Nuclear Import

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Nuclear Import Export
Nuclear structure Nuclear pore Import/ Export
Signals Receptors Ran FG Platform Regulation
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Nuclear Structure
Origin Relates to Topology Size 1-10µm, usually
1/cell Parts nuclear envelope inner
membrane outer membrane (continuous w/
ER) lumen (cont. w/ ER lumen) nuclear pore
complexes (NPCs) nucleolus (RNA synthesis,
ribosome assembly) nucleoplasm nuclear lamina
(M-phase disassembly) chromatin (hetero-
euchromatin) nuclear matrix (ill-defined
filamentous network)
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nuclear lamina intermediate filaments called
lamins binds integral nuclear membrane
proteins function assembly support also
connects to organizes chromatin
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nuclear lamina is a meshwork bound to nuclear
envelope
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Disassembly/reassembly cycle of nuclear envelope
during mitosis
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Nuclear Pore
125x106D, 120nm, 30 proteins called nucleoporins
(multiple copies), FG-repeats 8-fold symmetry,
3-4K pores/cell central plug nuclear
basket cytoplasmic fibers Passive diffusion 5K
no barrier, limit 40-60KD Channel 9nm x 15 nm
long Opens to 26 nm w/ signal gt gating
mechanism Bidirectional
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Transport

• Imported
• Polymerases
• Histones
• Transcription factors
• Ribosomal proteins

• Exported
• tRNAs
• mRNPs
• Ribosomal subunits
• Transcription factors

106 ribosgt560K ribo proteins imported/min
14,000 ribo subs exported/min 3-4K pores/cellgt
150 ribo proteins/min/pore Also 100
histones/min/pore etc.
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Signals
Not cleaved Folded proteins transported NLS
(nuclear localization signal) Defined by
transfection/microinjection experiments e.g.
stretch of basic residues SV40 TPKKKRKV drives
import of pyruvate kinase NES (nuclear export
signal) Defined by heterokaryon expt e.g.
leucine rich
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FRAP assay demonstrating shuttling
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11.4 A model for hnRNP-mediated export of mRNAs
from the nucleus
Figure 11-34
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Receptors
Identified with affinity chromatography permeab
ilized cell assays (Ran, import receptor,
NTF2) Large family of related Nuclear Import
Export Receptors Bind signals (direct or
indirect), porins RanGTP cargo binding
controlled by Ran destabilizes import
receptors stabilizes export receptors
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RCC1 stabilizes exchange intermediate RanGAP1,
RanBP1, RanBP2 gt105 fold cooperative RanBP1
binds Ran-gtdissoc. then RanGAP RanGAP sumo/
RanBP2 complex on pore NTF2 import receptor for
RanGDP
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Facilitated Diffusion
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
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.
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FG Platform
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Schematic view of Porin FG repeats
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Model of Transporter Passage Through pore (Gating
Mechanism)
Transporter
FG
FG
hydrophilic
NPC central channel
note FG meshwork
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11.4 HIV Rev protein regulates the transport of
unspliced viral mRNAs
Figure 11-38