MCB 252 Topic 8 Nucleo Cytoplasmic Transport part 2 Prof David Rivier MCB 252 Spring 2015 Reading Lodish figure 8 24 and the accompanying text pages 368 370 Lodish section 13 6 pages 615 621 same as topic 7 How is the nucleo cytoplasmic trafficking regulated Nucleo cytoplasmic trafficking Transporters the importin superfamily Transporter adapter Importin importin a Importin snurportin 1 Transportin 1 Importin 5 and 7 Transportin SR Cargo NLS containing proteins snRNPs hnRNPs A1 B D E F Ribosomal proteins SR proteins CRM1 NES containing proteins U snRNAs snurportin 1 CAS Exportin importin a tRNAs How is the nucleo cytoplasmic trafficking regulated Nucleo cytoplasmic trafficking CRM1 Chromosome Region Maintenance CRM1 was originally identified in yeast in a genetic screen unrelated to nuclear transport CRM1 protein is encoded by an essential gene and was shown to be the specific target of the cytotoxic drug leptomycin B LMB One of the first evidences that CRM1 is an exportin came from the observation that LMB blocks nuclear export of the HIV rev protein EARLY LATE PHASE PHASE Tap P15 Ran GTP Translation CRM1 NES REV Splicing RBD RRE Transcription HIV Ran GDP Ran GTP Ran GTP CRM1 NES REV RRE RBD RBD HIV Virion Ran GTP LMB No export of the full length HIV RNA CRM1 NES REV RRE RBD RBD How is the nucleo cytoplasmic trafficking regulated Nucleo cytoplasmic trafficking CRM1 RanGTP Adapted from Nature Structural Molecular Biology 17 1367 1376 2010 Ran GTP Ran GTP CRM1 NES CARGO Ran GDP No export of the cargo Ran GTP CRM1 LMB NES CARGO NES NES Ran GTP NES NES NES CRM1 NES No export of the cargo NES NES NES NES NES NES NES NES NES NES NES NES NES NES NES CARGO NES NES NES NES NES How is the nucleo cytoplasmic trafficking regulated Nucleo cytoplasmic trafficking C A C A A C A A C C B D B C C D A B D D B C D A B B C A C D C D Steady state C B Nucleoplasm C D C A A C D B A C C Cytoplasm A A C How is the nucleo cytoplasmic trafficking regulated Nucleo cytoplasmic trafficking Question How one can show experimentally that a protein shuttles between the nucleus and the cytoplasm If the protein is cytoplasmic then two main strategies are often used 1 LMB treatment 2 Deletion analysis LMB treatment results in the nuclear accumulation of the shuttling protein C C A C A A C A A C C C A C C C LMB A A C C C A C C A A Nucleoplasm C C A C Cytoplasm A C Deletion Analysis N NES P195 P195 NLS P195 P195 P195 P195 P195 P195 P195 P195 P195 P195 C Deletion Analysis N NES C P195 NLS P195 NLS P195 NLS P195 NLS P195 NLS P195 NLS P195 NLS P195 NLS P195 NLS P195 NLS Deletion Analysis N P195 NES P195 NES P195 NES P195 NES P195 NES P195 NES P195 NES NLS C How is the nucleo cytoplasmic trafficking regulated Nucleo cytoplasmic trafficking Question How one can show experimentally that a protein shuttles between the nucleus and the cytoplasm If the protein is nuclear then the main strategy is to produce heterokaryons Heterokaryons PolyEthyleneGlycol PEG Frog cell Human cell Heterokaryon fused cytoplasms Heterokaryons 1 Cycloheximide blocks translation 2 PEG Human cell Frog cell Nuclear protein type B No Shuttling Heterokaryon fused cytoplasms What would happen if cyclohemide was omitted 1 Cycloheximide blocks translation 2 PEG Human cell Frog cell Nuclear protein type B False Positive Heterokaryon fused cytoplasms Heterokaryons 1 Cycloheximide blocks translation 2 PEG Human cell Frog cell Nuclear protein type D Shuttling Heterokaryon fused cytoplasms How is the nucleo cytoplasmic trafficking regulated Nucleo cytoplasmic trafficking 1 1 3 2 1 HeLa Cell 2 Frog Cell 3 Heterokaryon 3 2 1 2 hnRNPC does not shuttle 3 hnRNPA shuttles How a signal is received and transmitted Plasma membrane Bio membranes Properties of phospholipid bilayers Membrane proteins Protein modifications Prenylation Ras MAPK NFkB P53 Phosphorylation dephosphorylation Protein kinases and phosphatases Poly ubiquitination E1 E2 E3 system Proteasome controlled protein degradation Nuclear envelope How the information enters the nucleus Nuclear Pore Complexes NPCs Nucleo cytoplasmic exchanges