Skip to main content

Investigating Alternative Transport of Integral Plasma Membrane Proteins from the ER to the Golgi: Lessons from the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

  • Protocol
  • First Online:
Unconventional Protein Secretion

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1459))

Abstract

Secretory traffic became a topical field because many important cell regulators are plasma membrane proteins (transporters, channels, receptors), being thus key targets in biomedicine and drug discovery. Cystic fibrosis (CF), caused by defects in a single gene encoding the CF transmembrane conductance regulator (CFTR), constitutes the most common of rare diseases and certainly a paradigmatic one.

Here we focus on five different approaches that allow biochemical and cellular characterization of CFTR from its co-translational insertion into the ER membrane to its delivery to the plasma membrane.

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

Access this chapter

eBook
USD 39.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 54.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 54.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Farinha CM, Matos P, Amaral MD (2013) Control of cystic fibrosis transmembrane conductance regulator membrane trafficking: not just from the endoplasmic reticulum to the Golgi. FEBS J 280:4396–4406

    Article  CAS  PubMed  Google Scholar 

  2. Bannykh SI, Bannykh GI, Fish KN, Moyer BD, Riordan JR, Balch WE (2000) Traffic pattern of cystic fibrosis transmembrane regulator through the early exocytic pathway. Traffic 1:852–870

    Article  CAS  PubMed  Google Scholar 

  3. Farinha CM, Amaral MD (2005) Most F508del-CFTR is targeted to degradation at an early folding checkpoint and independently of calnexin. Mol Cell Biol 25:5242–5252

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Chang XB, Cui L, Hou YX, Jensen TJ, Aleksandrov AA, Mengos A, Riordan JR (1999) Removal of multiple arginine-framed trafficking signals overcomes misprocessing of delta F508 CFTR present in most patients with cystic fibrosis. Mol Cell 4:137–142

    Article  CAS  PubMed  Google Scholar 

  5. Roxo-Rosa M, Xu Z, Schmidt A, Neto M, Cai Z, Soares CM, Sheppard DN, Amaral MD (2006) Revertant mutants G550E and 4RK rescue cystic fibrosis mutants in the first nucleotide-binding domain of CFTR by different mechanisms. Proc Natl Acad Sci U S A 103:17891–17896

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Farinha CM, King-Underwood J, Sousa M, Correia AR, Henriques BJ, Roxo-Rosa M, Da Paula AC, Williams J, Hirst S, Gomes CM, Amaral MD (2013) Revertants, low temperature, and correctors reveal the mechanism of F508del-CFTR rescue by VX-809 and suggest multiple agents for full correction. Chem Biol 20:943–955

    Article  CAS  PubMed  Google Scholar 

  7. Nishimura N, Balch WE (1997) A di-acidic signal required for selective export from the endoplasmic reticulum. Science 277:556–558

    Article  CAS  PubMed  Google Scholar 

  8. Yoo JS, Moyer BD, Bannykh S, Yoo HM, Riordan JR, Balch WE (2002) Non-conventional trafficking of the cystic fibrosis transmembrane conductance regulator through the early secretory pathway. J Biol Chem 277:11401–11409

    Article  CAS  PubMed  Google Scholar 

  9. Gee HY, Noh SH, Tang BL, Kim KH, Lee MG (2011) Rescue of DeltaF508-CFTR trafficking via a GRASP-dependent unconventional secretion pathway. Cell 146:746–760

    Article  CAS  PubMed  Google Scholar 

  10. Wang X, Venable J, LaPointe P, Hutt DM, Koulov AV, Coppinger J, Gurkan C, Kellner W, Matteson J, Plutner H, Riordan JR, Kelly JW, Yates JR 3rd, Balch WE (2006) Hsp90 cochaperone Aha1 downregulation rescues misfolding of CFTR in cystic fibrosis. Cell 127:803–815

    Article  CAS  PubMed  Google Scholar 

  11. Simpson JC, Joggerst B, Laketa V, Verissimo F, Cetin C, Erfle H, Bexiga MG, Singan VR, Heriche JK, Neumann B, Mateos A, Blake J, Bechtel S, Benes V, Wiemann S, Ellenberg J, Pepperkok R (2012) Genome-wide RNAi screening identifies human proteins with a regulatory function in the early secretory pathway. Nat Cell Biol 14:764–774

    Article  CAS  PubMed  Google Scholar 

  12. Botelho HM, Uliyakina I, Awatade NT, Proença MC, Tischer C, Sirianant L, Kunzelmann K, Pepperkok R, Amaral MD (2015) Protein traffic disorders: an effective high-throughput fluorescence microscopy pipeline for drug discovery. Sci Rep 5:9038

    Article  PubMed  PubMed Central  Google Scholar 

  13. Almaça J, Faria D, Sousa M, Uliyakina I, Conrad C, Sirianant L, Clarke LA, Martins JP, Santos M, Heriché JK, Huber W, Schreiber R, Pepperkok R, Kunzelmann K, Amaral MD (2013) High-content siRNA screen reveals global ENaC regulators and potential cystic fibrosis therapy targets. Cell 154:1390–1400

    Article  PubMed  Google Scholar 

  14. Van Goor F, Hadida S, Grootenhuis PD, Burton B, Cao D, Neuberger T, Turnbull A, Singh A, Joubran J, Hazlewood A, Zhou J, McCartney J, Arumugam V, Decker C, Yang J, Young C, Olson ER, Wine JJ, Frizzell RA, Ashlock M, Negulescu P (2009) Rescue of CF airway epithelial cell function in vitro by a CFTR potentiator, VX-770. Proc Natl Acad Sci U S A 106:18825–18830

    Article  PubMed  PubMed Central  Google Scholar 

  15. Erfle H, Neumann B, Liebel U, Rogers P, Held M, Walter T, Ellenberg J, Pepperkok R (2007) Reverse transfection on cell arrays for high content screening microscopy. Nat Protoc 2:392–399

    Article  CAS  PubMed  Google Scholar 

  16. Kamentsky L, Jones TR, Fraser A, Bray MA, Logan DJ, Madden KL, Ljosa V, Rueden C, Eliceiri KW, Carpenter AE (2011) Improved structure, function and compatibility for cell profiler: modular high-throughput image analysis software. Bioinformatics 27:1179–1180

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Amaral MD, Lukacs GL (2011) Introduction to section III: biochemical methods to study CFTR protein. Methods Mol Biol 741:213–218

    Article  CAS  PubMed  Google Scholar 

  18. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  19. Mendes AI, Matos P, Moniz S, Jordan P (2010) Protein kinase WNK1 promotes cell surface expression of glucose transporter GLUT1 by regulating a Tre-2/USP6-BUB2-Cdc16 domain family member 4 (TBC1D4)-Rab8A complex. J Biol Chem 285:39117–39126

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Mendes AI, Matos P, Moniz S, Luz S, Amaral MD, Farinha CM, Jordan P (2011) Antagonistic regulation of cystic fibrosis transmembrane conductance regulator cell surface expression by protein kinases WNK4 and spleen tyrosine kinase. Mol Cell Biol 31:4076–4086

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Moniz S, Sousa M, Moraes BJ, Mendes AI, Palma M, Barreto C, Fragata JI, Amaral MD, Matos P (2013) HGF stimulation of Rac1 signaling enhances pharmacological correction of the most prevalent cystic fibrosis mutant F508del-CFTR. ACS Chem Biol 8:432–442

    Article  CAS  PubMed  Google Scholar 

  22. Loureiro CA, Matos AM, Dias-Alves A, Pereira JF, Uliyakina I, Barros P, Amaral MD, Matos P (2015) A molecular switch in the scaffold NHERF1 enables misfolded CFTR to evade the peripheral quality control checkpoint. Sci Signal 8:48

    Article  Google Scholar 

Download references

Acknowledgements

Work supported by UID/MULTI/04046/2013 center grant (to BioISI) from FCT/MCTES/PIDDAC, Portugal. H.M.B. is recipient of SFRH/BPD/93017/2013 postdoctoral fellowship (FCT, Portugal) and P.M. is supported by grant IF/2012 (FCT, Portugal).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Margarida D. Amaral .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media New York

About this protocol

Cite this protocol

Amaral, M.D., Farinha, C.M., Matos, P., Botelho, H.M. (2016). Investigating Alternative Transport of Integral Plasma Membrane Proteins from the ER to the Golgi: Lessons from the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). In: Pompa, A., De Marchis, F. (eds) Unconventional Protein Secretion. Methods in Molecular Biology, vol 1459. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3804-9_7

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-3804-9_7

  • Published:

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3802-5

  • Online ISBN: 978-1-4939-3804-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics