Nucleocytoplasmic Trafficking

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Nucleocytoplasmic Trafficking

• Susan Wente
• Department of Cell and Developmental Biology
• 3120A Medical Research Building III
• 936-3443

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Nuclear Envelope -two double lipid bilayer
membrane structure -pores formed by the fusion
of the inner and outer membranes molecules
smaller than 40-60 kD freely diffusable larger
molecules traverse by active mechanism
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Nuclear Trafficking
Nucleus
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Estimates of Transport Flux through Nuclear Pore
Complexes

• HeLa Cell
• -10 million ribosomes generated for each
  cell cycle (24 hrs)
• 10 X 106 / 24 / 60 7000 ribosomes/min
• Per minute
• 560,000 ribosomal proteins imported
• 14,000 ribosomal subunits exported
• Through each NPC per minute
• 100 ribosomal proteins imported
• 3 ribosomal subunits exported
• In vitro Import Measurements (Ribbeck Gorlich,
  2001)
• 103 translocation events per NPC per second

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Nuclear Pore Complexes from Xenopus laevis
Landmarks in Cell Biology Unwin and Milligan J.
Cell Bio. 1982
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Nuclear Pore Complex Structure
Yeast S. cerevisiae
Vertebrate Xenopus
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John Heuser
Quick freeze, deep etch EM image of Dictyostelium
annulate lamellae
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Nuclear Pore Complexes
Cytoplasm
Nucleus
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NPC Proteins Nucleoporins Nups
Ribosome NPC 4 MDa 60 MDa 80 proteins 30
proteins
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NPC Proteins Nucleoporins Nups
Pore membrane
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NPC Proteins Nucleoporins Nups
Exclusively Cytoplasmic Face
Pore membrane
Exclusively Nuclear Face
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NPC Proteins Nucleoporins Nups
Exclusively Cytoplasmic Face
Symmetrical, On Both Faces
Pore membrane
Exclusively Nuclear Face
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General Strategy Attack at the Site of Entry
and Exit
-Nuclear Localization and the Signal
Hypothesis -Translocation Models -RanGTPase
cycle -Examples of Experimental
Strategies -mRNA Export
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Localization Signals Import and Export
-amino acid sequences that are both necessary and
sufficient for the import or export of a
protein -uncleaved and at variable points in each
individual protein -folded substrates -piggy-bac
king by a carrier
Multiple different signals Multiple distinct
receptors
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Family of Importin/Karyopherin b Transport Factors
-14 in S. cerevisiae gt21 in humans -each
specific for a given cargo
Ohno et al., 1998
Nucleoporins
Binding Domains
N
C
RanGTP
Adapter/cargo with NLS/NES
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Prototypical Protein Import Pathway
Shuttling Transporter
Cargo
SIGNALS

RAN GDP
Nuclear Localization Signal
RAN GTP
DOCKING
cytoplasm
TRANSLOCATE
nucleus

RAN GTP
RAN GTP
CARGO RELEASE
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Leucine-rich NES Protein Export Pathway
RAN GDP
CARGO RELEASE
RAN GTP
TRANSLOCATE
KAP?-DOCKING
RAN GTP
Crm1
SIGNALS
Leucine rich NES
Rev LPPLERLTL
RAN GTP
NES
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RanGTP Differentially ModulatesTransport
Factor-Cargo Interaction

RAN GDP
NLS
NLS

RAN GTP
RAN GTP
Import cargo release
Export cargo binding
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NPC Proteins Nucleoporins Nups
1/3rd of NPC mass from FG Nups
FG FXFG GLFG -non-tandem repeats, polar
spacer sequences -localized in all
substructures -docking sites for transport
factors
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FG Nucleoporins for Movement thru the NPC
GAP
RAN GDP
RAN GTP
FG
FG
cytoplasm
Asymmetric
FG
FG
Symmetric
FG
FG
nucleus
Asymmetric
FG
FG
GEF
RAN GTP
RAN GDP
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Strategies to Reveal Transport Mechanisms -Tissu
e Culture Cell Microinjection -Ectopic
Expression in Cells and Localization -Heterokar
yons to Analyze Shuttling -In vitro Transport
Assays Coupled with Biochemical Fractionation
(Flex time) -digitonin-permeabilized
cells Adams Gerace 1990 -Two-hybrid
Approach -Genomic Approach -searching for
RanGTP binding proteins -Genetic
Strategies -Screens for yeast mutants with
defective transport
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A) Cultured cells were stained by indirect
immunofluorescence for Pyruvate Kinase. B)
Localization of PK fused to the NLS SV40 Large T
Antigen.
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Heterokaryon Transport Studies
HeLa cells were fused with Xenopus laevis cells,
treated with actinomyosin D and stained for human
hnRNP A1(red) and hnRNP C (green). Unfused
HeLa cells, arrowhead. Unfused X.l. cells,
dotted arrow. Heterokaryon, solid arrow.
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Import of SV40 T Ag in permeabilized cells
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In situ Hybridization to Detect polyA RNA
Localization
polyARNA
DAPI
Wild type
Mutants
-Digoxigenin-Oligo (dT)30
-Anti-Dig FITC FAB
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Cell permeable AP-hGle1 Shuttling Domain inhibits
poly(A) RNA export and hGle1 localization at
the NPC
DAPI
hGle1 - IF
poly(A) RNA
3X exp
AP control
AP-SD
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RNA Transport NES Receptor RanGTP
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mRNA Export
cytoplasm
nucleus
Mex67 Mtr2 Dbp5 Gle1 Gle2
Dbp5
Mex67/Mtr2
Pre-mRNA splicing/processing
Modified based on C. Cole (2000)
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Structural Changes in the NPC and the hnRNP are
Observed During Export of Balbiani Ring
Particles
What mediates or signals these changes?
Kiseleva et al, 1996
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Survivors Guide to Nuclear Transport
Nomenclature
NUP nucleoporin NLS nuclear localization
sequence NES nuclear export sequence RAN
small GTPase (Gsp1) RanGEF RCC1, Prp20 RanGAP
Rna1 Import and Export factors
Importin/Karyopherin/Transportin (cargo)
-b and a/Kap95 - Kap60 (cNLS)
-transportin 1,Kapb2 (M9-NLS hnRNP A1)
Exportin/Karyopherin (example cargo)
-exportin/Crm1/Xpo1 (leucine rich-NES)
-CAS/Cse1 (alpha) -exportin-t/Los1
(tRNA) mRNA export factors (vertebrate/S.cerevisi
ae) TAP/Mex67 p15, NXT1/Mtr2 RAE1/Gle2 Gle
1 Dbp5