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.

The N-end rule pathway as a nitric oxide sensor controlling the levels of multiple regulators

Abstract

The conjugation of arginine to proteins is a part of the N-end rule pathway of protein degradation. Three amino (N)-terminal residues—aspartate, glutamate and cysteine—are arginylated by ATE1-encoded arginyl-transferases. Here we report that oxidation of N-terminal cysteine is essential for its arginylation. The in vivo oxidation of N-terminal cysteine, before its arginylation, is shown to require nitric oxide. We reconstituted this process in vitro as well. The levels of regulatory proteins bearing N-terminal cysteine, such as RGS4, RGS5 and RGS16, are greatly increased in mouse ATE1-/- embryos, which lack arginylation. Stabilization of these proteins, the first physiological substrates of mammalian N-end rule pathway, may underlie cardiovascular defects in ATE1-/- embryos. Our findings identify the N-end rule pathway as a new nitric oxide sensor that functions through its ability to destroy specific regulatory proteins bearing N-terminal cysteine, at rates controlled by nitric oxide and apparently by oxygen as well.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: N-terminal cysteine must be oxidized before its arginylation.
Figure 2: Strongly increased levels of RGS4, RGS5 and RGS16 proteins in ATE1 -/- embryos.
Figure 3: Decreasing NO concentration in vivo stabilizes RGS4 and RGS16.
Figure 4: In vitro reconstitution of nitric oxide-dependent arginylation of RGS4.

References

  1. Bachmair, A., Finley, D. & Varshavsky, A. In vivo half-life of a protein is a function of its amino-terminal residue. Science 234, 179–186 (1986)

    CAS  Article  ADS  Google Scholar 

  2. Varshavsky, A. The N-end rule: functions, mysteries, uses. Proc. Natl Acad. Sci. USA 93, 12142–12149 (1996)

    CAS  Article  ADS  Google Scholar 

  3. Varshavsky, A. The N-end rule and regulation of apoptosis. Nature Cell Biol. 5, 373–376 (2003)

    CAS  Article  Google Scholar 

  4. Kwon, Y. T. et al. Female lethality and apoptosis of spermatocytes in mice lacking the UBR2 ubiquitin ligase of the N-end rule pathway. Mol. Cell. Biol. 23, 8255–8271 (2003)

    CAS  Article  Google Scholar 

  5. Bachmair, A. & Varshavsky, A. The degradation signal in a short-lived protein. Cell 56, 1019–1032 (1989)

    CAS  Article  Google Scholar 

  6. Suzuki, T. & Varshavsky, A. Degradation signals in the lysine–asparagine sequence space. EMBO J. 18, 6017–6026 (1999)

    CAS  Article  Google Scholar 

  7. Hershko, A., Ciechanover, A. & Varshavsky, A. The ubiquitin system. Nature Med. 10, 1073–1081 (2000)

    Article  Google Scholar 

  8. Pickart, C. Back to the future with ubiquitin. Cell 116, 181–190 (2004)

    CAS  Article  Google Scholar 

  9. Baker, R. T. & Varshavsky, A. Yeast N-terminal amidase: a new enzyme and component of the N-end rule pathway. J. Biol. Chem. 270, 12065–12074 (1995)

    CAS  Article  Google Scholar 

  10. Kwon, Y. T. et al. Altered activity, social behaviour, and spatial memory in mice lacking the NTAN1 amidase and the asparagine branch of the N-end rule pathway. Mol. Cell. Biol. 20, 4135–4148 (2000)

    CAS  Article  Google Scholar 

  11. Kwon, Y. T., Kashina, A. S. & Varshavsky, A. Alternative splicing results in differential expression, activity, and localization of the two forms of arginyl-tRNA-protein transferase, a component of the N-end rule pathway. Mol. Cell. Biol. 19, 182–193 (1999)

    CAS  Article  Google Scholar 

  12. Kwon, Y. T. et al. An essential role of N-terminal arginylation in cardiovascular development. Science 297, 96–99 (2002)

    CAS  Article  ADS  Google Scholar 

  13. Du, F., Navarro-Garcia, F., Xia, Z., Tasaki, T. & Varshavsky, A. Pairs of dipeptides synergistically activate the binding of substrate by ubiquitin ligase through dissociation of its autoinhibitory domain. Proc. Natl Acad. Sci. USA 99, 14110–14115 (2002)

    CAS  Article  ADS  Google Scholar 

  14. Tasaki, T. et al. A family of mammalian E3 ubiquitin ligases that contain the UBR box motif and recognize N-degrons. Mol. Cell. Biol. 25, 7120–7136 (2005)

    CAS  Article  Google Scholar 

  15. Gonda, D. K. et al. Universality and structure of the N-end rule. J. Biol. Chem. 264, 16700–16712 (1989)

    CAS  PubMed  Google Scholar 

  16. Turner, G. C., Du, F. & Varshavsky, A. Peptides accelerate their uptake by activating a ubiquitin-dependent proteolytic pathway. Nature 405, 579–583 (2000)

    CAS  Article  ADS  Google Scholar 

  17. Ditzel, M. et al. Degradation of DIAP1 by the N-end rule pathway is essential for regulating apoptosis. Nature Cell Biol. 5, 467–473 (2003)

    CAS  Article  Google Scholar 

  18. Rao, H., Uhlmann, F., Nasmyth, K. & Varshavsky, A. Degradation of a cohesin subunit by the N-end rule pathway is essential for chromosome stability. Nature 410, 955–960 (2001)

    CAS  Article  ADS  Google Scholar 

  19. Ignarro, L. J. Nitric oxide as a unique signalling molecule in the vascular system: a historical overview. J. Physiol. Pharmacol. 53, 503–514 (2002)

    CAS  PubMed  Google Scholar 

  20. Boehning, D. & Snyder, S. H. Novel neural modulators. Annu. Rev. Neurosci. 26, 105–131 (2003)

    CAS  Article  Google Scholar 

  21. Hess, D. T., Matsumoto, A., Kim, S.-O., Marshall, H. E. & Stamler, J. S. Protein S-nitrosylation: purview and parameters. Nature Rev. Mol. Cell Biol. 6, 150–166 (2005)

    CAS  Article  Google Scholar 

  22. Nathan, C. Specificity of a third kind: reactive oxygen and nitrogen intermediates in cell signalling. J. Clin. Invest. 111, 769–778 (2003)

    CAS  Article  Google Scholar 

  23. Eu, J. P., Sun, J., Xu, L., Stamler, J. S. & Meissner, G. The skeletal muscle calcium release channel: coupled O2 sensor and NO signalling functions. Cell 102, 499–509 (2000)

    CAS  Article  Google Scholar 

  24. Packer, M. A. et al. Nitric oxide negatively regulates mammalian adult neurogenesis. Proc. Natl Acad. Sci. USA 100, 9566–9571 (2003)

    CAS  Article  ADS  Google Scholar 

  25. Feng, Q. et al. Development of heart failure and congenital septal defects in mice lacking endothelial nitric oxide synthase. Circulation 106, 873–879 (2002)

    CAS  Article  Google Scholar 

  26. Barouch, L. A. et al. Nitric oxide regulates the heart by spatial confinement of nitric oxide synthase isoforms. Nature 416, 337–340 (2002)

    CAS  Article  ADS  Google Scholar 

  27. van Coelln, R., Dawson, V. L. & Dawson, T. M. Parkin-associated Parkinson's disease. Cell Tissue Res. 318, 175–184 (2004)

    Article  Google Scholar 

  28. Yao, D. et al. Nitrosative stress linked to sporadic Parkinson's disease: S-nitrosylation of parkin regulates its E3 ubiquitin ligase activity. Proc. Natl Acad. Sci. USA 101, 10810–10814 (2004)

    CAS  Article  ADS  Google Scholar 

  29. Tanaka, K., Suzuki, T., Hattori, N. & Mizuno, Y. Ubiquitin, proteasome and parkin. Biochim. Biophys. Acta 1695, 226–238 (2004)

    Google Scholar 

  30. Wieland, T. & Mittman, C. Regulators of G-protein signalling: multifunctional proteins with impact on signalling in the cardiovascular system. Pharmacol. Therapeut. 97, 95–115 (2003)

    CAS  Article  Google Scholar 

  31. Rogers, J. S. et al. RGS4 reduces contractile dysfunction and hypertrophic gene induction in Gaq-overexpressing mice. J. Mol. Cell. Cardiol. 33, 209–218 (2001)

    CAS  Article  Google Scholar 

  32. Albig, A. R. & Schiemann, W. P. Identification and characterization of regulator of G protein signalling 4 (RGS4) as a novel inhibitor of tubulogenesis: RGS4 inhibits mitogen-activated protein kinases and vascular endothelial growth factor signalling. Mol. Biol. Cell 16, 609–625 (2005)

    CAS  Article  Google Scholar 

  33. Balzi, E., Choder, M., Chen, W., Varshavsky, A. & Goffeau, A. Cloning and functional analysis of the arginyl-tRNA-protein transferase gene ATE1 of Saccharomyces cerevisiae. J. Biol. Chem. 265, 7464–7471 (1990)

    CAS  PubMed  Google Scholar 

  34. Varshavsky, A. ‘Spalog’ and ‘sequelog’: neutral terms for spatial and sequence similarity. Curr. Biol. 14, R181–R183 (2004)

    CAS  Article  Google Scholar 

  35. Berman, D. M. & Gilman, A. G. Mammalian RGS proteins: Barbarians at the gate. J. Biol. Chem. 273, 1269–1272 (1998)

    CAS  Article  Google Scholar 

  36. Smotrys, J. E. & Linder, M. E. Palmitoylation of intracellular signalling proteins: regulation and function. Annu. Rev. Biochem. 73, 559–587 (2004)

    CAS  Article  Google Scholar 

  37. Krumins, A. M. et al. Differentially regulated expression of endogenous RGS4 and RGS7. J. Biol. Chem. 279, 2593–2599 (2004)

    CAS  Article  Google Scholar 

  38. Davydov, I. V. & Varshavsky, A. RGS4 is arginylated and degraded by the N-end rule pathway in vitro. J. Biol. Chem. 275, 22931–22941 (2000)

    CAS  Article  Google Scholar 

  39. Mülsch, A., Lurie, D. J., Seimenis, I., Fichtlscherer, B. & Foster, M. A. Detection of nitrosyl-iron complexes by proton-electron-double-resonance imaging. DNIC as endogenous NO carrier. Free Radic. Biol. Med. 27, 636–646 (1999)

    Article  Google Scholar 

  40. Becker, K., Savvides, S. N., Keese, M., Schirmer, R. H. & Karplus, P. A. Enzyme inactivation through sulfhydryl oxidation by physiologic NO-carriers. Nature Struct. Biol. 5, 267–271 (1998)

    CAS  Article  Google Scholar 

  41. Kempf, T. & Wollert, K. C. Nitric oxide and the enigma of heart hyperthrophy. Bioessays 26, 608–615 (2004)

    CAS  Article  Google Scholar 

  42. Bedford, M. T. & Richard, S. Arginine methylation: an emerging regulator of protein function. Mol. Cell 18, 263–272 (2005)

    CAS  Article  Google Scholar 

  43. Vossenaar, E. R., Zendman, A. J. W., van Venrooij, W. J. & Pruijn, G. J. M. PAD, a growing family of citrullinating enzymes: genes, features and involvement in disease. BioEssays 25, 1106–1118 (2003)

    CAS  Article  Google Scholar 

Download references

Acknowledgements

We thank G. Enikolopov, T. Michurina and J. M. Encinas for NOS1-/- mice; A. Mülsch for DNIC-[GSH]2; M. Shahgholi and J. Zhou for MS analyses of peptides; F. Rusnak and G. Hathaway for protein sequencing; J. Racs and S. Horvath for peptide synthesis; L. del Carmen Sandoval, B. W. Kennedy and S. Pease for advice and assistance with mouse mutants; Y. T. Kwon for [UBR1-/-UBR2-/-] cells; R. Baker for plasmids that enabled the USP2-based ubiquitin fusion technique; G. Eriani and F. Du for gifts of other plasmids; Z. Xia for USP2 enzyme; R. Roberts for use of his laboratory equipment; and E. Graciet and C. Brower for comments on the manuscript. Purification of mouse ATE1-1 was performed at CalTech's Protein Expression Center by I. K. Nangiana and the late P. Snow. We dedicate this paper to the memory of Dr Snow. This work was supported by grants from the NIH and the Ellison Medical Foundation to A.V.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Alexander Varshavsky.

Ethics declarations

Competing interests

Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests.

Supplementary information

Supplementary Notes

Contains Supplementary Methods, Supplmentary Discussion, Supplementary Figure Legends and additional references. (DOC 147 kb)

Supplementary Figure S1

N-terminal cysteine must be oxidized before its arginylation by S. cerevisiae R transferase. (PDF 1215 kb)

Supplementary Figure S2

Northern hybridization and immunoblotting with antibody to ATE1. (PDF 591 kb)

Supplementary Figure S3

RT–PCR of mRNAs encoding NO synthases. (PDF 1964 kb)

Supplementary Figure S4

Levels of RGS4 in NOS1-/- mice. (PDF 197 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Hu, RG., Sheng, J., Qi, X. et al. The N-end rule pathway as a nitric oxide sensor controlling the levels of multiple regulators. Nature 437, 981–986 (2005). https://doi.org/10.1038/nature04027

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature04027

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

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