CBIO 3400 Week 11 Study Questions 1 Describe the structure and function of the nuclear lamina How is it organized during mitosis Structure lines inner nuclear membrane nucleoplasmic membrane connected to chromatin by lamin associated proteins LAPs network meshwork of intermediate filaments Function During Mitosis mitosis strengthens nuclear envelope determines how DNA is organized in nucleus if organized incorrectly then DNA processing can t occur properly causes Progeria phosphorylations of LamB LAP2 cause nuclear lamina to break down at beginning of at end of mitosis phosphorylations go away nuclear lamina re assembles MPF is a CDK MPF must be shut down when anaphase starts phosphorylations start going away NE begins to re form 2 Describe the location structure and function of nuclear pore complexes Location Structure region where inner outer nuclear membranes join cylinder diameter 120 nm mass 125 MDa central part pore 45 nm diameter made up of 30 nucleoporins Nups 8 16 copies each per NPC originally described in thin section transmission EM as electron dense structure within NE central plug forms center surrounded by inner spoke ring has 8 equally spaced spokes emanating from it inner spoke ring 4 coaxial rings attach each spoke to its neighbors 1 inner spoke ring 2 outer spoke ring 3 cytoplasmic ring 4 nucleoplasmic ring 8 cytoplasmic filaments particles connected to coaxial ring nuclear basket anchored to nuclear ring 8 filaments attach to nuclear ring converge on a distal basket ring called a molecular cloud loaded w FG repeats can be released from NE w detergent Function permeable to molecules 5 000 Da when water molecules are micro injected into cytoplasm they go into nucleus super huge proteins 60 000 Da do not rapidly equilibrate nuclear import export passageway 1 3 What is a nuclear localization signal NLS If you add a NLS to a cytosolic protein where will you expect to find this protein in the cell NLS sequence on protein recognized by receptor transporter to initiate nuclear import short sequence of AAs 4 8 residues charged Lys Arg usually contains Proline sufficient to direct non nuclear protein to nucleus covalently attach NLS to non nuclear protein goes into nucleus 2 main types 1 SV40 T Antigen contains Pro 2 Bi Partite NLS can occur anywhere in protein more common 2 blocks of basic AAs separated by a 10 AA spacer discovered as mutant virus that would not cause tumor formation Lys converted to Thr shown that this is NLS shows NLS is necessary for nuclear import If I add a NLS to a cytosolic protein then I will expect to find that protein in the nucleoplasm b c the NLS is sufficient for nuclear import mutations in NLS sequence inhibit transport addition of competitor NLS polypeptide inhibits import of NLS protein nuclear import of NLS protein saturable 4 Describe the mechanism of nuclear protein import starting from a newly synthesized nuclear protein in the cytosol 1 NLS Nuclear Localization Signal brings nuclear protein near nuclear membrane 2 Receptor importin recognizes NLS recruits transporter importin Transporter binds protein cargo Sometimes the receptor transporter are the same sometimes they re not 3 Transporter cargo complex binds NPC Nuclear Pore Complex 4 Transporter chaperones cargo through hydrophobic FG repeats of nuclear pore 5 Ran GTP in nucleoplasm binds transporter kicks out cargo 6 Cargo releases gets trapped in nucleoplasm b c transporter left it so it can t get back 7 Transporter goes back through nuclear pore bound to Ran GTP to cytoplasm where it sees through nuclear pore Ran GAP bound to cytosolic fibrils activity cytoplasm binds new cargo 8 Ran GAP stimulates hydrolysis of Ran GTP to Ran GDP RanBP1 2 facilitates Ran GAP 9 RanBP1 2 dissociates Ran GTP from transporter karyopherin Transporter releases into 10 NTF2 Nuclear Transport Factor 2 binds Ran GDP in the cytoplasm associates with nucleoporins Nups at XFXFG repeats in the NPC to transport Ran GDP into nucleoplasm 11 Ran GEF RCC1 facilitated by chromatin stimulates nucleotide exchange from Ran GDP to Ran GTP in nucleoplasm This exchange kicks NTF2 off of Ran GTP NTF2 is recycled back to cytoplasm to be used again 2 RAN Ran GTPase Cycle Summary graphic depic ng all factors in Ran GTPase cycle Nuclear Import Export Ran GTPase Cycle 3
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