Biology:Nuclear localization sequence

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A nuclear localization signal or sequence (NLS) is an amino acid sequence that 'tags' a protein for import into the cell nucleus by nuclear transport.[1] Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface.[1] Different nuclear localized proteins may share the same NLS.[1] An NLS has the opposite function of a nuclear export signal (NES), which targets proteins out of the nucleus.

Types

Classical

These types of NLSs can be further classified as either monopartite or bipartite. The major structural differences between the two are that the two basic amino acid clusters in bipartite NLSs are separated by a relatively short spacer sequence (hence bipartite - 2 parts), while monopartite NLSs are not. The first NLS to be discovered was the sequence PKKKRKV in the SV40 Large T-antigen (a monopartite NLS).[2] The NLS of nucleoplasmin, KR[PAATKKAGQA]KKKK, is the prototype of the ubiquitous bipartite signal: two clusters of basic amino acids, separated by a spacer of about 10 amino acids.[3] Both signals are recognized by importin α. Importin α contains a bipartite NLS itself, which is specifically recognized by importin β. The latter can be considered the actual import mediator.

Chelsky et al. proposed the consensus sequence K-K/R-X-K/R for monopartite NLSs.[3] A Chelsky sequence may, therefore, be part of the downstream basic cluster of a bipartite NLS. Makkah et al. carried out comparative mutagenesis on the nuclear localization signals of SV40 T-Antigen (monopartite), C-myc (monopartite), and nucleoplasmin (bipartite), and showed amino acid features common to all three. The role of neutral and acidic amino acids was shown for the first time in contributing to the efficiency of the NLS.[4]

Rotello et al. compared the nuclear localization efficiencies of eGFP fused NLSs of SV40 Large T-Antigen, nucleoplasmin (AVKRPAATKKAGQAKKKKLD), EGL-13 (MSRRRKANPTKLSENAKKLAKEVEN), c-Myc (PAAKRVKLD) and TUS-protein (KLKIKRPVK) through rapid intracellular protein delivery. They found significantly higher nuclear localization efficiency of c-Myc NLS compared to that of SV40 NLS.[5]

Non-classical

There are many other types of NLS, such as the acidic M9 domain of hnRNP A1, the sequence KIPIK in yeast transcription repressor Matα2, and the complex signals of U snRNPs. Most of these NLSs appear to be recognized directly by specific receptors of the importin β family without the intervention of an importin α-like protein.[6]

A signal that appears to be specific for the massively produced and transported ribosomal proteins,[7][8] seems to come with a specialized set of importin β-like nuclear import receptors.[9]

Recently a class of NLSs known as PY-NLSs has been proposed, originally by Lee et al.[10] This PY-NLS motif, so named because of the proline-tyrosine amino acid pairing in it, allows the protein to bind to Importin β2 (also known as transportin or karyopherin β2), which then translocates the cargo protein into the nucleus. The structural basis for the binding of the PY-NLS contained in Importin β2 has been determined and an inhibitor of import designed.[11]

Discovery

The presence of the nuclear membrane that sequesters the cellular DNA is the defining feature of eukaryotic cells. The nuclear membrane, therefore, separates the nuclear processes of DNA replication and RNA transcription from the cytoplasmic process of protein production. Proteins required in the nucleus must be directed there by some mechanism. The first direct experimental examination of the ability of nuclear proteins to accumulate in the nucleus was carried out by John Gurdon when he showed that purified nuclear proteins accumulate in the nucleus of frog (Xenopus) oocytes after being micro-injected into the cytoplasm. These experiments were part of a series that subsequently led to studies of nuclear reprogramming, directly relevant to stem cell research.

The presence of several million pore complexes in the oocyte nuclear membrane and the fact that they appeared to admit many different molecules (insulin, bovine serum albumin, gold nanoparticles) led to the view that the pores are open channels and nuclear proteins freely enter the nucleus through the pore and must accumulate by binding to DNA or some other nuclear component. In other words, there was thought to be no specific transport mechanism.

This view was shown to be incorrect by Dingwall and Laskey in 1982. Using a protein called nucleoplasmin, the archetypal ‘molecular chaperone’, they identified a domain in the protein that acts as a signal for nuclear entry.[12] This work stimulated research in the area, and two years later the first NLS was identified in SV40 Large T-antigen (or SV40, for short). However, a functional NLS could not be identified in another nuclear protein simply on the basis of similarity to the SV40 NLS. In fact, only a small percentage of cellular (non-viral) nuclear proteins contained a sequence similar to the SV40 NLS. A detailed examination of nucleoplasmin identified a sequence with two elements made up of basic amino acids separated by a spacer arm. One of these elements was similar to the SV40 NLS but was not able to direct a protein to the cell nucleus when attached to a non-nuclear reporter protein. Both elements are required.[13] This kind of NLS has become known as a bipartite classical NLS. The bipartite NLS is now known to represent the major class of NLS found in cellular nuclear proteins[14] and structural analysis has revealed how the signal is recognized by a receptor (importin α) protein[15] (the structural basis of some monopartite NLSs is also known[16]). Many of the molecular details of nuclear protein import are now known. This was made possible by the demonstration that nuclear protein import is a two-step process; the nuclear protein binds to the nuclear pore complex in a process that does not require energy. This is followed by an energy-dependent translocation of the nuclear protein through the channel of the pore complex.[17][18] By establishing the presence of two distinct steps in the process the possibility of identifying the factors involved was established and led on to the identification of the importin family of NLS receptors and the GTPase Ran.

Mechanism of nuclear import

Proteins gain entry into the nucleus through the nuclear envelope. The nuclear envelope consists of concentric membranes, the outer and the inner membrane. The inner and outer membranes connect at multiple sites, forming channels between the cytoplasm and the nucleoplasm. These channels are occupied by nuclear pore complexes (NPCs), complex multiprotein structures that mediate the transport across the nuclear membrane.

A protein translated with an NLS will bind strongly to importin (aka karyopherin), and, together, the complex will move through the nuclear pore. At this point, Ran-GTP will bind to the importin-protein complex, and its binding will cause the importin to lose affinity for the protein. The protein is released, and now the Ran-GTP/importin complex will move back out of the nucleus through the nuclear pore. A GTPase-activating protein (GAP) in the cytoplasm hydrolyzes the Ran-GTP to GDP, and this causes a conformational change in Ran, ultimately reducing its affinity for importin. Importin is released and Ran-GDP is recycled back to the nucleus where a Guanine nucleotide exchange factor (GEF) exchanges its GDP back for GTP.

See also

References

  1. ↑ 1.0 1.1 1.2 Mahato, Ram I.; Smith, Louis C.; Rolland, Alain (1999-01-01), Hall, Jeffrey C.; Dunlap, Jay C.; Friedmann, Theodore et al., eds. (in en), 4 - Pharmaceutical Perspectives of Nonviral Gene Therapy, Advances in Genetics, 41, Academic Press, pp. 95–156, doi:10.1016/s0065-2660(08)60152-2, ISBN 9780120176410, PMID 10494618, http://www.sciencedirect.com/science/article/pii/S0065266008601522, retrieved 2020-12-17 
  2. ↑ "A short amino acid sequence able to specify nuclear location". Cell 39 (3 Pt 2): 499–509. 1984. doi:10.1016/0092-8674(84)90457-4. PMID 6096007. 
  3. ↑ 3.0 3.1 "The nucleoplasmin nuclear location sequence is larger and more complex than that of SV-40 large T antigen". J. Cell Biol. 107 (3): 841–9. Sep 1988. doi:10.1083/jcb.107.3.841. PMID 3417784. 
  4. ↑ "Comparative mutagenesis of nuclear localization signals reveals the importance of neutral and acidic amino acids". Curr. Biol. 6 (8): 1025–7. August 1996. doi:10.1016/S0960-9822(02)00648-6. PMID 8805337. 
  5. ↑ "Quantitative tracking of protein trafficking to the nucleus using cytosolic protein delivery by nanoparticle-stabilized nanocapsules". Bioconjug. Chem. 26 (6): 1004–7. 2015. doi:10.1021/acs.bioconjchem.5b00141. PMID 26011555. 
  6. ↑ "Nucleocytoplasmic transport: the soluble phase". Annu Rev Biochem 67 (1): 265–306. 1998. doi:10.1146/annurev.biochem.67.1.265. PMID 9759490. 
  7. ↑ "Nuclear and nucleolar localisation of Saccharomyces cerevisiae ribosomal proteins S22 and S25". FEBS Lett. 452 (3): 335–40. June 1999. doi:10.1016/S0014-5793(99)00669-9. PMID 10386617. 
  8. ↑ The Ribosome: Structure, Function, Antibiotics, and Cellular Interactions. ASM Press. 2000. ISBN 978-1-55581-184-6. http://estore.asm.org/viewItemDetails.asp?ItemID=277. 
  9. ↑ "A distinct nuclear import pathway used by ribosomal proteins". Cell 89 (5): 715–25. May 1997. doi:10.1016/S0092-8674(00)80254-8. PMID 9182759. 
  10. ↑ "Rules for nuclear localisation sequence recognition by karyopherin beta 2". Cell 126 (3): 543–58. August 2006. doi:10.1016/j.cell.2006.05.049. PMID 16901787. 
  11. ↑ "Structure-based design of a pathway-specific nuclear import inhibitor". Nature Structural & Molecular Biology 14 (5): 452–4. May 2007. doi:10.1038/nsmb1229. PMID 17435768. 
  12. ↑ "A polypeptide domain that specifies migration of nucleoplasmin into the nucleus". Cell 30 (2): 449–58. September 1982. doi:10.1016/0092-8674(82)90242-2. PMID 6814762. 
  13. ↑ "Nuclear targeting sequences--a consensus?". Trends in Biochemical Sciences 16 (12): 478–81. December 1991. doi:10.1016/0968-0004(91)90184-W. PMID 1664152. 
  14. ↑ Yanagawa, Hiroshi; Tomita, Masaru; Miyamoto-Sato, Etsuko; Takashima, Hideaki; Matsumura, Nobutaka; Hasebe, Masako; Kosugi, Shunichi (2009-01-02). "Six Classes of Nuclear Localization Signals Specific to Different Binding Grooves of Importin α" (in en). Journal of Biological Chemistry 284 (1): 478–485. doi:10.1074/jbc.M807017200. ISSN 0021-9258. PMID 19001369. 
  15. ↑ "Crystallographic analysis of the specific yet versatile recognition of distinct nuclear localisation signals by karyopherin alpha". Structure 8 (3): 329–38. March 2000. doi:10.1016/s0969-2126(00)00107-6. PMID 10745017. 
  16. ↑ "Crystallographic analysis of the recognition of a nuclear localisation signal by the nuclear import factor karyopherin alpha". Cell 94 (2): 193–204. July 1998. doi:10.1016/S0092-8674(00)81419-1. PMID 9695948. 
  17. ↑ "The nucleoplasmin nuclear location sequence is larger and more complex than that of SV-40 large T antigen". The Journal of Cell Biology 107 (3): 841–9. September 1988. doi:10.1083/jcb.107.3.841. PMID 3417784. 
  18. ↑ "Nuclear import can be separated into distinct steps in vitro: nuclear pore binding and translocation". Cell 52 (5): 641–53. March 1988. doi:10.1016/0092-8674(88)90402-3. PMID 3345567. 

Further reading

External links



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