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37-552: Karyopherins can act as importins (i.e. helping proteins get into the nucleus) or exportins (i.e. helping proteins get out of the nucleus). They belong to the nuclear pore complex family in the transporter classification database (TCDB). Energy for transport is derived from the Ran gradient. Upon stress, several karyopherins stop shuttling between the nucleus and the cytoplasm and are sequestered in stress granules , cytoplasmic aggregates of ribonucleoprotein complexes. Importin beta

74-485: A truncated form of importin-α in which the NLS binding domain is missing. In addition, importin-α has been shown to transport the tumour suppressor gene , BRCA1 (breast cancer type 1 susceptibility protein) , into the nucleus . The overexpression of importin-α has also been linked with poor survival rates seen in certain melanoma patients. Importin activity is also associated with some viral pathologies . For instance, in

111-601: A curved-shaped structure, which facilitates binding to the NLS of specific cargo proteins. The major NLS binding site is found towards the N-terminus , with a minor site being found at the C-terminus . As well as the ARM structures, Importin-α also contains a 90 amino acid N-terminal region, responsible for binding to Importin-β, known as the Importin-β binding (IBB)domain. This is also

148-403: 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

185-511: 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 (NES), which targets proteins out of the nucleus. These types of NLSs can be further classified as either monopartite or bipartite. The major structural differences between

222-495: A receptor for nuclear localization signals (NLS) , thus allowing transport into the nucleus . Since these initial discoveries in 1994 and 1995, a host of Importin genes, such as IPO4 and IPO7 , have been found that facilitate the import of slightly different cargo proteins, due to their differing structure and locality. A large proportion of the importin-α adaptor protein is made up of several armadillo repeats (ARM) arranged in tandem . These repeats can stack together to form

259-453: A site of autoinhibition, and is implicated in the release of cargo once importin-α reaches the nucleus . Importin-β is the typical structure of a larger superfamily of karyopherins . The basis of their structure is 18-20 tandem repeats of the HEAT motif. Each one of these repeats contains two antiparallel alpha helices linked by a turn , which stack together to form the overall structure of

296-466: 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. Makkah et al . carried out comparative mutagenesis on

333-420: 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

370-411: A whole, they actually represent larger families of proteins that share a similar structure and function. Various different genes have been identified for both α and β, with some of them listed below. Note that often karyopherin and importin are used interchangeably. Nuclear localization sequence A nuclear localization signal or sequence ( NLS ) is an amino acid sequence that 'tags'

407-458: Is a variety of karyopherin that facilitates the transport of cargo proteins into the nucleus. First, it is binding importin alpha – another type of karyopherin that binds the cargo protein in the cytoplasm—before the cargo protein is imported into the nucleus through the nuclear pore using energy derived from the Ran gradient . Once inside the nucleus, the cargo dissociates from the karyopherins. Importin beta can also carry proteins into

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444-511: Is free of importin-β, the cargo protein can be released into the nucleus . The N-terminal importin-β-binding (IBB) domain of importin-α contains an auto-regulatory region that mimics the NLS motif . The release of importin-β frees this region and allows it to loop back and compete for binding with the cargo protein at the major NLS-binding site . This competition leads to the release of the protein . In some cases, specific release factors such as Nup2 and Nup50 can be employed to help release

481-444: Is to mediate the translocation of proteins with nuclear localization signals into the nucleus , through nuclear pore complexes (NPC) , in a process known as the nuclear protein import cycle. The first step of this cycle is the binding of cargo. Importin can perform this function as a monomeric importin-β protein , but usually requires the presence of importin-α, which acts as an adaptor to cargo proteins (via interactions with

518-486: The NLS ). The NLS is a sequence of basic amino acids that tags the protein as cargo destined for the nucleus . A cargo protein can contain either one or two of these motifs , which will bind to the major and/or minor binding sites on importin-α. Once the cargo protein is bound, importin-β interacts with the NPC , and the complex diffuses into the nucleus from the cytoplasm . The rate of diffusion depends on both

555-401: The cytoplasm , Ran - GTP is hydrolysed by Ran GAP , forming Ran - GDP , and releasing the two importins for further activity. It is this hydrolysis of GTP that provides the energy for the cycle as a whole. In the nucleus , a GEF will charge Ran with a GTP molecule, which is then hydrolysed by a GAP in the cytoplasm , as stated above. It is this activity of Ran that allows for

592-418: 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

629-405: The protein . In order to transport cargo into the nucleus , importin-β must associate with the nuclear pore complexes . It does this by forming weak, transient bonds with nucleoporins at their various F G (Phe-Gly) motifs. Crystallographic analysis has shown that these motifs bind to importin-β at shallow hydrophobic pockets found on its surface. The primary function of importin

666-408: 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

703-471: 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

740-511: The binding of the PY-NLS contained in Importin β2 has been determined and an inhibitor of import designed. 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

777-421: The cargo as well. Finally, in order to return to the cytoplasm , importin-α must associate with a Ran-GTP / CAS (nuclear export factor) complex which facilitates its exit from the nucleus . CAS (cellular apoptosis susceptibility protein) is part of the importin-β superfamily of karyopherins and is defined as a nuclear export factor. Importin-β returns to the cytoplasm , still bound to Ran - GTP . Once in

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814-461: The concentration of importin-α present in the cytoplasm and also the binding affinity of importin-α to the cargo. Once inside the nucleus , the complex interacts with the Ras-family GTPase , Ran-GTP . This leads to the dissociation of the complex by altering the conformation of importin-β. Importin-β is left bound to Ran - GTP , ready to be recycled. Now that the importin-α/cargo complex

851-434: 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

888-559: 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

925-409: The importin-β family can bind and transport cargo by themselves, or can form heterodimers with importin-α. As part of a heterodimer , importin-β mediates interactions with the pore complex , while importin-α acts as an adaptor protein to bind the nuclear localization signal (NLS) on the cargo. The NLS-Importin α-Importin β trimer dissociates after binding to Ran GTP inside the nucleus , with

962-673: The infection pathway of the Ebola virus , a key step is the inhibition of the nuclear import of PY-STAT1 . This is achieved by the virus sequestering importin-α in the cytoplasm , meaning it can no longer bind its cargo at the NLS . As a result, importin cannot function and the cargo protein stays in the cytoplasm. Many different cargo proteins can be transported into the nucleus by importin. Often, different proteins will require different combinations of α and β in order to translocate. Some examples of different cargo are listed below. Although importin-α and importin-β are used to describe importin as

999-441: The key proteins involved had not been elucidated up until that point. A 60 kDa cytosolic protein, essential for protein import into the nucleus, and with a 44% sequence identity to SRP1p , was purified from Xenopus eggs. It was cloned, sequenced and expressed in E.coli and in order to completely reconstitute signal dependent transport, had to be combined with Ran (TC4). Other key stimulatory factors were also found in

1036-428: 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. By establishing the presence of two distinct steps in

1073-763: 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. 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. There are many other types of NLS, such as

1110-531: 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

1147-527: The nucleus without the aid of the importin alpha adapter protein. This protein -related article is a stub . You can help Misplaced Pages by expanding it . Importin 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). Importin has two subunits, importin α and importin β. Members of

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1184-468: 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 . 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

1221-407: The study. Importin-β, unlike importin-α, has no direct homologues in yeast, but was purified as a 90-95 kDa protein and found to form a heterodimer with importin-α in a number of different cases. These included a study led by Michael Rexach and further studies by Dirk Görlich . These groups found that importin-α requires another protein, importin-β to function, and that together they form

1258-500: 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). The NLS of nucleoplasmin , KR[PAATKKAGQA]KKKK, is the prototype of the ubiquitous bipartite signal: two clusters of basic amino acids, separated by

1295-464: The two importin proteins being recycled to the cytoplasm for further use. Importin can exist as either a heterodimer of importin-α/β or as a monomer of Importin-β. Importin-α was first isolated in 1994 by a group including Enno Hartmann , based at the Max Delbrück Center for Molecular Medicine . The process of nuclear protein import had already been characterised in previous reviews, but

1332-612: The unidirectional transport of proteins . There are several disease states and pathologies that are associated with mutations or changes in expression of importin-α and importin-β. Importins are vital regulatory proteins during the processes of gametogenesis and embryogenesis . As a result, a disruption in the expression patterns of importin-α has been shown to cause fertility defects in Drosophila melanogaster . There have also been studies that link altered importin-α to some cases of cancer . Breast cancer studies have implicated

1369-444: 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. 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

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