Nucleocytoplasmic Trafficking

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Nucleocytoplasmic Trafficking

• Susan Wente
• Department of Cell Developmental Biology
• U-3209 Medical Research Building III
• 936-3443
• susan.wente_at_vanderbilt.edu

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Lecture Readings 1) Text Molecular Biology
of the Cell Alberts et al., 4th Edition pg.
669-678 Molecular Cell Biology Lodish et
al., 4th Edition pg. 427-436 2) Recent
Review Suntharalingam and Wente Peering
through the Pore Nuclear Pore Complex
Structure, Assembly, and Function Dev. Cell
(2003) 4775-789 3) Assigned Paper Takano,
et al tRNA actively shuttles between
the nucleus and cytosol in yeast Science
(2005) 309140-142 Main paper plus Supplemental
Information
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Why are there membranes in the first place?
Segregation of protein function and genomic
material -specialized machinery to maintain
protein composition and communication
between compartments
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Nucleocytoplasmic Trafficking
Nucleus
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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Nucleocytoplasmic Trafficking
Plasma membrane
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Bidirectional Transport In and Out of the Nucleus
N
Protein Import
C
N
RNA Export
C
N
Shuttling Import Export
C
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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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Impacts on Disease and Development
Cancer -regulation of p53 tumor suppressor
import/export -FG Nups genes linked to
translocations in hematological malignancies,
myeloid leukemias -overexpression of Nups in
ovarian cancer tumors -mutation of a import
factor in breast cancer cell line Autoimmune
diseases -primary biliary cirrhosis Viral
pathogenesis
Kua et al., Nuclear transport and cancer from
mechanism to intervention Nat. Rev. Cancer
(2004) 4106-117 Cronshaw Matunis The nuclear
pore complex disease associations and
functional correlations Trends Endocrinol.
Metab. (2004) 1534-39
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Drugs that target transport pathways -Cyclosporin
A -Leptomycin B
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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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cytoplasm
NPC proteins Nucleoporins (Nups)
30 Nups 60 MDa
Ribosome 80 proteins 4 MDa
nucleus
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Higher Resolution Structural Analysis
-Cryo-electron tomography -X-ray structure
analysis of individual Nups -Mapping nearest
neighbor protein-protein interactions
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Movie of NPC Structure Modeling from MICHAEL
ROUT, Rockefeller University -      Have large
amount of data on whos next to who, and who
forms complexes with whom -      Using MODELLER,
protein modeling software -      Model proteins
as soft spheres -      Radius of sphere
corresponds to mass of protein -      Define
spatial restraints between the proteins taken
from our lists of NUP-NUP interactions and
isolated subcomplex compositions, include any
other info we have on position etc. of
NUPs -      Randomize protein positions -     
Then, allow model to reform, fulfilling all of
the restraints, if possible -      Do this 1000s
of times -      Look for models which best
fulfill the restraints (i.e., agree with all your
original interaction data) -      Heres MODELLER
generating one Model -      Starts with Formation
of the Model -      Then, Jiggling is
Optimization Process
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Structural Homology Between Nups and Vesicle Coat
Proteins
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Model for Common Ancestorial Link between Coated
Vesicles and NPCs
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General Strategy Attack at the Site of Entry
and Exit
-How do macromolecules move through? -How is the
NPC assembled? -How are transport and assembly
regulated?
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General Mechanisms for Protein Import into
Organelles
Cytoplasm
Cytoplasm
Cytoplasm

ER Mitochondria Chloroplasts
Peroxisomes Thylakoid, DpH Bacterial Export, Tat
Nucleus
Smith Schnell, 2001
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General Mechanisms for Protein Import into
Organelles
-Signal hypothesis Specific addresses
recognized by organelle specific receptors
Cytoplasm
Cytoplasm
Cytoplasm

ER Mitochondria Chloroplasts
Peroxisomes Thylakoid, DpH Bacterial Export, Tat
Nucleus
Smith Schnell, 2001
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Key Points of the Nucleocytoplasmic Transport
Mechanism
-Nuclear Localization and the Signal
Hypothesis -Translocation Models -RanGTPase
cycle -Examples of Experimental Strategies
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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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Experimental Strategy for Demonstrating an NLS
is Sufficient for Import Ectopic Expression
in Cultured Cells and Localization by Indirect
Immunofluorescence Microscopy
PK fused to the NLS SV40 Large T Antigen.
Pyruvate Kinase Alone
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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
RECYCLING
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Leucine-rich NES Protein Export Pathway
RAN GDP
CARGO RELEASE
RAN GTP
RECYCLING
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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(No Transcript)
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The Ran GTPase is required for-modulating cargo
binding to karyopherins-modulating karyopherin
interactions with FG nucleoporins -GTP
hydrolysis is not required for the actual
translocation step directionality
N
C
Steady-state Ran localization nuclear
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Experimental Strategy for Testing the
Nuclear Import Activity of A Transport
Factor Permeabilized Cells
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mRNA
TAP/Mex67
Adapted from Cullen, 2003
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Novel Shuttling Factors in the mRNA Export Pathway
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Experimental Strategy for Testing the Role of
a Factor in mRNA export In situ Hybridization
to Detect polyA RNA Localization in
Yeast Mutant Cells
polyARNA
DAPI
Wild type
Mutants
-Digoxigenin-Oligo (dT)30
-Anti-Dig FITC FAB
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Nuclear Entry and Exit The Rules and the
Players
-The Nups Translocon -The Shuttling
Transport Factors -Karyopherins -mRNA
export
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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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Yeast FG Nucleoporins
Nup159
Nup42
FG
Nup49
GLFG
Nup57
FxFG
Nsp1
Nup100
Nup145N
Nup116
Nup60
Nup2
Modified from Allen et al., 2001
Nup1
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(No Transcript)
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FG nups are binding sites for movement through
the NPC
1. Complex formation
FG
FG
2. Docking at NPC
cytoplasm
FG
FG
3. Translocation
Mechanism?
FG
FG
FG
nucleus
4. Release
FG
FG
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FG Nups in Models for NPC Translocation
Affinity Brownian Selective
Oily-Spaghetti Gradient (Virtual)
Phase Gating Partitioning
Modified from Weis, 2003
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Experimental Strategy for Testing Role of FG
Domains in Nuclear Transport Generating
Minimal FG NPCs in Yeast
Strawn et al., 2004 Nature Cell Biol. 6197-206
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Experimental Strategy for Analyzing Shuttling
(Import Export Cycles) 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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General Strategy Attack at the Site of Entry
and Exit
-How do macromolecules move through? -How is the
NPC assembled? -How are transport and assembly
regulated?
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Supplemental Lecture Information
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Formation of a pore and NPC in an intact nuclear
envelope
Intact double membranes
Hemi-fusion of inner leaflets
Full fusion to form pore
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Mitotic NPC Assembly
INTERPHASE
CYTOKINESIS
ANAPHASE
PROPHASE
METAPHASE
Burke and Ellenberg, 2002, Nat. Rev.
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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) -exportin-5/Msn5 (tRNA-eEF1A
complex, pre-miRNAs) mRNA export factors
(vertebrate/S.cerevisiae) TAP/Mex67 p15,
NXT1/Mtr2 RAE1/Gle2 Gle1 Dbp5
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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 Assigned
Reading -In vitro Transport Assays Coupled with
Biochemical Fractionation -digitonin-permea
bilized cells -Two-hybrid
Approach -Genomic Approach -searching for
RanGTP binding proteins -Genetic
Strategies -Screens for yeast mutants with
defective transport