Nuclear localization signal

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A nuclear localization signal or sequence (NLS) is an amino acid sequence which acts like a 'tag' on the exposed surface of a protein. This sequence is used to target the protein to the cell nucleus through the Nuclear Pore Complex and to direct a newly synthesized protein into the nucleus via its recognition by cytosolic nuclear transport receptors. Typically, this signal consists of one or more short sequences of positively charged lysines or arginines. Different nuclear localized proteins may share the same NLS. An NLS has the opposite function of a nuclear export signal, which targets proteins out of the nucleus.

Types of nuclear localization signals

The first NLS to be discovered is the sequence PKKKRKV in SV40 Large T-antigen.^[1] The NLS of nucleoplasmin, KR[PAATKKAGQA]KKKK, is the prototype of ubiquitous bipartite signal: two clusters of basic amino acids, separated by a spacer of about 10 amino acids.^[2] 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. Makkerh et al. carried out comparative mutagenesis on the nuclear localisation signals of SV40 T-Antigen (monopartite), C-myc (monopartite) and nucleoplasmin (bipartite), and showed amino acid features common to all three. Notably the role of neutral and acidic amino acids was shown for the first time in contributing to the efficiency of the NLS.^[4]

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 .^[5]

A signal that appears to be specific for the massively produced and transported ribosomal proteins,^[6][7] seems to come with a specialized set of importin β-like nuclear import receptors.^[8]

Nuclear Protein Import, background

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 were 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 which 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 which acted as a signal for nuclear entry (Dingwall, Sharnick and Laskey, 1982. Cell 30:449-458). This work stimulated research in the area and two years later the first nuclear localisation sequence (NLS) was identified in SV40 Large T antigen. 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 the Nucleoplasmin NLS 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 (Dingwall & Laskey, 1991. Trends Biochem Sci 16:478-481). The bipartite NLS is now known to represent the major class of NLS found in cellular nuclear proteins and structural analysis has revealed how the signal is recognised by a receptor (importin) molecule. 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 which does not require energy. This is followed by an energy dependent translocation of the nuclear protein through the channel of the pore complex (Richardson et al., 1988. Cell 52:655-664; Newmeyer & Forbes 1988, Cell 52: 641-653). 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. These are the gateways to the nucleus. The envelope consist of pores or large nuclear complexes.

A protein translated with a 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.

Nuclear export

Exporting proteins out of the nucleus is programmed by a nuclear export signal.

References

Additional reading

• Görlich D (Jun 1997). "Nuclear protein import". Curr Opin Cell Biol. 9 (3): 412–9. doi:10.1016/S0955-0674(97)80015-4. PMID 9159081. http://linkinghub.elsevier.com/retrieve/pii/S0955-0674(97)80015-4. 
• Lusk CP, Blobel G, King MC (May 2007). "Highway to the inner nuclear membrane: rules for the road". Nat Rev Mol Cell Biol. 8 (5): 414–20. doi:10.1038/nrm2165. PMID 17440484.