] Mondesert G. Reed S.I. ( 1996) BED1, a gene encoding a galactosyltransferase homologue, is required for polarized growth and efficient bud emergence in Saccharomyces cerevisiae. J. Cell Biol.  132, 137– 151. Google Scholar CrossRef Search ADS PubMed  [27] Ballou L. Hitzeman R.A. Lewis M.S. Ballou C.E. ( 1991) Vanadate-resistant yeast mutants are defective in protein glycosylation. Proc. Natl. Acad. Sci. USA  88, 3209– 3212. Google Scholar CrossRef Search ADS   [28] Fleer R. Chen X.J. Amellal N. Yeg P. Fournier A. Guinet F. Gault N. Faucher N. Folliard F. Fukuhara H. Mayaux J.F. ( 1991) High-level secretion of correctly processed recombinant interleukin-1β in Kluyveromyces lactis. Gene  107, 285– 295. Google Scholar CrossRef Search ADS PubMed  [29] Fleer R. Yeh P. Amellal N. Maury I. Fournier A. Bacchetta F. Baduel P. Jung G. L'Hote H. Becquart J. Fukuhara H. Mayaux J.F. ( 1991) Stable multicopy vectors for high-level secretion of human serum albumin by Kluyveromyces lactis. Biotechnology (NY)  9, 968– 975. Google Scholar CrossRef Search ADS   [30] Wesolowski-Louvel M. Breunig K.D. Fukuhara H. ( 1996) Kluyveromyces lactis. In: Nonconventional yeasts in biotechnology  ( Wolf K. Ed.), pp. 139– 201 Springer-Verlag, Berlin. [31] Swennen D. Paul M-F. Vernis L. Beckerich J.-M. Fournier A. Gaillardin C. ( 2002) Secretion of active anti-Ras single-chain Fv antibody by the yeast Yarrowia lipolytica and Kluyveromyces lactis. Microbiology  148, 41– 50. Google Scholar CrossRef Search ADS PubMed  [32] Orlean P. ( 1997) Biogenesis of yeast wall and surface components. In: The molecular and cellular biology of the yeast Saccharomyces cerevisiae. Cell cycle and biology  ( Pringle J.R. Broach J.R. Jones E.W. Eds.), pp. 229– 256 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. [33] Klis F.M. Mol P. Hellingwerf K. Brul S. ( 2002) Dynamic of cell wall structure in Saccharomyces cerevisiae. FEMS Microbiol. Rev.  26, 239– 256. Google Scholar CrossRef Search ADS PubMed  © 2004 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved.
TI - Disruption of the MNN10 gene enhances protein secretion in Kluyveromyces lactis and Saccharomyces cerevisiae
JF - FEMS Yeast Research
DO - 10.1016/j.femsyr.2004.03.001
DA - 2004-09-01
UR - https://www.deepdyve.com/lp/oxford-university-press/disruption-of-the-mnn10-gene-enhances-protein-secretion-in-zwIDjVFMgI
SP - 833
EP - 840
VL - 4
IS - 8
DP - DeepDyve
ER -