Skip to main content

Advertisement

SpringerLink
Log in

Intracellular interactome of secreted antibody Fab fragment in Pichia pastoris reveals its routes of secretion and degradation

  • Genomics, transcriptomics, proteomics
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Protein translation, translocation, folding, processing, and secretion in eukaryotic cells are complex and not always straightforward processes, e.g., different routes of secretion and degradation exist. Formation of malfolded proteins in the endoplasmic reticulum (ER) can be one of the major bottlenecks for recombinant protein production. In this regard, an in-depth analysis of the interactions of a secreted protein during its pathway through the cell may be beneficial, as realized in this study for the methylotrophic yeast Pichia pastoris. The antibody fragment Fab3H6 used here is the anti-idiotype to the HIV neutralizing antibody 2F5 and is known to be intracellularly degraded in significant amounts when expressed in P. pastoris. The interactome of Fab3H6 was analyzed by using a pull-down mass spectrometry approach, and 23 proteins were found to bind specifically to the antibody fragment. Those allowed concluding that Fab3H6 is post-translationally translocated into the ER and degraded via the proteasome as well as the vacuole. In line with this, the expression of Fab3H6 increased the proteasomal activities by over 20%. Partial inhibition of the proteasome resulted in a significant increase of extracellular Fab3H6. Thus, it seems that ER quality control overshoots its requirements for the recombinant protein expressed and that more than just terminally malfolded protein is degraded by ER-associated degradation. This work will further facilitate our understanding how recombinant proteins behave in the secretory pathway.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Aguilar PS, Fröhlich F, Rehman M, Shales M, Ulitsky M, Olivera-Couto A, Braberg H, Shamir R, Walter P, Mann M, Ejsing CS, Krogan NJ, Walther TC (2010) A plasma-membrane E-MAP reveals links of the eisosome with sphingolipid metabolism and endosomal trafficking. Nat Struct Mol Biol 17:901–908

    Article  CAS  Google Scholar 

  • Anelli T, Sitia R (2008) Protein quality control in the early secretory pathway. EMBO J 27:315–327

    Article  CAS  Google Scholar 

  • Austin AJ, Jones CE, Heeke GV (1998) Production of human tissue factor using the Pichia pastoris expression system. Protein Expr Purif 13:136–142

    Article  CAS  Google Scholar 

  • Barlowe C (1997) Coupled ER to Golgi transport reconstituted with purified cytosolic proteins. J Cell Biol 139:1097–1108

    Article  CAS  Google Scholar 

  • 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 proteins. Biotechnol Bioeng 100:177–183

    Article  CAS  Google Scholar 

  • Bonifacino J, Dell'Angelica E, Springer T (2001) Immunoprecipitation. Curr Protoc Mol Biol Chapter 10:Unit 10.16. Wiley-Blackwell, Hoboken

    Google Scholar 

  • Brückner A, Polge C, Lentze N, Auerbach D, Schlattner U (2009) Yeast two-hybrid, a powerful tool for systems biology. Int J Mol Sci 10:2763–2788

    Article  Google Scholar 

  • Buchetics M, Dragosits M, Maurer M, Rebnegger C, Porro D, Sauer M, Gasser B, Mattanovich D (2011) Reverse engineering of protein secretion by uncoupling of cell cycle phases from growth. Biotechnol Bioeng 108:2403–2412

    Article  CAS  Google Scholar 

  • Bush KT, Goldberg AL, Nigam SK (1997) Proteasome inhibition leads to a heat-shock response, induction of endoplasmic reticulum chaperones, and thermotolerance. J Biol Chem 272:9086–9092

    Article  CAS  Google Scholar 

  • Caplan AJ, Cyr DM, Douglas MG (1992) YDJ1p facilitates polypeptide translocation across different intracellular membranes by a conserved mechanism. Cell 71:1143–1155

    Article  CAS  Google Scholar 

  • Craven RA, Egerton M, Stirling CJ (1996) A novel Hsp70 of the yeast ER lumen is required for the efficient translocation of a number of protein precursors. EMBO J 15:2640–2650

    CAS  Google Scholar 

  • D'haene B, Vandesompele J, Hellemans J (2010) Accurate and objective copy number profiling using real-time quantitative PCR. Methods 50:262–270

    Article  Google Scholar 

  • de Bettignies G, Coux O (2010) Proteasome inhibitors: dozens of molecules and still counting. Biochimie 92:1530–1545

    Article  Google Scholar 

  • Delic M, Mattanovich D, Gasser B (2010) Monitoring intracellular redox conditions in the endoplasmic reticulum of living yeasts. FEMS Microbiol Lett 306:61–66

    Article  CAS  Google Scholar 

  • Dembla-Rajpal N, Seipelt R, Wang Q, Rymond BC (2004) Proteasome inhibition alters the transcription of multiple yeast genes. Biochim Biophys Acta 1680:34–45

    CAS  Google Scholar 

  • Dragosits M, Stadlmann J, Albiol J, Baumann K, Maurer M, Gasser B, Sauer M, Altmann F, Ferrer P, Mattanovich D (2009) The effect of temperature on the proteome of recombinant Pichia pastoris. J Proteome Res 8:1380–1392

    Article  CAS  Google Scholar 

  • Dragosits M, Stadlmann J, Graf A, Gasser B, Maurer M, Sauer M, Kreil DP, Altmann F, Mattanovich D (2010) The response to unfolded protein is involved in osmotolerance of Pichia pastoris. BMC Genomics 11:207

    Article  Google Scholar 

  • Dube DH, Li B, Greenblatt EJ, Nimer S, Raymond AK, Kohler JJ (2010) A two-hybrid assay to study protein interactions within the secretory pathway. PLoS One 5:e15648

    Article  CAS  Google Scholar 

  • Dudek J, Benedix J, Cappel S, Greiner M, Jalal C, Müller L, Zimmermann R (2009) Functions and pathologies of BiP and its interaction partners. Cell Mol Life Sci 66:1556–1569

    Article  CAS  Google Scholar 

  • Forgac M (1999) Structure and properties of the vacuolar (H+)-ATPases. J Biol Chem 274:12951–12954

    Article  CAS  Google Scholar 

  • Friedlander R, Jarosch E, Urban J, Volkwein C, Sommer T (2000) A regulatory link between ER-associated protein degradation and the unfolded-protein response. Nat Cell Biol 2:379–384

    Article  CAS  Google Scholar 

  • Gach J, Quendler H, Strobach S, Katinger H, Kunert R (2008) Structural analysis and in vivo administration of an anti-idiotypic antibody against mAb 2F5. Mol Immunol 45:1027–1034

    Article  CAS  Google Scholar 

  • Gagny B, Wiederkehr A, Dumoulin P, Winsor B, Riezman H, Haguenauer-Tsapis R (2000) A novel EH domain protein of Saccharomyces cerevisiae, Ede1p, involved in endocytosis. J Cell Sci 113(Pt 18):3309–3319

    CAS  Google Scholar 

  • Glickman MH, Rubin DM, Coux O, Wefes I, Pfeifer G, Cjeka Z, Baumeister W, Fried VA, Finley D (1998) A subcomplex of the proteasome regulatory particle required for ubiquitin-conjugate degradation and related to the COP9-signalosome and eIF3. Cell 94:615–623

    Article  CAS  Google Scholar 

  • Graham TR (2004) Flippases and vesicle-mediated protein transport. Trends Cell Biol 14:670–677

    Article  CAS  Google Scholar 

  • Guerfal M, Ryckaert S, Jacobs PP, Ameloot P, Van Craenenbroeck K, Derycke R, Callewaert N (2010) The HAC1 gene from Pichia pastoris: characterization and effect of its overexpression on the production of secreted, surface displayed and membrane proteins. Microb Cell Factories 9:49

    Article  Google Scholar 

  • Gurunathan S, David D, Gerst JE (2002) Dynamin and clathrin are required for the biogenesis of a distinct class of secretory vesicles in yeast. EMBO J 21:602–614

    Article  CAS  Google Scholar 

  • Harsay E, Bretscher A (1995) Parallel secretory pathways to the cell surface in yeast. J Cell Biol 131:297–310

    Article  CAS  Google Scholar 

  • Hirsch C, Gauss R, Horn S, Neuber O, Sommer T (2009) The ubiquitylation machinery of the endoplasmic reticulum. Nature 458:453–460

    Article  CAS  Google Scholar 

  • Holkeri H, Makarow M (1998) Different degradation pathways for heterologous glycoproteins in yeast. FEBS Lett 429:162–166

    Article  CAS  Google Scholar 

  • Idiris A, Tohda H, Sasaki M, Okada K, Kumagai H, Giga-Hama Y, Takegawa K (2010) Enhanced protein secretion from multiprotease-deficient fission yeast by modification of its vacuolar protein sorting pathway. Appl Microbiol Biotechnol 85:667–677

    Article  CAS  Google Scholar 

  • Kim HM, Yu Y, Cheng Y (2011) Structure characterization of the 26S proteasome. Biochim Biophys Acta 1809:67–79

    CAS  Google Scholar 

  • Kolarich D, Weber A, Turecek PL, Schwarz HP, Altmann F (2006) Comprehensive glyco-proteomic analysis of human α1-antitrypsin and its charge isoforms. Proteomics 6:3369–3380

    Article  CAS  Google Scholar 

  • Kunert R, Weik R, Siegler G, Katinger H (2005) Anti-idiotypic antibody inducing hiv-1 neutralizing antibodies. US Patent 2005080240

  • Lee DH, Goldberg AL (1998) Proteasome inhibitors cause induction of heat shock proteins and trehalose, which together confer thermotolerance in Saccharomyces cerevisiae. Mol Cell Biol 18:30–38

    CAS  Google Scholar 

  • Leonardo M, Damasceno KA, Anderson GR, Cregg JM, Old LJ, Batt CA (2007) Cooverexpression of chaperones for enhanced secretion of a single-chain antibody fragment in Pichia pastoris. Appl Genetic Mol Biotechnol 74:381–389

    Google Scholar 

  • Li SC, Kane PM (2009) The yeast lysosome-like vacuole: endpoint and crossroads. Biochim Biophys Acta 1793:650–663

    Article  CAS  Google Scholar 

  • Markham K, Bai Y, Schmitt-Ulms G (2007) Co-immunoprecipitations revisited: an update on experimental concepts and their implementation for sensitive interactome investigations of endogenous proteins. Anal Bioanal Chem 389:461–473

    Article  CAS  Google Scholar 

  • Marx H, Sauer M, Resina D, Vai M, Porro D, Valero F, Ferrer P, Mattanovich D (2006) Cloning, disruption and protein secretory phenotype of the GAS1 homologue of Pichia pastoris. FEMS Microbiol Lett 264:40–47

    Article  CAS  Google Scholar 

  • Matlack KE, Misselwitz B, Plath K, Rapoport TA (1999) BiP acts as a molecular ratchet during posttranslational transport of prepro-α factor across the ER membrane. Cell 97:553–564

    Article  CAS  Google Scholar 

  • Mattanovich D, Callewaert N, Rouzé P, Lin YC, Graf A, Redl A, Tiels P, Gasser B, De Schutter K (2009) Open access to sequence: browsing the Pichia pastoris genome. Microb Cell Factories 8:53

    Article  Google Scholar 

  • Mattanovich D, Branduardi P, Dato L, Gasser B, Sauer M, Porro D (2012) Recombinant protein production in yeasts. Methods Mol Biol 824:329–358

    Article  Google Scholar 

  • Moravec RA, O'Brien MA, Daily WJ, Scurria MA, Bernad L, Riss TL (2009) Cell-based bioluminescent assays for all three proteasome activities in a homogeneous format. Anal Biochem 387:294–302

    Article  CAS  Google Scholar 

  • Mouyna I, Fontaine T, Vai M, Monod M, Fonzi WA, Diaquin M, Popolo L, Hartland RP, Latgé JP (2000) Glycosylphosphatidylinositol-anchored glucanosyltransferases play an active role in the biosynthesis of the fungal cell wall. J Biol Chem 275:14882–14889

    Article  CAS  Google Scholar 

  • Nakajima H, Hirata A, Ogawa Y, Yonehara T, Yoda K, Yamasaki M (1991) A cytoskeleton-related gene, USO1, is required for intracellular protein transport in Saccharomyces cerevisiae. J Cell Biol 113:245–260

    Article  CAS  Google Scholar 

  • Ng DT, Brown JD, Walter P (1996) Signal sequences specify the targeting route to the endoplasmic reticulum membrane. J Cell Biol 134:269–278

    Article  CAS  Google Scholar 

  • Nijholt DA, de Graaf TR, van Haastert ES, Oliveira AO, Berkers CR, Zwart R, Ovaa H, Baas F, Hoozemans JJ, Scheper W (2011) Endoplasmic reticulum stress activates autophagy but not the proteasome in neuronal cells: implications for Alzheimer's disease. Cell Death Differ 18:1071–1081

    Article  CAS  Google Scholar 

  • Nyfeler B, Michnick SW, Hauri HP (2005) Capturing protein interactions in the secretory pathway of living cells. Proc Natl Acad Sci U S A 102:6350–6355

    Article  CAS  Google Scholar 

  • Papa L, Germain D (2011) Estrogen receptor mediates a distinct mitochondrial unfolded protein response. J Cell Sci 124:1396–1402

    Article  CAS  Google Scholar 

  • Papanikou E, Glick BS (2009) The yeast Golgi apparatus: insights and mysteries. FEBS Lett 583:3746–3751

    Article  CAS  Google Scholar 

  • Pelham HR, Hardwick KG, Lewis MJ (1988) Sorting of soluble ER proteins in yeast. EMBO J 7:1757–1762

    CAS  Google Scholar 

  • Pfeffer M, Maurer M, Kollensperger G, Hann S, Graf AB, Mattanovich D (2011) Modeling and measuring intracellular fluxes of secreted recombinant protein in Pichia pastoris with a novel 34S labeling procedure. Microb Cell Factories 10:47

    Article  CAS  Google Scholar 

  • Piper RC, Cooper AA, Yang H, Stevens TH (1995) VPS27 controls vacuolar and endocytic traffic through a prevacuolar compartment in Saccharomyces cerevisiae. J Cell Biol 131:603–617

    Article  CAS  Google Scholar 

  • Popolo L, Vai M (1999) The Gas1 glycoprotein, a putative wall polymer cross-linker. Biochim Biophys Acta 1426:385–400

    Article  CAS  Google Scholar 

  • Porro D, Sauer M, Branduardi P, Mattanovich D (2005) Recombinant protein production in yeasts. Mol Biotechnol 31:245–259

    Article  CAS  Google Scholar 

  • Rapoport T (2007) Protein translocation across the eukaryotic endoplasmic reticulum and bacterial plasma membranes. Nature 450:663–669

    Article  CAS  Google Scholar 

  • Rubio C, Pincus D, Korennykh A, Schuck S, El-Samad H, Walter P (2011) Homeostatic adaptation to endoplasmic reticulum stress depends on Ire1 kinase activity. J Cell Biol 193:171–184

    Article  CAS  Google Scholar 

  • Ruenwai R, Neiss A, Laoteng K, Vongsangnak K, Dalfard AB, Cheevadhanarak S, Petranovic D, Nielsen J (2011) Heterologous production of polyunsaturated fatty acids in Saccharomyces cerevisiae causes a global transcriptional response resulting in reduced proteasomal activity and increased oxidative stress. Biotechnol J 6:343–356

    Article  CAS  Google Scholar 

  • Santos SD, Manadas B, Duarte CB, Carvalho AL (2010) Proteomic analysis of an interactome for long-form AMPA receptor subunits. J Proteome Res 9:1670–1682

    Article  CAS  Google Scholar 

  • Shaner L, Wegele H, Buchner J, Morano KA (2005) The yeast Hsp110 Sse1 functionally interacts with the Hsp70 chaperones Ssa and Ssb. J Biol Chem 280:41262–41269

    Article  CAS  Google Scholar 

  • Stolz A, Wolf DH (2010) Endoplasmic reticulum associated protein degradation: a chaperone assisted journey to hell. Biochim Biophys Acta 1803:694–705

    Article  CAS  Google Scholar 

  • Swennen D, Henry C, Beckerich JM (2010) Folding proteome of Yarrowia lipolytica targeting with uracil permease mutants. J Proteome Res 9:6169–6179

    Article  CAS  Google Scholar 

  • Tang YC, Chang HC, Hayer-Hartl M, Hartl FU (2007) SnapShot: molecular chaperones, Part II. Cell 128:412

    Article  CAS  Google Scholar 

  • Travers KJ, Patil CK, Wodicka L, Lockhart DJ, Weissman S, Walter P (2000) Functional and genomic analyses reveal an essential coordination between the unfolded protein response and ER-associated degradation. Cell 101:249–258

    Article  CAS  Google Scholar 

  • van Anken E, Braakman I (2005) Versatility of the endoplasmic reticulum protein folding factory. Crit Rev Biochem Mol Biol 40:191–228

    Article  Google Scholar 

  • Whittaker MM, Whittaker JW (2005) Construction and characterization of Pichia pastoris strains for labeling aromatic amino acids in recombinant proteins. Protein Expr Purif 41(2):266–274

    Article  CAS  Google Scholar 

  • Yoon J, Aishan T, Maruyama J, Kitamoto K (2010) Enhanced production and secretion of heterologous proteins by the filamentous fungus Aspergillus oryzae via disruption of vacuolar protein sorting receptor gene Aovps10. Appl Environ Microbiol 76:5718–5727

    Article  CAS  Google Scholar 

  • Yuan W, Strømhaug PE, Dunn WA (1999) Glucose-induced autophagy of peroxisomes in Pichia pastoris requires a unique E1-like protein. Mol Biol Cell 10:1353–1366

    CAS  Google Scholar 

  • Zhang B, Chang A, Kjeldsen TB, Arvan P (2001) Intracellular retention of newly synthesized insulin in yeast is caused by endoproteolytic processing in the Golgi complex. J Cell Biol 153:1187–1198

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank Markus Buchetics and Nils Landes from ACIB and the University of Natural Resources and Life Sciences for discussion and support of cellular material. In addition, we thank Kathrin Benakovich for helping to set up the co-immunoprecipitation assay. This project was financially supported by Boehringer Ingelheim RCV (Vienna, Austria) with grants to BOKU and FH-Campus Vienna. Further support by the Federal Ministry of Economy, Family and Youth (BMWFJ), the Federal Ministry of Traffic, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol, and ZIT–Technology Agency of the City of Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG is acknowledged.

Author information

Authors and Affiliations

  1. Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU), 1190, Vienna, Austria

    Martin Pfeffer, Marizela Delic & Diethard Mattanovich

  2. School of Bioengineering, University of Applied Sciences FH-Campus Vienna, 1190, Vienna, Austria

    Michael Maurer

  3. Austrian Centre of Industrial Biotechnology (ACIB GmbH), 1190, Vienna, Austria

    Michael Maurer, Friedrich Altmann & Diethard Mattanovich

  4. Department of Chemistry, University of Natural Resources and Life Sciences, 1190, Vienna, Austria

    Johannes Stadlmann, Josephine Grass & Friedrich Altmann

Authors
  1. Martin Pfeffer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michael Maurer
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Johannes Stadlmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Josephine Grass
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Marizela Delic
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Friedrich Altmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Diethard Mattanovich
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Diethard Mattanovich.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary tables 1

(PDF 48 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Pfeffer, M., Maurer, M., Stadlmann, J. et al. Intracellular interactome of secreted antibody Fab fragment in Pichia pastoris reveals its routes of secretion and degradation. Appl Microbiol Biotechnol 93, 2503–2512 (2012). https://doi.org/10.1007/s00253-012-3933-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-012-3933-3

Keywords

Search

Navigation