Nuclear structure Nuclear pore Import/ Export Signals Receptors Ran (Directionality) FG Platform (Gating) Regulation Nuclear Import & Export Overview

Origin Relates to Topology Parts chromatin matrix nucleoli lamina (M-phase disassembly) pores inner membrane outer membrane lumen (peri-nuclear space) Nuclear Structure

Nuclear Lamina

EM of lamina

Mitotic Disassembly

Nuclear Pores

11.4 Signal-mediated transport through nuclear pore complexes (NPC) Figure The nuclear pore complex

Imported Polymerases Histones Transcription factors Ribosomal proteins Exported tRNAs mRNPs Ribosomal subunits Transcription factors 10 6 ribos=>560K ribo proteins imported/min 14,000 ribo subs exported/min 3-4K pores/cell=> 150 ribo proteins/min/pore Also 100 histones/min/pore etc. Nuclear Cargo

125MD octagonal symmetry, 3-4K pores/cell central plug nuclear basket cytoplasmic fibers About 30 (?) proteins “Nucleoporins” (FG repeats) Passive diffusion, limit 40-60KD Channel 9nm x 15 nm long Opens w/ signal: gating mech. unknown Bidirectional Nuclear Pore

9nm (40-60kDa) 40nm (up to 50MDa) Size Exlcusion Limits

Model for passage of mRNPs through nuclear pore complexes

Not cleaved Folded proteins transported NLS (nuclear localization signal) Defined by transfection/microinjection experiments e.g. stretch of basic residues SV40 T=PKKKRKV drives import of pyruvate kinase NES (nuclear export signal) Defined by heterokaryon expt e.g. leucine rich Signals

11.4 Heterokaryon assay demonstrating that human hnRNP A1 protein (red) can cycle in and out of the cytoplasm but human hnRNP protein C (grn) cannot Figure 11-32

3 Types Importin beta (classic) NTF2 (for Ran import) TAP/p15 (mRNA export) Identified with affinity chromatography permeabilized cell assays Importin beta (karyopherins) Large family of related Import & Export Receptors Bind signals (direct or indirect), porins & RanGTP cargo binding controlled by Ran destabilizes import receptors stabilizes export receptors Receptors

Cytosol lost NPCs intact Permeabilized Cells

Nuclear transport requires cytosolic factors and ATP

Importin beta structure

RCC1: stabilizes exchange intermediate RanGAP1, RanBP1, RanBP2: >10 5 fold cooperative RanBP1 binds Ran->dissoc. then RanGAP RanGAP sumo/ RanBP2 complex on pore NTF2: import receptor for RanGDP

On its own, translocation per se would be a fully reversible process that would only allow an equilibration of cargoes between nucleus and cytoplasm. Directional transport is achieved by Ran-regulated cargo loading and unloading of the transport receptors. This also constitutes the input of energy in the form of one molecule of GTP hydrolyzed per transport cycle. Transport cycles of exportins and importins use the chemical potential of a primary gradient, namely that of RanGTP, to drive the directed transport of cargo molecules

11.4 A model for hnRNP-mediated export of mRNAs from the nucleus Figure 11-34

Calineurin Masks NES

11.4 HIV Rev protein regulates the transport of unspliced viral mRNAs Figure 11-38