Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

Genetic isolation of transport signals directing cell surface expression

Nature Cell Biology volume 7, pages 985–992 (2005)Cite this article

Abstract

Membrane proteins represent approximately 30% of the proteome in both prokaryotes and eukaryotes1. The spatial localization of membrane-bound proteins is often determined by specific sequence motifs2 that may be regulated in response to physiological changes, such as protein interactions3 and receptor signalling4. Identification of signalling motifs is therefore important for understanding membrane protein expression, function and transport mechanisms. We report a genetic isolation of novel motifs that confer surface expression. Further characterization showed that SWTY, one class of these isolated motifs with homology to previously reported forward transport motifs5, has the ability to both override the RKR endoplasmic reticulum localization signal and potentiate steady-state surface expression. The genetically isolated SWTY motif is functionally interchangeable with a known motif in cardiac potassium channels and an identified motif in an HIV coreceptor, and operates by recruiting 14-3-3 proteins. This study expands the repertoire of and enables a screening method for membrane trafficking signals.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Design of a genetic screen system.
Figure 2: Interaction of the SWTY motif with 14-3-3 proteins.
Figure 3: Overriding of RKR ER localization by SWTY, R18 and fused 14-3-3.
Figure 4: Phosphorylation-dependent interaction between 14-3-3 and SWTY-related motifs.
Figure 5: Native SWTY-like sequences.

Similar content being viewed by others

Accession codes

Accessions

BINDPlus

References

  1. Wallin, E. & von Heijne, G. Genome-wide analysis of integral membrane proteins from eubacterial, archaean, and eukaryotic organisms. Protein Sci. 7, 1029–1038 (1998).

    Article  CAS  Google Scholar 

  2. Bonifacino, J. S. & Glick, B. S. The mechanisms of vesicle budding and fusion. Cell 116, 153–166 (2004).

    Article  CAS  Google Scholar 

  3. Zerangue, N., Schwappach, B., Jan, Y. N. & Jan, L. Y. A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane K(ATP) channels. Neuron 22, 537–548 (1999).

    Article  CAS  Google Scholar 

  4. Viard, P. et al. PI3K promotes voltage-dependent calcium channel trafficking to the plasma membrane. Nature Neurosci. 7, 939–946 (2004).

    Article  CAS  Google Scholar 

  5. O'Kelly, I., Butler, M. H., Zilberberg, N. & Goldstein, S. A. Forward transport. 14-3-3 binding overcomes retention in endoplasmic reticulum by dibasic signals. Cell 111, 577–588 (2002).

    Article  CAS  Google Scholar 

  6. Schwappach, B., Zerangue, N., Jan, Y. N. & Jan, L. Y. Molecular basis for K(ATP) assembly: transmembrane interactions mediate association of a K+ channel with an ABC transporter. Neuron 26, 155–167 (2000).

    Article  CAS  Google Scholar 

  7. Shikano, S. & Li, M. Membrane receptor trafficking: Evidence of proximal and distal zones conferred by two independent endoplasmic reticulum localization signals. Proc. Natl Acad. Sci. USA 100, 5783–5788 (2003).

    Article  CAS  Google Scholar 

  8. Nishimura, N. & Balch, W. E. A di-acidic signal required for selective export from the endoplasmic reticulum. Science 277, 556–558 (1997).

    Article  CAS  Google Scholar 

  9. Sevier, C. S., Weisz, O. A., Davis, M. & Machamer, C. E. Efficient export of the vesicular stomatitis virus G protein from the endoplasmic reticulum requires a signal in the cytoplasmic tail that includes both tyrosine-based and di-acidic motifs. Mol. Biol. Cell 11, 13–22 (2000).

    Article  CAS  Google Scholar 

  10. Ma, D. et al. Role of ER export signals in controlling surface potassium channel numbers. Science 291, 316–319 (2001).

    Article  CAS  Google Scholar 

  11. Nilsson, T., Jackson, M. & Peterson, P. A. Short cytoplasmic sequences serve as retention signals for transmembrane proteins in the endoplasmic reticulum. Cell 58, 707–718 (1989).

    Article  CAS  Google Scholar 

  12. Tang, W. et al. Functional expression of a vertebrate inwardly rectifying K+ channel in yeast. Mol. Biol. Cell 6, 1231–1240 (1995).

    Article  CAS  Google Scholar 

  13. Chung, J. J., Shikano, S., Hanyu, Y. & Li, M. Functional diversity of protein C-termini: more than zipcoding? Trends Cell Biol. 12, 146–150 (2002).

    Article  CAS  Google Scholar 

  14. Li, M., Unwin, N., Stauffer, K. A., Jan, Y. N. & Jan, L. Y. Images of purified Shaker potassium channels. Curr. Biol. 4, 110–115 (1994).

    Article  CAS  Google Scholar 

  15. Moore, B. & Perez, V. J. Specific Acidic Proteins of the Nervous System (ed. Carlson, F. D.) 343–359 (Prentice-Hall, Englewood Cliffs, NJ, 1967).

    Google Scholar 

  16. Wang, B. et al. Isolation of high-affinity peptide antagonists of 14-3-3 proteins by phage display. Biochemistry 38, 12499–12504 (1999).

    Article  CAS  Google Scholar 

  17. Sun, H., Shikano, S., Xiong, Q. & Li, M. Function recovery after chemobleaching (FRAC): evidence for activity silent membrane receptors on cell surface. Proc. Natl Acad. Sci. USA 101, 16964–16969 (2004).

    Article  CAS  Google Scholar 

  18. Fu, H., Subramanian, R. R. & Masters, S. C. 14-3-3 proteins: structure, function, and regulation. Annu. Rev. Pharmacol. Toxicol. 40, 617–647 (2000).

    Article  CAS  Google Scholar 

  19. Yaffe, M. B. How do 14-3-3 proteins work? — Gatekeeper phosphorylation and the molecular anvil hypothesis. FEBS Lett. 513, 53–57 (2002).

    Article  CAS  Google Scholar 

  20. Ganguly, S. et al. Melatonin synthesis: 14-3-3-dependent activation and inhibition of arylalkylamine N-acetyltransferase mediated by phosphoserine-205. Proc. Natl Acad. Sci. USA 102, 1222–1227 (2005).

    Article  CAS  Google Scholar 

  21. Lopes, C. M., Gallagher, P. G., Buck, M. E., Butler, M. H. & Goldstein, S. A. Proton block and voltage gating are potassium-dependent in the cardiac leak channel Kcnk3. J. Biol. Chem. 275, 16969–16978 (2000).

    Article  CAS  Google Scholar 

  22. Maresca, M. et al. The virotoxin model of HIV-1 enteropathy: involvement of GPR15/Bob and galactosylceramide in the cytopathic effects induced by HIV-1 gp120 in the HT-29-D4 intestinal cell line. J. Biomed. Sci. 10, 156–166 (2003).

    Article  CAS  Google Scholar 

  23. Wade-Evans, A. M., Russell, J., Jenkins, A. & Javan, C. Cloning and sequencing of cynomolgus macaque CCR3, GPR15, and STRL33: potential coreceptors for HIV type 1, HIV type 2, and SIV. AIDS Res. Hum. Retroviruses 17, 371–375 (2001).

    Article  CAS  Google Scholar 

  24. Yuan, H., Michelsen, K. & Schwappach, B. 14-3-3 dimers probe the assembly status of multimeric membrane proteins. Curr. Biol. 13, 638–646 (2003).

    Article  CAS  Google Scholar 

  25. Yaffe, M. B. et al. A motif-based profile scanning approach for genome-wide prediction of signaling pathways. Nature Biotechnol. 19, 348–353 (2001).

    Article  CAS  Google Scholar 

  26. Dougherty, M. K. & Morrison, D. K. Unlocking the code of 14-3-3. J. Cell Sci. 117, 1875–1884 (2004).

    Article  CAS  Google Scholar 

  27. Kagan, A., Melman, Y. F., Krumerman, A. & McDonald, T. V. 14-3-3 amplifies and prolongs adrenergic stimulation of HERG K+ channel activity. EMBO J. 21, 1889–1898 (2002).

    Article  CAS  Google Scholar 

  28. Mossessova, E., Bickford, L. C. & Goldberg, J. SNARE selectivity of the COPII coat. Cell 114, 483–495 (2003).

    Article  CAS  Google Scholar 

  29. Miller, E. A. et al. Multiple cargo binding sites on the COPII subunit Sec24p ensure capture of diverse membrane proteins into transport vesicles. Cell 114, 497–509 (2003).

    Article  CAS  Google Scholar 

  30. Chung, J. J., Yang, H. & Li, M. Genome-wide analyses of carboxyl-terminal sequences. Mol. Cell. Proteomics 2, 173–181 (2003).

    Article  CAS  Google Scholar 

  31. Coblitz, B. et al. Carboxyl-terminal recognition by 14-3-3 proteins for surface expression of membrane receptors. J. Biol. Chem. (in the press).

  32. Ma, D. et al. Diverse trafficking patterns due to multiple traffic motifs in G protein-activated inwardly rectifying potassium channels from brain and heart. Neuron 33, 715–729 (2002).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank L. Jan for providing Kir2.1 and Kir6.2 cDNAs, S. Goldstein for KCNK3 cDNA, H. Fu for providing 14-3-3 constructs and many insightful discussions, R. Doms for GPR15 cDNA, M. Spieker and R. Sheng for helping with DNA recovery and sequencing, and the members of the Li laboratory and C. Montell and C. Machamer for helpful comments on this manuscript. We are indebted to S. Goldstein for his invaluable advice on this manuscript. The work is supported by grants from the National Institutes of Health (GM70959 and NS33324 to M.L.); pre-doctoral (to B.C.) and postdoctoral (to H.S.) fellowship awards from the American Heart Association.

Author information

Authors and Affiliations

  1. Department of Neuroscience and High Throughput Biology Center, Johns Hopkins University School of Medicine, 733 North Broadway, Baltimore, 21205, MD, USA

    Sojin Shikano, Brian Coblitz, Haiyan Sun & Min Li

Authors
  1. Sojin Shikano
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Brian Coblitz
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Haiyan Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Min Li
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Min Li.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary figures S1 and S2 (PDF 113 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Shikano, S., Coblitz, B., Sun, H. et al. Genetic isolation of transport signals directing cell surface expression. Nat Cell Biol 7, 985–992 (2005). https://doi.org/10.1038/ncb1297

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb1297

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing