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Volume 161, Issue 7

Multistep processing of the secretion leader of the extracellular protein Epx1 in and implications for protein localization Free

Abstract

Secretion leaders are required to direct nascent proteins to the secretory pathway. They are of interest in the study of intracellular protein transport, and are required for the production of secretory recombinant proteins. Secretion leaders are processed in two steps in the endoplasmic reticulum and Golgi. Although yeast cells typically contain about 150 proteins entering the secretory pathway, only a low number of proteins are actually secreted to the cell supernatant. Analysis of the secretome of the yeast revealed that the most abundant secretory protein, which we named Epx1, belongs to the cysteine-rich secretory protein family CRISP. Surprisingly, the Epx1 secretion leader undergoes a three-step processing on its way to the cell exterior instead of the usual two-step processing. The Kex2 cleavage site within the Epx1 leader is not conserved in the homologues of most other yeasts. We studied the effect of exchanging the Kex2-cleavage motif on the secretory behaviour of reporter proteins fused to variants of the Epx1 leader sequence, and observed mistargeting for some but not all of the variants using fluorescence microscopy. By targeting several recombinant human proteins for secretion, we revealed that a short variant of the leader sequence, as well as the Epx1 signal sequence alone, resulted in the correct N-termini of the secreted proteins. Both leader variants proved to be very efficient, even exceeding the secretion levels obtained with commonly used secretion leaders. Taken together, the novel Epx1 secretion leader sequences are a valuable tool for recombinant protein production as well as basic research of intracellular transport.

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© 2015 The Authors
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2015-07-01
2024-04-18
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References

  1. Almeida M.S., Cabral K.S., de Medeiros L.N., Valente A.P., Almeida F.C.L., Kurtenbach E. (2001). cDNA cloning and heterologous expression of functional cysteine-rich antifungal protein Psd1 in the yeast Pichia pastoris Arch Biochem Biophys 395199–207 [View Article][PubMed]. [Google Scholar]
  2. Bader O., Krauke Y., Hube B. (2008). Processing of predicted substrates of fungal Kex2 proteinases from Candida albicans C. glabrata Saccharomyces cerevisiae Pichia pastoris BMC Microbiol 8116 [View Article][PubMed]. [Google Scholar]
  3. Ballensiefen W., Schmitt H.D. (1997). Periplasmic Bar1 protease of Saccharomyces cerevisiae is active before reaching its extracellular destinationEur J Biochem 247142–147 [View Article][PubMed]. [Google Scholar]
  4. Baumann K., Maurer M., Dragosits M., Cos O., Ferrer P., Mattanovich D. (2008). Hypoxic fed-batch cultivation of Pichia pastoris increases specific and volumetric productivity of recombinant proteinsBiotechnol Bioeng 100177–183 [View Article][PubMed]. [Google Scholar]
  5. Bryant N.J., Boyd A. (1993). Immunoisolation of Kex2p-containing organelles from yeast demonstrates colocalisation of three processing proteinases to a single Golgi compartmentJ Cell Sci 106815–822[PubMed]. [Google Scholar]
  6. Bryant N.J., Stevens T.H. (1998). Vacuole biogenesis in Saccharomyces cerevisiae: protein transport pathways to the yeast vacuoleMicrobiol Mol Biol Rev 62230–247[PubMed]. [Google Scholar]
  7. Choudhary V., Schneiter R. (2012). Pathogen-related yeast (PRY) proteins and members of the CAP superfamily are secreted sterol-binding proteinsProc Natl Acad Sci U S A 10916882–16887 [View Article][PubMed]. [Google Scholar]
  8. Coughlan C.M., Walker J.L., Cochran J.C., Wittrup K.D., Brodsky J.L. (2004). Degradation of mutated bovine pancreatic trypsin inhibitor in the yeast vacuole suggests post-endoplasmic reticulum protein quality controlJ Biol Chem 27915289–15297 [View Article][PubMed]. [Google Scholar]
  9. Daly R., Hearn M.T. (2005). Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and productionJ Mol Recognit 18119–138 [View Article][PubMed]. [Google Scholar]
  10. Damasceno L.M., Huang C.J., Batt C.A. (2012). Protein secretion in Pichia pastoris and advances in protein productionAppl Microbiol Biotechnol 9331–39 [View Article][PubMed]. [Google Scholar]
  11. Delic M., Rebnegger C., Wanka F., Puxbaum V., Haberhauer-Troyer C., Hann S., Köllensperger G., Mattanovich D., Gasser B. (2012). Oxidative protein folding and unfolded protein response elicit differing redox regulation in endoplasmic reticulum and cytosol of yeastFree Radic Biol Med 522000–2012 [View Article][PubMed]. [Google Scholar]
  12. Delic M., Valli M., Graf A.B., Pfeffer M., Mattanovich D., Gasser B. (2013). The secretory pathway: exploring yeast diversityFEMS Microbiol Rev 37872–914[PubMed].[CrossRef] [Google Scholar]
  13. Devi L. (1991). Consensus sequence for processing of peptide precursors at monobasic sitesFEBS Lett 280189–194 [View Article][PubMed]. [Google Scholar]
  14. Dujon B. (2010). Yeast evolutionary genomicsNat Rev Genet 11512–524 [View Article][PubMed]. [Google Scholar]
  15. Dunn W.A. Jr, Cregg J.M., Kiel J.A.K.W., van der Klei I.J., Oku M., Sakai Y., Sibirny A.A., Stasyk O.V., Veenhuis M. (2005). Pexophagy: the selective autophagy of peroxisomesAutophagy 175–83 [View Article][PubMed]. [Google Scholar]
  16. Eiden-Plach A., Zagorc T., Heintel T., Carius Y., Breinig F., Schmitt M.J. (2004). Viral preprotoxin signal sequence allows efficient secretion of green fluorescent protein by Candida glabrata Pichia pastoris Saccharomyces cerevisiae, Schizosaccharomyces pombe Appl Environ Microbiol 70961–966 [View Article][PubMed]. [Google Scholar]
  17. Emr S.D., Schekman R., Flessel M.C., Thorner J. (1983). An MFα1-SUC2 (α-factor-invertase) gene fusion for study of protein localization and gene expression in yeastProc Natl Acad Sci U S A 807080–7084 [View Article][PubMed]. [Google Scholar]
  18. Fitzgerald I., Glick B.S. (2014). Secretion of a foreign protein from budding yeasts is enhanced by cotranslational translocation and by suppression of vacuolar targetingMicrob Cell Fact 13125 [View Article][PubMed]. [Google Scholar]
  19. Fuller R.S., Brake A.J., Thorner J. (1989). Intracellular targeting and structural conservation of a prohormone-processing endoproteaseScience 246482–486 [View Article][PubMed]. [Google Scholar]
  20. Gagnon-Arsenault I., Tremblay J., Bourbonnais Y. (2006). Fungal yapsins and cell wall: a unique family of aspartic peptidases for a distinctive cellular functionFEMS Yeast Res 6966–978 [View Article][PubMed]. [Google Scholar]
  21. Gasser B., Prielhofer R., Marx H., Maurer M., Nocon J., Steiger M., Puxbaum V., Sauer M., Mattanovich D. (2013). Pichia pastoris: protein production host and model organism for biomedical researchFuture Microbiol 8191–208 [View Article][PubMed]. [Google Scholar]
  22. Ghosalkar A., Sahai V., Srivastava A. (2008). Secretory expression of interferon-alpha 2b in recombinant Pichia pastoris using three different secretion signalsProtein Expr Purif 60103–109 [View Article][PubMed]. [Google Scholar]
  23. Govindappa N., Hanumanthappa M., Venkatarangaiah K., Periyasamy S., Sreenivas S., Soni R., Sastry K. (2014). A new signal sequence for recombinant protein secretion in Pichia pastoris J Microbiol Biotechnol 24337–345 [View Article][PubMed]. [Google Scholar]
  24. Hashimoto Y., Koyabu N., Imoto T. (1998). Effects of signal sequences on the secretion of hen lysozyme by yeast: construction of four secretion cassette vectorsProtein Eng 1175–77 [View Article][PubMed]. [Google Scholar]
  25. Heiss S., Maurer M., Hahn R., Mattanovich D., Gasser B. (2013). Identification and deletion of the major secreted protein of Pichia pastoris Appl Microbiol Biotechnol 971241–1249 [View Article][PubMed]. [Google Scholar]
  26. Holkeri H., Makarow M. (1998). Different degradation pathways for heterologous glycoproteins in yeastFEBS Lett 429162–166 [View Article][PubMed]. [Google Scholar]
  27. Johnson L.M., Bankaitis V.A., Emr S.D. (1987). Distinct sequence determinants direct intracellular sorting and modification of a yeast vacuolar proteaseCell 48875–885 [View Article][PubMed]. [Google Scholar]
  28. Julius D., Schekman R., Thorner J. (1984). Glycosylation and processing of prepro-alpha-factor through the yeast secretory pathwayCell 36309–318 [View Article][PubMed]. [Google Scholar]
  29. Kleizen B., Braakman I. (2004). Protein folding and quality control in the endoplasmic reticulumCurr Opin Cell Biol 16343–349 [View Article][PubMed]. [Google Scholar]
  30. Klionsky D.J., Herman P.K., Emr S.D. (1990). The fungal vacuole: composition, function, and biogenesisMicrobiol Rev 54266–292[PubMed]. [Google Scholar]
  31. Kobayashi K. (2006). Summary of recombinant human serum albumin developmentBiologicals 3455–59 [View Article][PubMed]. [Google Scholar]
  32. Kobayashi K., Kuwae S., Ohya T., Ohda T., Ohyama M., Tomomitsu K. (2000). High level secretion of recombinant human serum albumin by fed-batch fermentation of the methylotrophic yeast, Pichia pastoris, based on optimal methanol feeding strategyJ Biosci Bioeng 90280–288 [View Article][PubMed]. [Google Scholar]
  33. Kottmeier K., Ostermann K., Bley T., Rödel G. (2011). Hydrophobin signal sequence mediates efficient secretion of recombinant proteins in Pichia pastoris Appl Microbiol Biotechnol 91133–141 [View Article][PubMed]. [Google Scholar]
  34. Kunze I., Hensel G., Adler K., Bernard J., Neubohn B., Nilsson C., Stoltenburg R., Kohlwein S.D., Kunze G. (1999). The green fluorescent protein targets secretory proteins to the yeast vacuoleBiochim Biophys Acta 1410287–298 [View Article][PubMed]. [Google Scholar]
  35. Kurtzman C.P. (2005). Description of Komagataella phaffii sp. nov. and the transfer of Pichia pseudopastoris to the methylotrophic yeast genus Komagataella Int J Syst Evol Microbiol 55973–976 [View Article][PubMed]. [Google Scholar]
  36. Lin-Cereghino G.P., Stark C.M., Kim D., Chang J., Shaheen N., Poerwanto H., Agari K., Moua P., Low L.K., other authors. (2013). The effect of α-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris Gene 519311–317 [View Article][PubMed]. [Google Scholar]
  37. Macauley-Patrick S., Fazenda M.L., McNeil B., Harvey L.M. (2005). Heterologous protein production using the Pichia pastoris expression systemYeast 22249–270 [View Article][PubMed]. [Google Scholar]
  38. Martoglio B., Dobberstein B. (1998). Signal sequences: more than just greasy peptidesTrends Cell Biol 8410–415 [View Article][PubMed]. [Google Scholar]
  39. Mattanovich D., Graf A., Stadlmann J., Dragosits M., Redl A., Maurer M., Kleinheinz M., Sauer M., Altmann F., Gasser B. (2009). Genome, secretome and glucose transport highlight unique features of the protein production host Pichia pastoris Microb Cell Fact 829 [View Article][PubMed]. [Google Scholar]
  40. Mattanovich D., Branduardi P., Dato L., Gasser B., Sauer M., Porro D. (2012). Recombinant protein production in yeastsMethods Mol Biol 824329–358 [View Article][PubMed]. [Google Scholar]
  41. Ng D.T., Brown J.D., Walter P. (1996). Signal sequences specify the targeting route to the endoplasmic reticulum membraneJ Cell Biol 134269–278 [View Article][PubMed]. [Google Scholar]
  42. Parr C.L., Keates R.A., Bryksa B.C., Ogawa M., Yada R.Y. (2007). The structure and function of Saccharomyces cerevisiae proteinase AYeast 24467–480 [View Article][PubMed]. [Google Scholar]
  43. Prielhofer R., Maurer M., Klein J., Wenger J., Kiziak C., Gasser B., Mattanovich D. (2013). Induction without methanol: novel regulated promoters enable high-level expression in Pichia pastoris Microb Cell Fact 125 [View Article][PubMed]. [Google Scholar]
  44. Rockwell N.C., Krysan D.J., Komiyama T., Fuller R.S. (2002). Precursor processing by kex2/furin proteasesChem Rev 1024525–4548 [View Article][PubMed]. [Google Scholar]
  45. Rossanese O.W., Soderholm J., Bevis B.J., Sears I.B., O'Connor J., Williamson E.K., Glick B.S. (1999). Golgi structure correlates with transitional endoplasmic reticulum organization in Pichia pastoris Saccharomyces cerevisiae J Cell Biol 14569–81 [View Article][PubMed]. [Google Scholar]
  46. Ruggiano A., Foresti O., Carvalho P. (2014). Quality control: ER-associated degradation: protein quality control and beyondJ Cell Biol 204869–879 [View Article][PubMed]. [Google Scholar]
  47. Salunkhe S., Soorapaneni S., Prasad K.S., Raiker V.A., Padmanabhan S. (2010). Strategies to maximize expression of rightly processed human interferon alpha2b in Pichia pastoris Protein Expr Purif 71139–146 [View Article][PubMed]. [Google Scholar]
  48. Schaefer J.V., Plückthun A. (2012). Engineering aggregation resistance in IgG by two independent mechanisms: lessons from comparison of Pichia pastoris and mammalian cell expressionJ Mol Biol 417309–335 [View Article][PubMed]. [Google Scholar]
  49. Stadlmayr G., Mecklenbräuker A., Rothmüller M., Maurer M., Sauer M., Mattanovich D., Gasser B. (2010). Identification and characterisation of novel Pichia pastoris promoters for heterologous protein productionJ Biotechnol 150519–529 [View Article][PubMed]. [Google Scholar]
  50. Steinlein L.M., Graf T.N., Ikeda R.A. (1995). Production and purification of N-terminal half-transferrin in Pichia pastoris Protein Expr Purif 6619–624 [View Article][PubMed]. [Google Scholar]
  51. Suda Y., Nakano A. (2012). The yeast Golgi apparatusTraffic 13505–510 [View Article][PubMed]. [Google Scholar]
  52. Thibault G., Ng D.T.W. (2012). The endoplasmic reticulum-associated degradation pathways of budding yeastCold Spring Harb Perspect Biol 4a013193 [View Article][PubMed]. [Google Scholar]
  53. Tschopp J.F., Brust P.F., Cregg J.M., Stillman C.A., Gingeras T.R. (1987). Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris Nucleic Acids Res 153859–3876 [View Article][PubMed]. [Google Scholar]
  54. Valls L.A., Hunter C.P., Rothman J.H., Stevens T.H. (1987). Protein sorting in yeast: the localization determinant of yeast vacuolar carboxypeptidase Y resides in the propeptideCell 48887–897 [View Article][PubMed]. [Google Scholar]
  55. Vida T.A., Emr S.D. (1995). A new vital stain for visualizing vacuolar membrane dynamics and endocytosis in yeastJ Cell Biol 128779–792 [View Article][PubMed]. [Google Scholar]
  56. von Heijne G. (1984). Analysis of the distribution of charged residues in the N-terminal region of signal sequences: implications for protein export in prokaryotic and eukaryotic cellsEMBO J 32315–2318[PubMed]. [Google Scholar]
  57. Wang P., Huang L., Jiang H., Tian J., Chu X., Wu N. (2014). Improving the secretion of a methyl parathion hydrolase in Pichia pastoris by modifying its N-terminal sequencePLoS One 9e96974 [View Article][PubMed]. [Google Scholar]
  58. Waters M.G., Evans E.A., Blobel G. (1988). Prepro-alpha-factor has a cleavable signal sequenceJ Biol Chem 2636209–6214[PubMed]. [Google Scholar]
  59. Zhang B., Chang A., Kjeldsen T.B., Arvan P. (2001). Intracellular retention of newly synthesized insulin in yeast is caused by endoproteolytic processing in the Golgi complexJ Cell Biol 1531187–1198 [View Article][PubMed]. [Google Scholar]
  60. Zhao H.L., He Q., Xue C., Sun B., Yao X.Q., Liu Z.M. (2009). Secretory expression of glycosylated and aglycosylated mutein of onconase from Pichia pastoris using different secretion signals and their purification and characterizationFEMS Yeast Res 9591–599 [View Article][PubMed]. [Google Scholar]
  61. Zimmermann R., Eyrisch S., Ahmad M., Helms V. (2011). Protein translocation across the ER membraneBiochim Biophys Acta 1808912–924 [View Article][PubMed]. [Google Scholar]
  62. Zsebo K.M., Lu H.S., Fieschko J.C., Goldstein L., Davis J., Duker K., Suggs S.V., Lai P.H., Bitter G.A. (1986). Protein secretion from Saccharomyces cerevisiae directed by the prepro-alpha-factor leader regionJ Biol Chem 2615858–5865[PubMed]. [Google Scholar]
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Multistep processing of the secretion leader of the extracellular protein Epx1 in Pichia pastoris and implications for protein localization
Microbiology 161, 1356 (2015); https://doi.org/10.1099/mic.0.000105
/content/journal/micro/10.1099/mic.0.000105
/content/journal/micro/10.1099/mic.0.000105
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