Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Science. Author manuscript; available in PMC Aug 24, 2012.
Published in final edited form as:
PMCID: PMC3296478

Extremely Long-lived Nuclear Pore Proteins in the Rat Brain+


To combat the functional decline of the proteome, cells use the process of protein turnover to replace potentially impaired polypeptides with new functional copies. Here we found that extremely long-lived proteins (ELLPs) did not turn over in post-mitotic cells of the rat central nervous system. These ELLPs were associated with chromatin and the nuclear pore complex, the central transport channels that mediate all molecular trafficking in and out of the nucleus. The longevity of these proteins would be expected to expose them to potentially harmful metabolites putting them at risk of accumulating damage over extended periods of time. Thus, it is possible that failure to maintain proper levels and functional integrity of ELLPs in non-proliferative cells might contribute to age-related deterioration in cell and tissue function.

Functional deterioration and accumulation of damage to the proteome is an inevitable consequence of cellular aging. This damage is largely repaired through protein turnover where potentially impaired polypeptides are replaced with new, functional copies. These turnover mechanisms are particularly important in post-mitotic cells, such as neurons, because they cannot dilute potentially toxic species through cell division. As such, nearly all proteins within the human proteome are recycled in less than a few days (1, 2). However, a few exceptional cases of extremely long-lived proteins (ELLPs) with half-lives on the order of months have been identified (3, 4), including eye lens crystalline, collagen and myelin basic protein (MBP), a key structural component of myelination of nerve cells (4). Additional ELLPs probably remain to be discovered. For example, a recent study showed that a subset of nuclear pore complex (NPC) proteins, which form the transport channels responsible for mediating all nuclear trafficking (5), are present but no longer expressed in differentiated cells (6). Furthermore, in the nematode, C. elegans, all NPCs present in the adult animal are assembled during embryogenesis. Thus at least a subset of nucleoporins (Nups), are not, or are only very slowly, replaced during adulthood. However, because worms have a relatively short life span of a few weeks, it remains unclear if NPC components remain incorporated in the nuclear membrane over years, particularly in the central nervous system of mammals, which contain non-dividing cells that are as old as the organism itself (7).

To explore this question, we performed pulse chase labeling of whole rats with the stable isotope 15N followed by mass spectrometry to monitor global protein turnover on a timescale of years (the average life span of a lab rat is 2 years). Two female Sprague Dawley rats and their progeny were fed a 15N-enriched algal cell diet, and at 6 weeks all progeny rats were switched to a 14N diet. Fully 15N labeled (t=0) rats were immediately sacrificed and their tissues (brain, liver, plasma, skeletal muscle, heart, kidney, lung, duodenum) harvested. Nuclei from liver, an organ that turns over within 4–6 months, and brain were purified, digested with trypsin, and analyzed by MudPIT (multidimensional protein identification technology) LCLC-MS/MS (multidimensional liquid chromatography-tandem mass spectrometry). At time=0, we calculated 15N isotopic protein labeling efficiency of >98% and identified more than 3,400 fully 15N proteins (20,754 peptides) and only 9 14N proteins (14 peptides). Two additional animals were sacrificed at 6 and 12 months and 15N/14N ratios were determined for more than 3,500 unique proteins. Only 7 heavy (15N) proteins (11 peptides) were found in the liver after 6 months, consistent with the relatively rapid turn-over of hepatocytes. In contrast, the brain contained a large number of heavy peptides (92 peptides) even after 12 months (Fig. 1B, Fig. S1E). These peptides corresponded to 25 proteins and included MBP and histones, the latter having reported half-lives of ~220 days in mouse brain (8), confirming the validity of our approach (Fig. 1A). All the other heavy proteins identified were components of the two essential core modules of the NPC, the pentameric Nup205 complex and the nonameric Nup107-160 complex (6) (Fig. 1B and Table 1). This represents an essential intracellular protein machine with protein components in excess of a year in age.

Fig. 1
Identification of NUPs and histones as extremely long-lived proteins in mammalian brain

Detailed analysis of 15N spectral counts and 15N/14N MS1 ratios revealed that in contrast to the stable scaffold, the peripheral Nups and components of the central transport channel were devoid of heavy peptides, suggesting they were completely replaced after 6 months (Fig. 1C). Thus, unlike other large protein complexes, such as the proteasome or ribosome, in which all components have similar turn-over values (1, 2), the individual components of NPCs have very different lifetimes. This supports the idea that NPCs are built to last the entire lifespan of the cell and are not completely removed and assembled anew in post-mitotic cells. Rather, in the absence of the key regulators that orchestrate the complex NPC disassembly process in mitosis, NPC maintenance in non-dividing cells relies on the non- or extremely slow- exchange of scaffold and rapid replacement of peripheral Nups.

A lack of protein turnover exposes the proteome to an increased risk of aberrant chemical modifications and oxidative damage during aging, and thus might represent an Achilles heel of protein homeostasis in post-mitotic tissues. Indeed, healthy rats exhibit age-dependent decline of NPC function (6). Our results may thus provide a molecular explanation for the observed NPC deterioration and suggest that ELLPs represent a diverse class of proteins that regulate essential cellular functions and could be linked directly to the decline of the aging proteome.

Supplementary Material



B.H.T is supported by the Hewitt Foundation; M.W.H by the Ellison Medical Foundation and Glenn Foundation; J.N.S is supported by the NIH/NIA fellowship F32AG039127; JRY and JNS are supported by NIH P41 RR011823, P01 AG031097, and R01 MH067880. The RAW files and parameter files will be publically available at http://fields.scripps.edu/published/ellnpp2011/ upon publication.


+This manuscript has been accepted for publication in Science. This version has not undergone final editing. Please refer to the complete version of record at http://www.sciencemag.org/. The manuscript may not be reproduced or used in any manner that does not fall within the fair use provisions of the Copyright Act without the prior, written permission of AAAS.”

References and Notes

1. Cambridge SB. Systems-wide Proteomic Analysis in Mammalian Cells Reveals Conserved, Functional Protein Turnover. J Proteome Res. 2011;10:5275–5284. [PubMed]
2. Price JC, Guan S. Analysis of proteome dynamics in the mouse brain. Proc Natl Acad Sci U S A. 2010 Aug 10;107:14508–14513. [PMC free article] [PubMed]
3. Fischer CA, Morell P. Turnover of proteins in myelin and myelin-like material of mouse brain. Brain Res. 1974 Jul 5;74:51–56. [PubMed]
4. Shapira R, Wilhelmi MR. Turnover of myelin proteins of rat brain, determined in fractions separated by sedimentation in a continuous sucrose gradient. J Neurochem. 1981 Apr;36:1427–1431. [PubMed]
5. Capelson M, Doucet C. Nuclear pore complexes: guardians of the nuclear genome. Cold Spring Harb Symp Quant Biol. 2010;75:585–597. [PMC free article] [PubMed]
6. D'Angelo MA, Raices M. Age-dependent deterioration of nuclear pore complexes causes a loss of nuclear integrity in postmitotic cells. Cell. 2009 Jan 23;136:284–295. [PMC free article] [PubMed]
7. Spalding KL, Bhardwaj RD, Buchholz BA, Druid H, Frisen J. Retrospective birth dating of cells in humans. Cell. 2005 Jul 15;122:133–139. [PubMed]
8. Commerford SL, Carsten AL. Histone turnover within nonproliferating cells. Proc Natl Acad Sci U S A. 1982 Feb;79:1163–168. [PMC free article] [PubMed]
9. McClatchy DB, Dong MQ. 15N metabolic labeling of mammalian tissue with slow protein turnover. J Proteome Res. 2007 May;6:2005. [PMC free article] [PubMed]
10. MacCoss MJ, Wu CC. Measurement of the isotope enrichment of stable isotope-labeled proteins using high-resolution mass spectra of peptides. Anal Chem. 2005 Dec 1;77:7646. [PubMed]
11. Blobel G, Potter VR. Nuclei from rat liver: isolation method that combines purity with high yield. Science. 1966 Dec 30;154:1662. [PubMed]
12. Link AJ, et al. Direct analysis of protein complexes using mass spectrometry. Nat Biotechnol. 1999 Jul;17:676. [PubMed]
13. Washburn MP, Wolters D. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol. 2001 Mar;19:242. [PubMed]
14. McDonald WH, et al. MS1, MS2, and SQT-three unified, compact, and easily parsed file formats for the storage of shotgun proteomic spectra and identifications. Rapid Commun Mass Spectrom. 2004;18:2162. [PubMed]
15. Peng J, Elias JE. Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome. J Proteome Res. 2003 Jan–Feb;2:43. [PubMed]
16. Eng MAJ, Yates JR., 3rd J Am Soc Mass Spectrom. 1994;5:976. [PubMed]
17. Tabb DL, McDonald WH, Yates JR., 3rd DTASelect and Contrast: tools for assembling and comparing protein identifications from shotgun proteomics. J Proteome Res. 2002 Jan–Feb;1:21. [PMC free article] [PubMed]
18. Cociorva D, LTD, Yates JR. Validation of tandem mass spectrometry database search results using DTASelect. Curr Protoc Bioinformatics Chapter. 2007 Jan;13(Unit 13):4. [PubMed]
19. Park SK, Venable JDT. A quantitative analysis software tool for mass spectrometry-based proteomics. Nat Methods. 2008 Apr;5:319. [PMC free article] [PubMed]
PubReader format: click here to try


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • PubMed
    PubMed citations for these articles

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...