N-end rule

The N-end rule is a rule that governs the rate of protein degradation through recognition of the N-terminal residue of proteins. The rule states that the N-terminal amino acid of a protein determines its half-life (time after which half of the total amount of a given polypeptide is degraded). The rule applies to both eukaryotic and prokaryotic organisms, but with different strength, rules, and outcome.[1] In eukaryotic cells, these N-terminal residues are recognized and targeted by ubiquitin ligases, mediating ubiquitination thereby marking the protein for degradation.[2] The rule was initially discovered by Alexander Varshavsky and co-workers in 1986.[3] However, only rough estimations of protein half-life can be deduced from this 'rule', as N-terminal amino acid modification can lead to variability and anomalies, whilst amino acid impact can also change from organism to organism. Other degradation signals, known as degrons, can also be found in sequence.

Rules in different organisms

The rule may operate differently in different organisms.

Yeast

N-terminal residues - approximate half-life of proteins for S. cerevisiae[3]

  • Met, Gly, Ala, Ser, Thr, Val, Pro - > 20 hrs (stabilizing)
  • Ile, Glu - approx. 30 min (stabilizing)
  • Tyr, Gln - approx. 10 min (destabilizing)
  • Leu, Phe, Asp, Lys - approx. 3 min (destabilizing)
  • Arg - approx. 2 min (destabilizing)

Mammals

"N"-terminal residues - approximate half-life of proteins in mammalian systems [4]

  • Val -> 100h
  • Met, Gly -> 30h
  • Pro -> 20h
  • Ile -> 20h
  • Thr -> 7.2h
  • Leu -> 5.5h
  • Ala -> 4.4h
  • His -> 3.5h
  • Trp -> 2.8h
  • Tyr -> 2.8h
  • Ser -> 1.9h
  • Asn -> 1.4h
  • Lys -> 1.3h
  • Cys -> 1.2h
  • Asp -> 1.1h
  • Phe -> 1.1h
  • Glu -> 1.0h
  • Arg -> 1.0h
  • Gln -> 0.8h

Bacteria

In Escherichia coli, positively-charged and some aliphatic and aromatic residues on the N-terminus, such as arginine, lysine, leucine, phenylalanine, tyrosine, and tryptophan, have short half-lives of around 2-minutes and are rapidly degraded. Other amino acids on the other hand may have half-lives of more than 10 hours when added to the N-terminal of the same protein.[5] However, a complicating issue is that the first residue of bacterial proteins is normally expressed with an N-terminal formylmethionine (f-Met). The formyl group of this methionine is quickly removed, and the methionine itself is then removed by methionyl aminopeptidase. The removal of the methionine is more efficient when the second residue is small and uncharged (for example alanine), but inefficient when it is bulky and charged such as arginine. Once the f-Met is removed, the second residue becomes the N-terminal residue and are subject to the N-end rule. Residues with middle sized side-chains such as leucine as the second residue therefore may have a short half-life.[6]

Possible Existence of the N-End Rule in Chloroplasts

There are several reasons why it is possible that the N-end rule functions in the chloroplast organelle of plant cells as well.[7] The first piece of evidence comes from the endosymbiotic theory which encompasses the idea that chloroplasts are derived from cyanobacteria, photosynthetic organisms that can convert light into energy.[8][9] It is thought that the chloroplast developed from an endosymbiosis between a eukaryotic cell and a cyanobacterium, because chloroplasts share several features with the bacterium, including photosynthetic capabilities.[8][9] The bacterial N-end rule is already well documented; it involves the Clp protease system which consists of the adaptor protein ClpS and the ClpA/P chaperone and protease core.[5][10] A similar Clp system is present in the chloroplast stroma, suggesting that the N-end rule might function similarly in chloroplasts and bacteria.[7][11]

Additionally, a 2013 study in Arabidopsis thaliana revealed the protein ClpS1, a possible plastid homolog of the bacterial ClpS recognin.[12] ClpS is a bacterial adaptor protein that is responsible for recognizing protein substrates via their N-terminal residues and delivering them to a protease core for degradation.[13] This study suggests that ClpS1 is functionally similar to ClpS, also playing a role in substrate recognition via specific N-terminal residues (degrons) like its bacterial counterpart.[12] It is posited that upon recognition, ClpS1 binds to these substrate proteins and brings them to the ClpC chaperone of the protease core machinery to initiate degradation.[12]

In another study, Arabidopsis thaliana stromal proteins were analyzed to determine the relative abundance of specific N-terminal residues.[14] This study revealed that Alanine, Serine, Threonine, and Valine were the most abundant N-terminal residues, while Leucine, Phenylalanine, Tryptophan, and Tyrosine (all triggers for degradation in bacteria) were among the residues that were rarely detected.[14]

Furthermore, an affinity assay using ClpS1 and N-terminal residues was performed to determine whether ClpS1 did indeed have specific binding partners.[15] This study revealed that Phenylalanine and Tryptophan bind specifically to ClpS1, making them prime candidates for N-degrons in chloroplasts.[15]

Further research is currently being conducted to confirm whether the N-end rule operates in chloroplasts.[7][15]

References
  1. Varshavsky A. (1997). "The N-end rule pathway of protein degradation". Genes to Cells. 2 (1): 13–28. doi:10.1046/j.1365-2443.1997.1020301.x. PMID 9112437.
  2. Takafumi Tasaki, Shashikanth M. Sriram, Kyong Soo Park, and Yong Tae Kwon (2012). "The N-End Rule Pathway". Annual Review of Biochemistry. 81: 261–289. doi:10.1146/annurev-biochem-051710-093308. PMC 3610525. PMID 22524314.CS1 maint: uses authors parameter (link)
  3. Bachmair A, Finley D, Varshavsky A (1986). "In vivo half-life of a protein is a function of its amino-terminal residue". Science. 234 (4773): 179–186. doi:10.1126/science.3018930. PMID 3018930.
  4. David K. Gonda; et al. (1989). "Universality and Structure of the N-end Rule". Journal of Biological Chemistry. 264 (28): 16700–16712. PMID 2506181.
  5. Tobias JW, Shrader TE, Rocap G, Varshavsky A (November 29, 1991). "The N-end rule in bacteria". Science. 254 (5036): 1374–7. doi:10.1126/science.1962196. PMID 1962196.CS1 maint: uses authors parameter (link)
  6. P H Hirel, M J Schmitter, P Dessen, G Fayat, and S Blanquet (November 1989). "Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid". Proc Natl Acad Sci U S A. 86 (21): 8247–51. doi:10.1073/pnas.86.21.8247. PMC 298257. PMID 2682640.CS1 maint: uses authors parameter (link)
  7. Bouchnak, I.; van Wijk, K. J. (July 2019). "N-Degron Pathways in Plastids". Trends in Plant Science. 9 (19): 30160–30168. doi:10.1016/j.tplants.2019.06.013. PMID 31300194.
  8. Archibald, J. M. (5 October 2015). "Endosymbiosis and Eukaryotic Cell Evolution". Current Biology. 25 (19): R911–R921. doi:10.1016/j.cub.2015.07.055. ISSN 0960-9822. PMID 26439354.
  9. McFadden, G. I. (1 January 2001). "Chloroplast Origin and Integration". Plant Physiology. 125 (1): 50–53. doi:10.1104/pp.125.1.50. ISSN 0032-0889. PMC 1539323. PMID 11154294.
  10. Dougan, D.A.; Micevski, D.; Truscott, K.N. (January 2012). "The N-end rule pathway: From recognition by N-recognins, to destruction by AAA+proteases". Biochimica et Biophysica Acta (BBA) - Molecular Cell Research. 1823 (1): 83–91. doi:10.1016/j.bbamcr.2011.07.002. PMID 21781991.
  11. Nishimura, K.; van Wijk, K. J. (September 2015). "Organization, function and substrates of the essential Clp protease system in plastids". Biochimica et Biophysica Acta (BBA) - Bioenergetics. 1847 (9): 915–930. doi:10.1016/j.bbabio.2014.11.012. PMID 25482260.
  12. Nishimura, K.; Asakura, Y.; Friso, G.; Kim, J.; Oh, S.H.; Rutschow, H.; Ponnala, L.; van Wijk, K. J. (28 June 2013). "ClpS1 Is a Conserved Substrate Selector for the Chloroplast Clp Protease System in Arabidopsis". The Plant Cell. 25 (6): 2276–2301. doi:10.1105/tpc.113.112557. PMC 3723626. PMID 23898032.
  13. Erbse, A.; Schmidt, R.; Bornemann, T.; Schneider-Mergener, J.; Mogk, A.; Zahn, R.; Dougan, D. A.; Bukau, B. (February 2006). "ClpS is an essential component of the N-end rule pathway in Escherichia coli". Nature. 439 (7077): 753–756. doi:10.1038/nature04412. PMID 16467841.
  14. Rowland, E.; Kim, J.; Bhuiyan, N. H.; van Wijk, K. J. (14 September 2015). "The Arabidopsis Chloroplast stromal N-terminome; complexities of N-terminal protein maturation and stability". Plant Physiology. 169 (3): 1881–96. doi:10.1104/pp.15.01214. PMC 4634096. PMID 26371235.
  15. Montandon, C.; Dougan, D. A.; van Wijk, K. J. (20 April 2019). "N‐degron specificity of chloroplast ClpS1 in plants". FEBS Letters. 593 (9): 962–970. doi:10.1002/1873-3468.13378. PMID 30953344.
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