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Regulating the Golgi apparatus sorting of proteinase A to decrease its excretion in Saccharomyces cerevisiae

  • Lulu Song
  • Yefu ChenEmail author
  • Qinghuan Guo
  • Siyao Huang
  • Xuewu Guo
  • Dongguang Xiao
Fermentation, Cell Culture and Bioengineering - Original Paper
  • 17 Downloads

Abstract

Beer foam stability, a key factor in evaluating overall beer quality, is influenced by proteinase A (PrA). Actin-severing protein cofilin and Golgi apparatus-localized Ca2+ ATPase Pmr1 are involved in protein sorting at the trans-Golgi network (TGN) in yeast Curwin et al. (Mol Biol Cell 23:2327–2338, 2012). To reduce PrA excretion into the beer fermentation broth, we regulated the Golgi apparatus sorting of PrA, thereby facilitating the delivery of more PrA to the vacuoles in the yeast cells. In the present study, the cofilin-coding gene COF1 and the Pmr1-coding gene PMR1 were overexpressed in the parental strain W303-1A and designated as W + COF1 and W + PMR1, respectively. The relative expression levels of COF1 in W + COF1 and PMR1 in W + PMR1 were 5.26- and 19.76-fold higher than those in the parental strain. After increases in the expression levels of cofilin and Pmr1 were confirmed, the PrA activities in the wort broth fermented with W + COF1, W + PMR1, and W303-1A were measured. Results showed that the extracellular PrA activities of W + COF1 and W + PMR1 were decreased by 9.24% and 13.83%, respectively, at the end of the main fermentation compared with that of W303-1A. Meanwhile, no apparent differences were found on the fermentation performance of recombinant and parental strains. The research uncovers an effective strategy for decreasing PrA excretion in Saccharomyces cerevisiae.

Keywords

Proteinase A (PrA) Beer foam COF1 PMR1 trans-Golgi network sorting 

Notes

Acknowledgements

The current study was financially supported by the program for the National Natural Science Foundation of China (No. 31271916).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.

References

  1. 1.
    Antebi A, Fink GR (1992) The yeast Ca(2 +)-ATPase homologue, PMR1, is required for normal Golgi function and localizes in a novel Golgi-like distribution. Mol Biol Cell 3:633–654.  https://doi.org/10.1091/mbc.3.6.633 CrossRefGoogle Scholar
  2. 2.
    Baranski TJ, Faust PL, Kornfeld S (1990) Generation of a lysosomal enzyme targeting signal in the secretory protein pepsinogen. Cell 63:281–291.  https://doi.org/10.1016/0092-8674(90)90161-7 CrossRefGoogle Scholar
  3. 3.
    Bonangelino CJ, Chavez EM, Bonifacino JS (2002) Genomic screen for vacuolar protein sorting genes in Saccharomyces cerevisiae. Mol Biol Cell 13:2486–2501.  https://doi.org/10.1091/mbc.02-01-0005 CrossRefGoogle Scholar
  4. 4.
    Bonifacino JS, Glick BS (2004) The mechanisms of vesicle budding and fusion. Cell 116:153–166.  https://doi.org/10.1016/S0092-8674(03)01079-1 CrossRefGoogle Scholar
  5. 5.
    Chen Q, Liu X, Fu M, Zhang H (2010) Effect of PrA encoding gene-PEP4 deletion in industrial S. cerevisiae WZ65 on key enzymes in relation to the glycolytic pathway. Eur Food Res Technol 231:943–950.  https://doi.org/10.1007/s00217-010-1355-y CrossRefGoogle Scholar
  6. 6.
    Chen Y, Song L, Han Y et al (2017) Decreased proteinase a excretion by strengthening its vacuolar sorting and weakening its constitutive secretion in Saccharomyces cerevisiae. J Ind Microbiol Biotechnol 44:149–159.  https://doi.org/10.1007/s10295-016-1868-x CrossRefGoogle Scholar
  7. 7.
    Cooper AA, Stevens TH (1996) Vps10p cycles between the late-Golgi and prevacuolar compartments in its function as the sorting receptor for multiple yeast vacuolar hydrolases. J Cell Biol 133:529–541.  https://doi.org/10.1083/jcb.133.3.529 CrossRefGoogle Scholar
  8. 8.
    Cooper DJ, Stewart GG, Bryce JH (2000) Yeast proteolytic activity during high and low gravity wort fermentations and its effect on head retention. J Inst Brew 106:197–201.  https://doi.org/10.1002/j.2050-0416.2000.tb00057.x CrossRefGoogle Scholar
  9. 9.
    Curwin AJ, von Blume J, Malhotra V (2012) Cofilin-mediated sorting and export of specific cargo from the Golgi apparatus in yeast. Mol Biol Cell 23:2327–2338.  https://doi.org/10.1091/mbc.e11-09-0826 CrossRefGoogle Scholar
  10. 10.
    Dreyer T, Halkier B, Svendsen I et al (1986) Primary structure of the aspartic proteinase a from Saccharomyces cerevisiae. Carlsberg Res Commun 51:27–41.  https://doi.org/10.1007/BF02907993 CrossRefGoogle Scholar
  11. 11.
    Dürr G, Strayle J, Plemper R et al (1998) The medial-Golgi ion pump Pmr1 supplies the yeast secretory pathway with Ca2+ and Mn2+ required for glycosylation, sorting, and endoplasmic reticulum associated protein degradation. Mol Biol Cell 9:1149–1162.  https://doi.org/10.1091/mbc.9.5.1149 CrossRefGoogle Scholar
  12. 12.
    Gietz RD, Woods RA (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350:87–96.  https://doi.org/10.1016/S0076-6879(02)50957-5 CrossRefGoogle Scholar
  13. 13.
    Gueldener U, Heinisch J, Koehler GJ et al (2002) A second set of loxP marker cassettes for cre-mediated multiple gene knockouts in budding yeast. Nucl Acid Res 30:e23.  https://doi.org/10.1093/nar/30.6.e2 CrossRefGoogle Scholar
  14. 14.
    Harris SD, Cotter DA (1987) Vacuolar (lysosomal) trehalase of Saccharomyces cerevisiae. Curr Microbiol 15:247–249.  https://doi.org/10.1007/BF01589375 CrossRefGoogle Scholar
  15. 15.
    Hata T, Hayashi R, Doi E (1967) Purifcation of yeast proteinases: part III. Isolation and physicochemical properties of yeast proteinase A and C. Agric Biol Chem 31:357–367.  https://doi.org/10.1271/bbb1961.31.150 Google Scholar
  16. 16.
    He GQ, Wang ZY, Liu ZS et al (2006) Relationship of proteinase activity, foam proteins, and head retention in unpasteurized beer. J Am Soc Brew Chem 64:33–38Google Scholar
  17. 17.
    Jones EW (1984) The synthesis and function of proteases in saccharomyces: genetic approaches. Ann Rev Gene 18:233–270.  https://doi.org/10.1146/annurev.ge.18.120184.001313 CrossRefGoogle Scholar
  18. 18.
    Kienzle C, Basnet N, Crevenna AH et al (2014) Cofilin recruits F-actin to SPCA1 and promotes Ca2+-mediated secretory cargo sorting. J Cell Biol 206:635.  https://doi.org/10.1083/jcb.201311052 CrossRefGoogle Scholar
  19. 19.
    Kienzle C, von Blume J (2014) Secretory cargo sorting at the trans-Golgi network. Trends Cell Biol 24:584–593.  https://doi.org/10.1016/j.tcb.2014.04.007 CrossRefGoogle Scholar
  20. 20.
    Klionsky DJ, Cueva R, Yaver DS (1992) Aminopeptidase I of Saccharomyces cerevisiae is localized to the vacuole independent of the secretory pathway. J Cell Biol 119:287–299.  https://doi.org/10.1083/jcb.119.2.287 CrossRefGoogle Scholar
  21. 21.
    Kondo H, Yomo H, Furukubo S et al (1999) Advanced method for measuring proteinase A in beer and application to brewing. J Inst Brew 105:293–300.  https://doi.org/10.1002/j.2050-0416.1999.tb00523.x CrossRefGoogle Scholar
  22. 22.
    Lee MC, Miller EA, Goldberg J et al (2004) Bi-directional protein transport between the ER and Golgi. Annu Rev Cell Dev Biol 20:87–123.  https://doi.org/10.1006/dbio.2001.0416 CrossRefGoogle Scholar
  23. 23.
    Lenney JF, Dalbec JM (1967) Purifcation and properties of two proteinases from Saccharomyces cerevisiae. Arch Biochem Biophys 120:42–48.  https://doi.org/10.1016/0003-9861(67)90595-4 CrossRefGoogle Scholar
  24. 24.
    Lilly M, Lambrechts MG, Pretorius IS (2000) Effect of increased yeast alcohol acetyltransferase activity on flavor profiles of wine and distillates. Appl Environ Microbiol 66:744–753.  https://doi.org/10.1128/AEM.66.2.744-753.20 CrossRefGoogle Scholar
  25. 25.
    Lu J, Dong J, Wu D et al (2012) Construction of recombinant industrial brewer’s yeast with lower diacetyl production and proteinase A activity. Eur Food Res Technol 235:951–961.  https://doi.org/10.1007/s00217-012-1821-9 CrossRefGoogle Scholar
  26. 26.
    Marcusson EG, Horazdovsky BF, Cereghino JL et al (1994) The sorting receptor for yeast vacuolar carboxypeptidase Y is encoded by the VPS10 gene. Cell 77:579–586.  https://doi.org/10.1016/0092-8674(94)90219-4 CrossRefGoogle Scholar
  27. 27.
    Okreglak V, Drubin DG (2007) Coflin recruitment and function during actinmediated endocytosis dictated by actin nucleotide state. J Cell Biol 178:1251–1264.  https://doi.org/10.1083/jcb.200703092 CrossRefGoogle Scholar
  28. 28.
    Paravicini G, Horazdovsky BF, Emr SD (1992) Alternative pathways for the sorting of soluble vacuolar proteins in yeast: a vps35 null mutant missorts and secretes only a subset of vacuolar hydrolases. Mol Biol Cell 3:415–427.  https://doi.org/10.1091/mbc.3.4.415 CrossRefGoogle Scholar
  29. 29.
    Parr CL, Keates RAB, Bryksa BC et al (2007) The structure and function of Saccharomyces cerevisiae proteinase A. Yeast 24:467–480.  https://doi.org/10.1002/yea.1485 CrossRefGoogle Scholar
  30. 30.
    Pryer NK, Wuestehube LJ, Schekman R (1992) Vesicle-mediated protein sorting. Annu Rev Biochem 61:471–516.  https://doi.org/10.1146/annurev.bi.61.070192.002351 CrossRefGoogle Scholar
  31. 31.
    Rudolph HK, Antebi A, Fink GR et al (1989) The yeast secretory pathway is perturbed by mutations in PMR1, a member of a Ca2+ ATPase family. Cell 58:133–145.  https://doi.org/10.1016/0092-8674(89)90410-8 CrossRefGoogle Scholar
  32. 32.
    Shao Z, Zhao H, Zhao H (2009) DNA assembler, an in vivo genetic method for rapid construction of biochemical pathways. Nucleic Acids Res 37:e16.  https://doi.org/10.1093/nar/gkn991 CrossRefGoogle Scholar
  33. 33.
    Simões I, Faro C (2004) Structure and function of plant aspartic proteinases. Eur J Biochem 271:2067–2075.  https://doi.org/10.1111/j.1432-1033.2004.04136.x CrossRefGoogle Scholar
  34. 34.
    Song L, Chen Y, Dong Y et al (2017) Saccharomyces cerevisiae proteinase A excretion and wine making. World J Microbiol Biotechnol 33:210.  https://doi.org/10.1007/s11274-017-2361-z CrossRefGoogle Scholar
  35. 35.
    Teichert U, Mechlere B, Müller H et al (1989) Lysosomal (vacuolar) proteinases of yeast are essential catalysts for protein degradation, dierentiation, and cell survival. J Biol Chem 264:16037–16045Google Scholar
  36. 36.
    Teste MA, Duquenne M, Francois JM, Parrou JL (2009) Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae. BMC Mol Biol 10:99.  https://doi.org/10.1186/1471-2199-10-99 CrossRefGoogle Scholar
  37. 37.
    van den Hazel HB, Kielland-brandt MC, Winther JR (1992) Autoactivation of proteinase A initiates activation of yeast vacuolar zymogens. Eur J Biochem 207:277–283.  https://doi.org/10.1111/j.1432-1033.1992.tb17048.x CrossRefGoogle Scholar
  38. 38.
    Van Nierop SNE, Evans DE, Axcell BC et al (2004) Impact of different wort boiling temperatures on the beer foam stabilizing properties of lipid transfer protein 1. J Agric Food Chem 52:3120–3129.  https://doi.org/10.1021/jf035125c CrossRefGoogle Scholar
  39. 39.
    von Blume J, Alleaume AM, Cantero-Recasens G et al (2011) ADF/coflin regulates secretory cargo sorting at the TGN via the Ca2+ ATPase SPCA1. Dev Cell 20:652–662.  https://doi.org/10.1016/j.devcel.2011.03.014 CrossRefGoogle Scholar
  40. 40.
    von Blume J, Alleaume AM, Kienzle C et al (2012) Cab45 is required for Ca2+-dependent secretory cargo sorting at the trans-Golgi network. J Cell Biol 199:1057–1066.  https://doi.org/10.1083/jcb.201207180 CrossRefGoogle Scholar
  41. 41.
    von Blume J, Duran JM, Forlanelli E et al (2009) Actin remodeling by ADF/coflin is required for cargo sorting at the trans-Golgi network. J Cell Biol 187:1055–1069.  https://doi.org/10.1083/jcb.200908040 CrossRefGoogle Scholar
  42. 42.
    Wan K, Yabuki Y, Mizuta K (2015) Roles of Ebp2 and ribosomal protein L36 in ribosome biogenesis in Saccharomyces cerevisiae. Curr Genet 61:31–41.  https://doi.org/10.1007/s00294-014-0442-1 CrossRefGoogle Scholar
  43. 43.
    Wang ZY, He GQ, Liu ZS et al (2005) Purifcation of yeast proteinase A from fresh beer and its specifcity on foam proteins. Int J Food Sci Technol 40:835–840.  https://doi.org/10.1111/j.1365-2621.2005.01000.x CrossRefGoogle Scholar
  44. 44.
    Westphal V, Marcusson EG, Winther JR et al (1996) Multiple pathways for vacuolar sorting of yeast proteinase A. J Biol Chem 271:11865–11870.  https://doi.org/10.1074/jbc.271.20.11865 CrossRefGoogle Scholar
  45. 45.
    Whyte JRC, Munro S (2001) A yeast homolog of the mammalian mannose 6-phosphate receptors contributes to the sorting of vacuolar hydrolases. Curr Biol 11:1074–1078.  https://doi.org/10.1016/S0960-9822(01)00273-1 CrossRefGoogle Scholar
  46. 46.
    Zhang HB, Ruan H, Li WF et al (2011) Construction of recombinant industrial S. cerevisiae strain with barley lipid-transfer protein 1 secretion capability and lower PrA activity. Eur Food Res Technol 233:707–716.  https://doi.org/10.1007/s00217-011-1559-9 CrossRefGoogle Scholar
  47. 47.
    Zhu B, Cai G, Hall EO, Freeman GJ (2007) In-fusion™ assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations. Biotechniques 43:354–359.  https://doi.org/10.2144/000112536 CrossRefGoogle Scholar
  48. 48.
    Zubenko GS, Park FJ, Jones EW (1982) Genetic properties of mutations at the PEP4 locus in Saccharomyces cerevisiae. Genetics 102:679–690Google Scholar

Copyright information

© Society for Industrial Microbiology and Biotechnology 2019

Authors and Affiliations

  1. 1.Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin Industrial Microbiology Key Laboratory, College of BiotechnologyTianjin University of Science and TechnologyTianjinPeople’s Republic of China

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