Nuclear localization signal
Encyclopedia
A nuclear localization signal or sequence (NLS) is an amino acid
sequence which 'tags' a protein for import into the cell nucleus
by nuclear transport
. Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface. 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.
(a monopartite NLS). 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. 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. 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 localization 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.
A signal that appears to be specific for the massively produced and transported ribosomal proteins, seems to come with a specialized set of importin β-like nuclear import receptors.
Recently a class of NLSs known as PY-NLSs has been proposed, originally by Lee et. al. 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.
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. 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. 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 and structural analysis has revealed how the signal is recognized by a receptor (importin α
) protein (the structural basis of some monopartite NLSs is also known). 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. 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
.
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.
Amino acid
Amino acids are molecules containing an amine group, a carboxylic acid group and a side-chain that varies between different amino acids. The key elements of an amino acid are carbon, hydrogen, oxygen, and nitrogen...
sequence which 'tags' a protein for import into the cell nucleus
Cell nucleus
In cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...
by nuclear transport
Nuclear transport
The entry and exit of large molecules from the cell nucleus is tightly controlled by the nuclear pore complexes . Although small molecules can enter the nucleus without regulation, macromolecules such as RNA and proteins require association with karyopherins called importins to enter the nucleus...
. Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface. Different nuclear localized proteins may share the same NLS. An NLS has the opposite function of a nuclear export signal
Nuclear export signal
A nuclear export signal is a short amino acid sequence of 4 hydrophobic residues in a protein that targets it for export from the cell nucleus to the cytoplasm through the nuclear pore complex using nuclear transport. It has the opposite effect of a nuclear localization signal, which targets a...
, which targets proteins out of the nucleus.
Classical NLSs
Classical NLSs can be further classified as either monopartite or bipartite. The first NLS to be discovered was the sequence PKKKRKV in the SV40 Large T-antigenSV40 Large T-antigen
SV40 large T antigen is a hexamer protein that is a proto-oncogene derived from the polyomavirus SV40 which is capable of transforming a variety of cell types. The transforming activity of TAg is due in large part to its perturbation of the retinoblastoma and p53 tumor suppressor proteins...
(a monopartite NLS). 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. Both signals are recognized by importin α
Importin α
Importin alpha, or karyopherin alpha proteins are involved in the import of proteins into the cell nucleus. They are a sub-family of karyopherin proteins....
. 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. 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 localization 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.
Non-classical NLSs
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 .A signal that appears to be specific for the massively produced and transported ribosomal proteins, seems to come with a specialized set of importin β-like nuclear import receptors.
Recently a class of NLSs known as PY-NLSs has been proposed, originally by Lee et. al. This PY-NLS motif, so named because of the proline
Proline
Proline is an α-amino acid, one of the twenty DNA-encoded amino acids. Its codons are CCU, CCC, CCA, and CCG. It is not an essential amino acid, which means that the human body can synthesize it. It is unique among the 20 protein-forming amino acids in that the α-amino group is secondary...
-tyrosine
Tyrosine
Tyrosine or 4-hydroxyphenylalanine, is one of the 22 amino acids that are used by cells to synthesize proteins. Its codons are UAC and UAU. It is a non-essential amino acid with a polar side group...
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.
Discovery of nuclear localization signals
The presence of the nuclear membrane that sequesters the cellular DNADNA
Deoxyribonucleic acid is a nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms . The DNA segments that carry this genetic information are called genes, but other DNA sequences have structural purposes, or are involved in...
is the defining feature of eukaryotic cells. The nuclear membrane therefore separates the nuclear processes of DNA replication and RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
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. This work stimulated research in the area and two years later the first NLS was identified in SV40 Large T-antigen
SV40 Large T-antigen
SV40 large T antigen is a hexamer protein that is a proto-oncogene derived from the polyomavirus SV40 which is capable of transforming a variety of cell types. The transforming activity of TAg is due in large part to its perturbation of the retinoblastoma and p53 tumor suppressor proteins...
(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. 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 and structural analysis has revealed how the signal is recognized by a receptor (importin α
Importin α
Importin alpha, or karyopherin alpha proteins are involved in the import of proteins into the cell nucleus. They are a sub-family of karyopherin proteins....
) protein (the structural basis of some monopartite NLSs is also known). 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. 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
GTPase
GTPases are a large family of hydrolase enzymes that can bind and hydrolyze guanosine triphosphate . The GTP binding and hydrolysis takes place in the highly conserved G domain common to all GTPases.-Functions:...
Ran
Ran (biology)
Ran is a small 25Kda 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....
.
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
Importin
Importin is a type of protein that moves other protein molecules into the nucleus by binding to a specific recognition sequence, called the nuclear localization signal . Importin is classified as a karyopherin....
(aka karyopherin), and together, the complex will move through the nuclear pore. At this point, Ran-GTP
Ran (biology)
Ran is a small 25Kda 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....
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
GTPase activating protein
GTPase-Activating Proteins, or GAPs, or GTPase-Accelerating Proteins are a family of regulatory proteins whose members can bind to activated G proteins and stimulate their GTPase activity, with the result of terminating the signaling event...
(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
Guanine nucleotide exchange factor
Guanine nucleotide exchange factors activate monomeric GTPases by stimulating the release of guanosine diphosphate to allow binding of guanosine triphosphate . A variety of unrelated structural domains have been shown to exhibit guanine nucleotide exchange activity...
(GEF) exchanges its GDP back for GTP.
See also
- A Nuclear export signalNuclear export signalA nuclear export signal is a short amino acid sequence of 4 hydrophobic residues in a protein that targets it for export from the cell nucleus to the cytoplasm through the nuclear pore complex using nuclear transport. It has the opposite effect of a nuclear localization signal, which targets a...
(NES) can direct a protein to be exported from the nucleus.