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In eukaryotic cells, a lot of proteins are selectively imported from the cytosol to the nucleus through nuclear pores. | In eukaryotic cells, a lot of proteins are selectively imported from the cytosol to the nucleus through nuclear pores. | ||
'''NPC'''s ( | '''NPC'''s (''Nuclear Pore Complexes'') are composed of many [[Nucleoporin|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. <ref name="ALBERTS">ALBERTS,B. JOHNSON,A. WALTER,P. LEWIS,J. RAFF,M. ROBERTS,K. (2007). Molecular Biology of the Cell (p. 704-712)</ref> | Thus, proteins over 60 kDa have trouble to pass through them that’s why they need complementary proteins : '''[[Importin|importins]]''' to go in the nucleus and '''[[Exportin|exportins]]''' to go out of the nucleus. <ref name="ALBERTS">ALBERTS,B. JOHNSON,A. WALTER,P. LEWIS,J. RAFF,M. ROBERTS,K. (2007). Molecular Biology of the Cell (p. 704-712)</ref> | ||
===Selectivity of nuclear import=== | ===Selectivity of nuclear import=== | ||
Proteins which have to go in the nucleus all possess a '''NLS''' ( | 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 : | A NLS is a basic residue-rich sequence with the following consensus sequence : | ||
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===Cytoplasm to nucleus=== | ===Cytoplasm to nucleus=== | ||
[[Image:Nuclear_import_protein.png | | [[Image:Nuclear_import_protein.png | 550px | right |thumb|Nuclear import of proteins through importins α and β]] | ||
To initiate the transport to the nucleus, most of the NLS-containing proteins (or cargos) have to be recognized by a Nuclear Import | 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|importin β]]'''. | ||
Import through NPC follows different steps : | Import through NPC follows different steps : | ||
* '''Step 1 :''' The NLS of the protein binds the NLS-binding site of importin α. | * '''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 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.<ref name="ALBERTS"/> | * '''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.<ref name="ALBERTS"/> | ||
===Cargo release and protein recycling=== | ===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. | 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 4 :''' Cargo release is driven by '''[[Ran|Ran]]''', a '''[[G-protein|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 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 6 :''' '''[[Nup50|Nup50]]''' (also called '''[[Nup50|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 7 :''' Importin α can’t leave the nucleus only with the help of Ran-GTP. It needs also interaction with '''[[CAS|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.<ref>PMID:16222336</ref> | * '''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'''.<ref name ="NUP50">PMID:16222336</ref> | ||
==Importin α structure== | ==Importin α structure== | ||
''(<scene name='Sandbox_Reserved_709/Importin_default/1'>Back to the default scene</scene>)'' | |||
<Structure load='1ejy' size=' | <Structure load='1ejy' size='350' color='white' frame='true' align='right' caption='Mouse importin α bound to a NLS of a peptide (PDB 1ejy)' scene='Sandbox_Reserved_709/Importin_default/1' /> | ||
'''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 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: | 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. | * '''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.<ref>http://prosite.expasy.org/PDOC51214</ref> | ||
Here, as the structure begins at the amino acid number 70, we cannot see the IBB which is located in the 2-60 region. | 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''' <scene name='Sandbox_Reserved_709/Importin_nlsbs/ | * '''A 50kDa C-terminal''' <scene name='Sandbox_Reserved_709/Importin_nlsbs/2'>NLS binding sites</scene> contained in <scene name='Sandbox_Reserved_709/Importin_arm/5'>10 tandem armadillo (Arm) repeats</scene>. 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).<ref>PMID:22248489</ref> | ||
Here you can see a <scene name='Sandbox_Reserved_709/Nls_basic/ | Here you can see a <scene name='Sandbox_Reserved_709/Nls_basic/2'>NLS </scene>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. <ref>PMID:8692858</ref> | * '''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. <ref>PMID:8692858</ref> | ||
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* '''A CAS-binding site.''' | * '''A CAS-binding site.''' | ||
<scene name='Sandbox_Reserved_709/ | ==Importin α : Nup50 complex== | ||
<Structure load='2c1m' size='350' color='white' frame='true' align='right' scene='Sandbox_Reserved_709/Impnup_default/2' caption='Mouse importin α:Nup50 complex (PDB 2c1m) /> | |||
''(<scene name='Sandbox_Reserved_709/Impnup_default/2'>Back to Default scene </scene>)'' | |||
'''Nup50''' (''Nucleoporin 50 kDa'', about 450 aminoacids) is a part of the '''NPCs''', in the '''nucleoplasmic side'''. It is able to bind to <scene name='Sandbox_Reserved_709/Importin/2'>importin importin α</scene> thanks to <scene name='Sandbox_Reserved_709/Nup50/3'>two binding segments</scene> 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''' <scene name='Sandbox_Reserved_709/Nup_bindingnls/1'>Lys3 and Arg4</scene> 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. <ref name ="NUP50"/> | |||
* 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 <scene name='Sandbox_Reserved_709/Nup50_bindingcas/1'>acidic surface</scene> 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'''.<ref name ="NUP50"/> | |||
The chain between the two binding segments is rich in acidic residues, and seems to work as a flexible linker between those two sites. | |||
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[[Category: Mus musculus]] | [[Category: Mus musculus]] | ||
[[Category: Importin-alpha]] | [[Category: Importin-alpha]] | ||
[[Category: Nuclear protein]] | [[Category: Nuclear protein]] | ||
[[Category: Nucleoporin nup50]] | [[Category: Nucleoporin nup50]] | ||
[[Category: Protein transport]] | [[Category: Protein transport]] | ||
[[Category: Protein transport-membrane protein complex]] | [[Category: Protein transport-membrane protein complex]] | ||
[[Category: Protein transport/membrane protein]] | [[Category: Protein transport/membrane protein]] | ||
[[Category: Importin aplpha/karyopherin alpha]] | |||
[[Category: Protein binding]] | |||
[[Category: Nucleoplasmin]] | |||
<ref group="xtra">PMID:22847741</ref><references group="xtra"/> | <ref group="xtra">PMID:22847741</ref><references group="xtra"/> | ||
<ref group="xtra">PMID:22510057</ref><references group="xtra"/> | <ref group="xtra">PMID:22510057</ref><references group="xtra"/> | ||
