Nuclear localization sequence

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A nuclear localization signalorsequence (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.

Contents

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

Related Research Articles

<span class="mw-page-title-main">Nuclear pore</span> Openings in nuclear envelope of eukaryotic cells

A nuclear pore is a channel as part of the nuclear pore complex (NPC), a large protein complex found in the nuclear envelope of eukaryotic cells. The nuclear envelope (NE) surrounds the cell nucleus containing DNA and facilitates the selective membrane transport of various molecules.

Protein targeting or protein sorting is the biological mechanism by which proteins are transported to their appropriate destinations within or outside the cell. Proteins can be targeted to the inner space of an organelle, different intracellular membranes, the plasma membrane, or to the exterior of the cell via secretion. Information contained in the protein itself directs this delivery process. Correct sorting is crucial for the cell; errors or dysfunction in sorting have been linked to multiple diseases.

<span class="mw-page-title-main">SV40 large T antigen</span> Proto-oncogene derived from polyomavirus SV40

SV40 large T antigen is a hexamer protein that is a dominant-acting oncoprotein derived from the polyomavirus SV40. TAg is capable of inducing malignant transformation of a variety of cell types. The transforming activity of TAg is due in large part to its perturbation of the retinoblastoma (pRb) and p53 tumor suppressor proteins. In addition, TAg binds to several other cellular factors, including the transcriptional co-activators p300 and CBP, which may contribute to its transformation function. Similar proteins from related viruses are known as large tumor antigen in general.

Karyopherins are proteins involved in transporting molecules between the cytoplasm and the nucleus of a eukaryotic cell. The inside of the nucleus is called the karyoplasm. Generally, karyopherin-mediated transport occurs through nuclear pores which act as a gateway into and out of the nucleus. Most proteins require karyopherins to traverse the nuclear pore.

Importin is a type of karyopherin that transports protein molecules from the cell's cytoplasm to the nucleus. It does so by binding to specific recognition sequences, called nuclear localization sequences (NLS).

<span class="mw-page-title-main">Ran (protein)</span> GTPase functioning in nuclear transport

Ran also known as GTP-binding nuclear protein Ran is a protein that in humans is encoded by the RAN gene. Ran is a small 25 kDa protein that is involved in transport into and out of the cell nucleus during interphase and also involved in mitosis. It is a member of the Ras superfamily.

Nuclear transport refers to the mechanisms by which molecules move across the nuclear membrane of a cell. The entry and exit of large molecules from the cell nucleus is tightly controlled by the nuclear pore complexes (NPCs). Although small molecules can enter the nucleus without regulation, macromolecules such as RNA and proteins require association with transport factors known as nuclear transport receptors, like karyopherins called importins to enter the nucleus and exportins to exit.

<span class="mw-page-title-main">Nuclear pore glycoprotein p62</span> Protein-coding gene in the species Homo sapiens

Nuclear pore glycoprotein p62 is a protein complex associated with the nuclear envelope. The p62 protein remains associated with the nuclear pore complex-lamina fraction. p62 is synthesized as a soluble cytoplasmic precursor of 61 kDa followed by modification that involve addition of N-acetylglucosamine residues, followed by association with other complex proteins. In humans it is encoded by the NUP62 gene.

<span class="mw-page-title-main">Nucleoporin</span> Family of proteins that form the nuclear pore complex

Nucleoporins are a family of proteins which are the constituent building blocks of the nuclear pore complex (NPC). The nuclear pore complex is a massive structure embedded in the nuclear envelope at sites where the inner and outer nuclear membranes fuse, forming a gateway that regulates the flow of macromolecules between the cell nucleus and the cytoplasm. Nuclear pores enable the passive and facilitated transport of molecules across the nuclear envelope. Nucleoporins, a family of around 30 proteins, are the main components of the nuclear pore complex in eukaryotic cells. Nucleoporin 62 is the most abundant member of this family. Nucleoporins are able to transport molecules across the nuclear envelope at a very high rate. A single NPC is able to transport 60,000 protein molecules across the nuclear envelope every minute.

<span class="mw-page-title-main">Importin subunit alpha-1</span> Protein-coding gene in the species Homo sapiens

Importin subunit alpha-1 is a protein that in humans is encoded by the KPNA2 gene.

<span class="mw-page-title-main">KPNB1</span> Protein-coding gene in the species Homo sapiens

Importin subunit beta-1 is a protein that in humans is encoded by the KPNB1 gene.

<span class="mw-page-title-main">Importin subunit alpha-4</span> Protein-coding gene in the species Homo sapiens

Importin subunit alpha-4 also known as karyopherin subunit alpha-3 is a protein that in humans is encoded by the KPNA3 gene.

<span class="mw-page-title-main">Importin subunit alpha-7</span> Protein-coding gene in the species Homo sapiens

Importin subunit alpha-7 is a protein that in humans is encoded by the KPNA6 gene.

<span class="mw-page-title-main">Importin subunit alpha-3</span> Protein-coding gene in the species Homo sapiens

Importin subunit alpha-3, also known as karyopherin subunit alpha-4, is a protein that in humans is encoded by the KPNA4 gene.

<span class="mw-page-title-main">IPO5</span> Protein-coding gene in the species Homo sapiens

Importin-5 is a protein that in humans is encoded by the IPO5 gene. The protein encoded by this gene is a member of the importin beta family. Structurally, the protein adopts the shape of a right hand solenoid and is composed of 24 HEAT repeats.

<span class="mw-page-title-main">Importin subunit alpha-6</span> Protein-coding gene in the species Homo sapiens

Importin subunit alpha-6 is a protein that in humans is encoded by the KPNA5 gene.

<span class="mw-page-title-main">Transportin 1</span> Protein-coding gene in the species Homo sapiens

Transportin-1 is a protein that in humans is encoded by the TNPO1 gene.

<span class="mw-page-title-main">Late protein</span>

A late protein is a viral protein that is formed after replication of the virus. One example is VP4 from simian virus 40 (SV40).

Importin alpha, or karyopherin alpha refers to a class of adaptor proteins that are involved in the import of proteins into the cell nucleus. They are a sub-family of karyopherin proteins.

A target peptide is a short peptide chain that directs the transport of a protein to a specific region in the cell, including the nucleus, mitochondria, endoplasmic reticulum (ER), chloroplast, apoplast, peroxisome and plasma membrane. Some target peptides are cleaved from the protein by signal peptidases after the proteins are transported.

References

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  10. ↑ Lee BJ, Cansizoglu AE, Süel KE, Louis TH, Zhang Z, Chook YM (August 2006). "Rules for nuclear localisation sequence recognition by karyopherin beta 2". Cell . 126 (3): 543–58. doi:10.1016/j.cell.2006.05.049. PMC   3442361 . PMID   16901787.
  11. ↑ Cansizoglu AE, Lee BJ, Zhang ZC, Fontoura BM, Chook YM (May 2007). "Structure-based design of a pathway-specific nuclear import inhibitor". Nature Structural & Molecular Biology . 14 (5): 452–4. doi:10.1038/nsmb1229. PMC   3437620 . PMID   17435768.
  12. ↑ Dingwall C, Sharnick SV, Laskey RA (September 1982). "A polypeptide domain that specifies migration of nucleoplasmin into the nucleus". Cell . 30 (2): 449–58. doi:10.1016/0092-8674(82)90242-2. PMID   6814762. S2CID   39465973.
  13. ↑ Dingwall C, Laskey RA (December 1991). "Nuclear targeting sequences--a consensus?". Trends in Biochemical Sciences . 16 (12): 478–81. 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". Journal of Biological Chemistry. 284 (1): 478–485. doi: 10.1074/jbc.M807017200 . ISSN   0021-9258. PMID   19001369.
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  18. ↑ Newmeyer DD, Forbes DJ (March 1988). "Nuclear import can be separated into distinct steps in vitro: nuclear pore binding and translocation". Cell . 52 (5): 641–53. doi:10.1016/0092-8674(88)90402-3. PMID   3345567. S2CID   45889419.

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