Handout 5 - Nucleo-cytoplasmic trafficking

Handout 5 - Nucleo-cytoplasmic trafficking - MCB 252 Cell...

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MCB 252 Cell Biology, Spring 2008 Handout 05 New Questions • How is a protein poly-ubiquitinated? • How does a protein enter the nucleus? More generally,how is the nucleo-cytoplasmic trafficking regulated? • How is a poly-ubiquitinated protein degraded? • How is a poly-ubiquitinated protein degraded?
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MCB 252 Cell Biology, Spring 2008 Handout 05 • Nucleo-cytoplasmic trafficking What are the molecular mechanisms that regulate nuclear import and export? Questions: What defines the directionality of nucleo- cytoplasmic exchanges? Cytoplasm Nucleoplasm Nuclear Pore Complex
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MCB 252 Cell Biology, Spring 2008 Handout 05 •Nuclear envelope • Nucleo-cytoplasmic trafficking Electron micrograph of the interphasic cell nucleus • Nucleolus Obvious nuclear domains: • Euchromatin • Heterochromatin
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MCB 252 Cell Biology, Spring 2008 Handout 05 • Nucleo-cytoplasmic trafficking Nucleus Cytoplasm Heterochromatin IM OM } Nuclear envelope Nuclear pore complex Nuclear lamina
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MCB 252 Cell Biology, Spring 2008 Handout 05 • Nucleo-cytoplasmic trafficking Nuclear side Cytoplasmic side
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MCB 252 Cell Biology, Spring 2008 Handout 05 ribosome RER Cytoplasm nuclear pore complex perinuclear space outer membrane inner membrane nuclear lamina chromatin • The Nucleopore Complex
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MCB 252 Cell Biology, Spring 2008 Handout 05 spoke cytoplasmic filaments basket filament central ring of NPC is fused inside the membrane, so nothing can pass on the outside cytoplasmic ring nuclear ring terminal ring • The Nucleopore Complex
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MCB 252 Cell Biology, Spring 2008 Handout 05 Vertebrate ribosome ~4 MDa ~125MDa ~50MDa 80 different proteins only 30 different proteins • The Nucleopore Complex These proteins are named nucleoporins
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MCB 252 Cell Biology, Spring 2008 Handout 05 In the mid 1990s a large scale purification of Nucleopore Complexes was done in yeast and was the basis for proteomic studies. • The Nucleopore Complex • All nucleoporins were identified • The relative distribution of nucleoporins within a nucleopore was defined by immuno-Electron Microscopy
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MCB 252 Cell Biology, Spring 2008 Handout 05 NPCs contain multiple copies of the nucleoporins. 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. Many nucleoporins are glycosylated. The sugar residues (N-acetylglucosamine) are directly exposed to the cytosol. In yeast most nucleoporins were estimated to be present in either 16 or 32 copies per NPC. • The Nucleopore Complex
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MCB 252 Cell Biology, Spring 2008 Handout 05 1. Purified NPC were fixed onto a grid The relative distribution of nucleoporins within a nucleopore was defined as follows: 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. Gold particles are clustered on the cytoplasmic side.
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This note was uploaded on 06/12/2010 for the course MCB 252 taught by Professor Prasanth during the Fall '08 term at University of Illinois at Urbana–Champaign.

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Handout 5 - Nucleo-cytoplasmic trafficking - MCB 252 Cell...

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