Cell Nucleus Import Export through Nuclear Pores Lecture 2 NUCLEAR IMPORT protein transla on ONLY occurs in CYTOPLASM nearly all nuclear proteins must be imported from cytoplasm major substrate types 1 2 3 4 nuclear proteins histones chroma n sca old proteins pols transcrip on factors snRNPs ribosomal subunit components aIer mitosis NE re forms all nuclear proteins need cket to get back into nucleus never cut o signal on nuclear protein compared to mitochondrial protein whose signal gets cut o when it enters mitochondria transla on in cytoplasm protein import 4 major substrate types don t cut o nuclear signal cket Nuclear Import NUCLEAR IMPORT small proteins enter nucleus via simple di usion large non nuclear proteins excluded from nucleus EX lysozyme conjugated to uorescein physiological temp 370C 40C cold slower di usion but s8ll happens can t enter b c don t have cket signal to show receptor EX bovine serum albumin conjugated to uorescein 370C large nuclear proteins transported to nucleus via an ac ve enzyma c process can t occur cold temp EX large T an gen of SV40 virus 370C 40C small simple di usion large non nuclear excluded large nuclear transported Nuclear Import Protein Types NUCLEAR LOCALIZATION SIGNAL short charged Pro su cient 2 types SV40 T AnEgen Pro Bi ParEte NLS anywhere short sequence of AAs 4 8 residues charged Lys Arg usually contains Proline Pro su cient to direct non nuclear protein to nucleus covalently aaach NLS to non nuclear protein goes into nucleus 2 main types 1 SV40 T AnEgen contains Pro 2 Bi ParEte NLS can occur anywhere in protein doesn t have to be N or C terminus more common 2 blocks of 2 4 basic AAs separated by a 10 AA spacer charged Lys Arg su cient 2 types SV40 T An gen Bi Par te NLS common anywhere Nuclear Import NLS NLS SV40 LARGE T ANTIGEN immuno uorescence assay for nuclear targeEng 5 steps NLS discovery T an gen in nucleus NLS necessary for nuclear import immuno uorescence assay for nuclear targeEng 1 Infect cells w SV40 virus 2 Fix cells Permeabilize w detergent 3 Add primary an body against T an gen 4 Add uorescent secondary an body 5 View w uorescence microscope NLS discovered as mutant virus that would not cause tumor forma on show T an gen in nucleus Lys converts to Thr shown that this is NLS shows NLS is necessary for nuclear import nuclear proteins produced in cytoplasm ac vely imported immuno uorescence assay for nuclear targe ng NLS necessary for import Nuclear Import NLS NUCLEAR PORE COMPLEX Structure total mass 125 MDa composed of 30 proteins nucleoporins Nups 8 16 copies each per NPC 8 spokes form central domain cytoplasmic nuclear rings 8 brils emanate from cytoplasmic ring a basket is anchored to nuclear ring cytoplasmic domain 32 MDa nuclear ring 21 MDa pore diameter 45 nm in central part BIG hole mass 125 MDa 30 Nups 8 16 copies each per NPC central pore diameter 45 nm Nuclear Import NPC Structure NPC Permeability ProperEes NPC has aqueous channels permeable to molecules 5 000 Da water molecules enter nucleus when micro injected super HUGE 60 000 Da protein will not rapidly into cytoplasm equilibrate called a molecular cloud NPCs loaded w FG repeats aqueous permeable 5 000 Da NPC molecular cloud Nuclear Import NPC Permeability PERMEABILIZED CELL MODELS digitonin mild detergent that disrupts PM but not NE most likely b c PM contains cholesterol destabilizes PM while leaving organelle membranes intact digitonin releases 20 of total proteins digitonin DOES NOT change gross morphology of cells cell residues obtained w digitonin permeabilized cell models done on DEAD cells EX nuclear pores puri ed from amphibian oocytes digitonin detergent permeabilized cell models cell residues obtained w digitonin Nuclear Import Permeabilized Cell Models NUCLEAR IMPORT Steps 1 2 3 4 5 NLS recognized by receptor transporter in cytoplasm need receptor transporter some mes same some mes not Transporter cargo complex binds NPC peripheraliza on Transporter chaperones cargo through nuclear pore get into molecular cloud Cargo released trapped in nucleoplasm cargo trapped b c transporter leI it it can t get back through nuclear pore Transporter returns to cytoplasm transporter recycles Players in Nuclear Protein Import cargo NPC receptor Ran GDP Ran GTP Ran GAP Ran GEF NTF2 transporter sees NLS complex binds NPC nuclear pore cargo released transporter recycles Nuclear Import Steps NUCLEAR IMPORT Requires GTP Cytosolic Factors only works w 5 things all present happening 1 warm up cell 2 add cytoplasm 3 add GTP 4 GTP hydrolysis 5 add G protein large nuclear protein 37oC cytosol GTP 4oC cytosol GTP 37oC cytosol only permeabilized cell model 37oC GTP only 37oC cytosol GTP S warmth cytoplasm GTP GTP hydrolysis G protein Nuclear Import Condi on Requirements NLS Nuclear Import of NLS Proteins is Mediated by a Cytosolic Receptor only proteins w NLS get ac vely imported into nucleus muta ons in NLS sequence inhibit transport addi on of NLS polypep de compe tor inhibits import of NLS protein b c NLS gets bound up w pep de instead of protein nuclear import of NLS protein saturable b c receptor is required for import NLS receptor NLS NLS required for import muta ons in NLS compe on inhibit NLS protein import saturable Nuclear Import NLS NLS Puri caEon of a Cytosolic NLS Receptor 2 controls 1 2 Remove NLS Add excessive soluble protein imporEn receptor karyopherin imporEn transporter karyopherin steps 1 2 3 Protein w NLS binds impor n Impor n receptor binds impor n transporter All 3 take a ride together cytosolic proteins NLS pep de 125I biochemical frac ona on chromatography chemical crosslinker crosslinks things that get very very close together frac ons containing radioac ve proteins 2 controls receptor transporter graphic w purple boxes Nuclear Import NLS NLS NLS Adaptor in Xenopus imporEn an NLS adaptor in Xenopus NLS adaptor links cargo to a transporter IBB Impor n Binding in impor n imporEn IAB Impor n Binding in impor n NLS adaptor links cargo transporter IBB IAB Nuclear Import NLS RAN Ras Related Nuclear Protein 25 kDa GTP binding protein cycles b tw cytoplasm nucleus binds karyopherins GTP binding cycles b tw cytoplasm nucleus binds karyopherins cytoplasm GDP GTP nucleoplasm GDP GTP ENERGY control by localizing GAP GEF e ect on karyopherin depends on bound nucleo de CYTOPLASM high Ran GDP Ran GDP DOES NOT bind karyopherins NUCLEOPLASM high Ran GTP Ran GTP DOES bind karyopherins provides energy for transport cells control Ran by localizing GAP GEF GTP