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From Proteopedia
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Nuclear import of proteins
In eukaryotic cells, a lot of proteins are selectively imported from the cytosol to the nucleus through nuclear pores.
NPCs (Nuclear Pore Complexes) are composed of many nucleoporins and are heavy proteins assembly. These complexes allow the passive diffusion of ions and the active transport of large molecules such as proteins in both directions. Because of the particular shape of NPCs, transport of large proteins take a lot of time. Thus, proteins over 60 kDa have trouble to pass through them that’s why they need complementary proteins : importins to go in the nucleus and exportins to go out of the nucleus. [1]
Selectivity of nuclear import
Proteins which have to go in the nucleus all possess a NLS (Nuclear Localization Signal) which is responsible for the selectivity of the active transport of proteins through NPCs.
A NLS is a basic residue-rich sequence with the following consensus sequence :
- Two adjacent basic amino acids (Arg or Lys).
- A spacer region of 10 residues.
- At least three basic residues (Arg or Lys) in the five positions after the spacer region.[2]
NLS can be found at any place of the amino acid sequence and often have a loop structure at the surface of proteins.
The transport through NPC is not the same as transmembrane transport in organelles because in this case, it is an aquifer pore whereas transmembrane transport in organelles involves transmembrane proteins. Thus, fully folded nuclear proteins can pass through NPCs. Nevertheless, it seems that really large proteins undergo a compression when they pass through NPC. [1]
Cytoplasm to nucleus
To initiate the transport to the nucleus, most of the NLS-containing proteins (or cargos) have to be recognized by a Nuclear Import Receptor. These are soluble cytosolic proteins such as importin α and importin β.
Import through NPC follows different steps :
- Step 1 : The NLS of the protein binds the NLS-binding site of importin α.
- Step 2 : Importin α binds to importin β because of its importin β binding domain (IBB). Importin β binds to some fibrils of the NPC. These fibrils contain a lot of short amino-acid repeats that contain phenylalanine and glycine and are therefore called FG-repeats.
- Step 3 : The complex cargo:importin α:importin β move along the NPC by repeatedly binding, dissociating and re-binding to adjacent FG-repeat sequences.[1]
Cargo release and protein recycling
Once the import complex enters the nucleus, it must be dissociated to release the cargo protein. Then, importin α must be recycled to the cytoplasm.
- Step 4 : Cargo release is driven by Ran, a G-protein that is found in Ran-GTP form in the nucleus due to a higher rate of RanGEF than RanGAP in chromatin. It binds to importin β, causing an important change in shape and reducing importin β affinity for importin α IBB.
- Step 5 : Importin β can pass through the NPC thanks to Ran-GTP. A Ran-BP (Ran-Binding Protein) is a major component of the NPC’s cytoplasmic filaments. Thus, Ran-GTP is not released in the cytoplasm, but is still bound with importin β to the NPC. Ran-BP is also able to bind to a RanGAP. Finally, RanGAP locally promotes hydrolysis of Ran-GTP into Ran-GDP, leading to its own release, and importin β release by the same.
- Step 6 : Nup50 (also called Npap60), a nucleoplasmin, weakens the link between importin α and the NLS of the cargo. Thus the cargo protein is now free in the nucleoplasm.
- Step 7 : Importin α can’t leave the nucleus only with the help of Ran-GTP. It needs also interaction with CAS (Cellular Apoptosis Susceptibility protein), an exportin which is similar in shape to importin β. Binding between CAS and importin α is favoured by interactions with Nup50.
- Step 8 :The newly formed importin α:CAS:Ran-GTP complex is able to interact with nucleoplasmins of NPC, and once in the cytoplasm, Ran-GTP is hydrolysed into Ran-GDP (see Step 5 for details). The complex splits, and importin α is now ready for a new import cycle.[3]
Importin α structure
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Importin α is a soluble adaptor protein also known as karyopherin α. Its function is to bind a protein containing a cNLS (classical Nuclear Localization Signal) and then to bind an importin β in order to help the import of this protein in the nucleus.
Importin α is composed of different domains:
- A flexible and hydrophilic 10kDa N-terminal Importin β binding domain (IBB domain). The IBB domain is a L-shapped molecule with an N-terminal extended moiety and a C-terminal helix running in mutually perpendicular directions. Because this domain is highly positively charged, it can binds to the inner surface of importin-β that contains many acidic residues. It has been shown that importin α contains a determinant which is sufficient for binding importin β. The consensus sequence of this determinant is "KFRLLSKE". The serine contained in this sequence is present in all importin α which shows its importance. However, the upstream region is sufficient for binding importin β too. Nowadays, we think that this upstream region contribute to the strength of the bond. This could explain the fact that the binding between importin α and β is stronger when α contains these two determinants.[4]
Here, as the structure begins at the amino acid number 70, we cannot see the IBB which is located in the 2-60 region.
- A 50kDa C-terminal contained in . These arm repeat domains have an elongated superhelical structure and each of them contains 3 α-helices (H1, H2, H3). Together, H3 helices define the inner concave surface of the protein and the NLS-binding site. The main chain of the cNLs runs antiparallel to the direction of the importin α superhelix, with the cNLS backbone interacting with an array of conserved asparagine residues in consecutive Arm repeats on the importin α surface.In fact there are two NLS-binding sites : a major one and a minor one. Thus, monopartite cNLSs are able to bind at two distinct sites on importin α (with a preference for the major site). Bipartite cNLSs simultaneously interact with both binding sites, with the larger C-terminal basic cluster binding to the major site (located in Arm repeats 2-4) and the smaller upstream cluster binding to the minor site (located in Arm repeats 6-8).[5]
Here you can see a bound to the NLS-binding site of importin α.
- A NLS. Thus, importin α belongs to the group of proteins containing both a ligand (NLS) and a cognate receptor (NLS-binding site). That’s why it could have a possibility of autologous ligand-receptor interactions. Nevertheless, it has been shown that NLS of importin α overlaps with the IBB. Thereby, binding of importin β to importin α covers the NLS of importin α preventing autologous ligand receptor interactions. [6]
As for the IBB, this NLS domain is not visible in this structure as it is located in the 45-54 region. However, the overlap of the NLS by the IBB is proved by this information.
- A CAS-binding site.
Importin α : Nup50 complex
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Nup50 (Nucleoporin 50 kDa, about 450 aminoacids) is a part of the NPCs, in the nucleoplasmic side. It is able to bind to thanks to on its N-terminus (only 46 first aminoacids represented on the structure).
- The first one (1-15) binds to the minor NLS binding site of importin α. This binding is allowed by the two basic aminoacids of Nup50. In fact, the positive charge will establish bonds with acidic aminoacids of the NLS binding site, just as an NLS-containing cargo would do. In presence of Nup50, the complex importin α:NLS dissociation rate is higher than the one of spontaneous dissociation : there is a competition between Nup50 and NLS, whose affinities for the NLS minor binding site are in the same order of magnitude. [3]
- The second one (24-46) binds to the C-terminus of importin α. Nup50 basic residues 41-46 (KKAKRR) are directed by the formation of two turns of an α helix (31-36 SEEVMK) towards an on ARM10. Those electrostatic interactions are essential for the binding of Nup50. Indeed, replacing residues 41-46 by Ala makes the binding undetectable. Furthermore, this binding overlaps the CAS and the Ran binding sites, explaining why interaction between Nup50 and importin is crucial for building the export complex.[3]
The chain between the two binding segments is rich in acidic residues, and seems to work as a flexible linker between those two sites.
See Also
Contributors
Marmin Lucas/ Talide Loïc
ESBS 1A 2012
Reference
- ↑ 1.0 1.1 1.2 ALBERTS,B. JOHNSON,A. WALTER,P. LEWIS,J. RAFF,M. ROBERTS,K. (2007). Molecular Biology of the Cell (p. 704-712)
- ↑ http://prosite.expasy.org/PDOC00015
- ↑ 3.0 3.1 3.2 Matsuura Y, Stewart M. Nup50/Npap60 function in nuclear protein import complex disassembly and importin recycling. EMBO J. 2005 Nov 2;24(21):3681-9. Epub 2005 Oct 13. PMID:16222336
- ↑ http://prosite.expasy.org/PDOC51214
- ↑ Marfori M, Lonhienne TG, Forwood JK, Kobe B. Structural Basis of High-Affinity Nuclear Localization Signal Interactions with Importin-alpha Traffic. 2012 Jan 16. doi: 10.1111/j.1600-0854.2012.01329.x. PMID:22248489 doi:10.1111/j.1600-0854.2012.01329.x
- ↑ Moroianu J, Blobel G, Radu A. The binding site of karyopherin alpha for karyopherin beta overlaps with a nuclear localization sequence. Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6572-6. PMID:8692858
- Lu Q, Lu Z, Liu Q, Guo L, Ren H, Fu J, Jiang Q, Clarke PR, Zhang C. Chromatin-bound NLS proteins recruit membrane vesicles and nucleoporins for nuclear envelope assembly via importin-alpha/beta. Cell Res. 2012 Nov;22(11):1562-75. doi: 10.1038/cr.2012.113. Epub 2012 Jul 31. PMID:22847741 doi:10.1038/cr.2012.113
- Ogawa Y, Miyamoto Y, Oka M, Yoneda Y. The interaction between importin-alpha and Nup153 promotes importin-alpha/beta-mediated nuclear import. Traffic. 2012 Jul;13(7):934-46. doi: 10.1111/j.1600-0854.2012.01367.x. Epub 2012 , May 14. PMID:22510057 doi:10.1111/j.1600-0854.2012.01367.x
