MCB 252 Topic 7 Regulation of Nucleo Cytoplasmic Transport Prof David Rivier MCB 252 Spring 2015 Reading Lodish section 13 6 pages 615 621 NFKB a master regulator of txn in cells of the immune system New Questions How is a protein poly ubiquitinated How is a poly ubiquitinated protein degraded How does a protein enter the nucleus More generally how is nucleo cytoplasmic trafficking regulated How is the nucleo cytoplasmic trafficking regulated ONM INM Nuclear Envelope Cytoplasm What Whatdefines definesthe thedirectionality directionalityof of nucleo cytoplasmic nucleo cytoplasmicexchanges exchanges What Whatare arethe themolecular molecularmechanisms mechanisms that thatregulate regulatenuclear nuclearimport importand and export export Nucleus The Thenuclear nuclearpore porecomplex complex How is the nucleo cytoplasmic trafficking regulated RER Heterochromatin Euchromatin Nucleolus Nuclear Envelope How is the nucleo cytoplasmic trafficking regulated Nucleoplasm NPC INM ONM Nuclear Envelope Cytoplasm NPC Nuclear Pore Complex How is the nucleo cytoplasmic trafficking regulated The Nuclear Pore Complex Cytoplasmic side Nucleoplasmic side How is the nucleo cytoplasmic trafficking regulated The Nuclear Pore Complex Cytoplasmic Cytoplasmic filaments filaments Nuclear Nuclear Basket Basket NPC is 125 MDa How is the nucleo cytoplasmic trafficking regulated The Nuclear Pore Complex NPC is composed of only 30 different proteins named Nucleoporins Most nucleoporins were estimated to be present in either 16 or 32 copies per NPC The relative distribution of nucleoporins within a nucleopore was defined Nucleoporins contain regions of repeated amino acid sequences that end in FG Phenylalanine Glycine or XFXFG This is the most highly conserved feature between yeast and mammalian nucleoporins Some nucleoporins are glycosylated How is the nucleo cytoplasmic trafficking regulated The Nuclear Pore Complex The relative distribution of nucleoporins within a NPC was defined by immuno EM 1 Purified NPC were fixed onto a grid 2 An antibody against the nucleoporin of interest was applied to the sample The antibody was conjugated to gold particles 3 After incubation and extensive washes of the unbound antibody the sample was analyzed by EM How is the nucleo cytoplasmic trafficking regulated The Nuclear Pore Complex How is the nucleo cytoplasmic trafficking regulated The Nuclear Pore Complex How is the nucleo cytoplasmic trafficking regulated The Nuclear Pore Complex Samir S Patel 2005 How is the nucleo cytoplasmic trafficking regulated The Nuclear Pore Complex NPC is composed of only 30 different proteins named Nucleoporins Most nucleoporins were estimated to be present in either 16 or 32 copies per NPC The relative distribution of nucleoporins within a nucleopore was defined Nucleoporins contain regions of repeated amino acid sequences that end in FG phenylalanine Glycine or XFXFG This is the most highly conserved feature between yeast and mammalian nucleoporins Some nucleoporins are glycosylated How is the nucleo cytoplasmic trafficking regulated The Nuclear Pore Complex F Phenylalanine G Glycine How is the nucleo cytoplasmic trafficking regulated The Nuclear Pore Complex Samir S Patel 2005 How is the nucleo cytoplasmic trafficking regulated The Nuclear Pore Complex FG repeats create a hydrophobic environment Samir S Patel 2005 The passage of a protein through the NPC involves multiple interactions with FG repeats How is the nucleo cytoplasmic trafficking regulated The Nuclear Pore Complex NPC is composed of only 30 different proteins named Nucleoporins Most nucleoporins were estimated to be present in either 16 or 32 copies per NPC The relative distribution of nucleoporins within a nucleopore was defined Nucleoporins contain regions of repeated amino acid sequences that end in FG phenylalanine Glycine or XFXFG This is the most highly conserved feature between yeast and mammalian nucleoporins Some nucleoporins are glycosylated How is the nucleo cytoplasmic trafficking regulated The Nuclear Pore Complex WGA WGA Some nucleoporins are glycosylated Lectins such as WGA can be used to block nucleo cytoplasmic exchanges How is the nucleo cytoplasmic trafficking regulated The Nuclear Pore Complex What defines the directionality of nucleocytoplasmic exchanges What are the molecular mechanisms that regulate nuclear import and export How is the nucleo cytoplasmic trafficking regulated Nucleo cytoplasmic trafficking Extremely active nucleo cytoplasmic exchanges 3000 5000 NPCs cell 106 molecules are exchanged every minute How is the nucleo cytoplasmic trafficking regulated Nucleo cytoplasmic trafficking The passive diffusion channel of the NPC has an effective diameter of 9nm Proteins MW Diffusion through the NPC Less than 40 KDa Reasonably fast More than 40 KDa Ovalbumin 46KDa 6 nm Very slow almost negligible Above 60 Kda BSA 68Kda 7nm Exceedingly slow There must be nucleo cytoplasmic exchanges based on an active transport mechanism How is the nucleo cytoplasmic trafficking regulated Nucleo cytoplasmic trafficking Specific transport signals The first evidence comes from the study of a nuclear protein named Nucleoplasmin Head Tail Large pentameric protein complex of 150 KDa How is the nucleo cytoplasmic trafficking regulated Nucleo cytoplasmic trafficking Frog oocyte 1 5 mm Nucleus micropipette Cytoplasm How is the nucleo cytoplasmic trafficking regulated Nucleo cytoplasmic trafficking Core Head Tail Mild proteolysis Tail Core 1 tail Tail Tail How is the nucleo cytoplasmic trafficking regulated Nucleo cytoplasmic trafficking Core Core Tail Core 1 tail How is the nucleo cytoplasmic trafficking regulated Nucleo cytoplasmic trafficking Core Core Core 1tail tail Injected into the cytoplasm Pentamer Injected into the cytoplasm Injected into the nucleus Injected into the cytoplasm Injected into the cytoplasm Remains in the cytoplasm Remains in the nucleus Accumulates Accumulates Accumulates into the nucleus into the nucleus into the nucleus The tail domain contains a Nuclear Localization Signal NLS How is the nucleo cytoplasmic trafficking regulated Nucleo cytoplasmic trafficking The first Nuclear Localization Sequence NLS was characterized on the simian virus 40 SV40 large T antigen as a short basic peptide NLS PKKKRKV To qualify as an NLS a sequence should be necessary and sufficient for nuclear localization of a protein How is the nucleo cytoplasmic trafficking regulated Nucleo cytoplasmic trafficking PKKKRKV Pyruvate