Sandbox Reserved 709: Difference between revisions
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===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|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 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|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 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|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 name ="NUP50">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== | ||
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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 <scene name='Sandbox_Reserved_709/Importin/2'> importin α </scene>thanks to <scene name='Sandbox_Reserved_709/Nup50/3'>two binding segments</scene> on its N-terminus (''only | '''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 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 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"/> | ||
