Block III Lecture 7

1
Block III Lecture 7 Nucleocytoplasmic
transport February 16, 2005
Maria L. Zapp, Ph.D. Program in Molecular
Medicine and The UMass Center for AIDS Research
2
Regulation of gene expression at the level of
nucleocytoplasmic transport
Prokaryotes
Eukaryotes
Compartmentalization and the need for nuclear
transport
One distinct characteristic of eukaryotic cells
is the existence of nuclear and cytoplasmic
compartments separated by a nuclear envelope
(NE). The NE is a double membrane that is
continuous with the ER and is perforated by
nuclear pore complexes (NPCs).
Adapted from Lewin, 1988
3
Problem
Cellular mRNAs, tRNAs, and rRNAs are transcribed
in the nucleus and must be exported to the
cytoplasm for protein translation. Conversely,
nuclear proteins such as histones, pre-mRNA
splicing and transcription factors are
synthesized in the cytoplasm and must be imported
into the nucleus to perform their functions.
Solution
Cellular RNAs and proteins are transported
bidirectionally across the NE through the NPCs.
This cellular process is known as
nuclear-cytoplasmic or nucleocytoplasmic
transport. Nucleocytoplasmic transport has two
distinct components nuclear import and nuclear
export
4
Nuclear Envelope (NE) Nuclear pore complexes,
nuclear lamina, and lipid membranes
Nuclear pore complexes (NPCs)
Nuclear lamina
5
Possible pathways for molecular movement into the
nucleus
NPC
1. Direct passage through the nuclear pores
2. Synthesis on the outer nuclear membrane
(ONM) or contiguous ER followed by
passage through the inner nuclear membrane (INM)
3. Synthesis in the nucleoplasm 4. Passage
by diffusion through the ONM and INM 5.
Passage by active transport through the ONM and
the INM 6. Passage in vesicles that form from
the ONM and subsequently fuse with the
INM 7. Passage in vesicles formed from both
nuclear membranes 8. Passage through holes in
the NM (i.e. at mitosis)
Adapted from Maul, G. 1777
6
What is the permeability of the nuclear envelope?
Microinjection assay using X. laevis oocytes
Representative 100 mM sections
Oocyte
Minus tracer
Plus tracer
Experiment Inject a radiolabeled tracer into
the cytoplasm of oocytes. Incubate for various
times. Quench oocytes by placing at -190oC.
Prepare 100mm sections (- 50oC). Determine
the intracellular concentrations of tracer by
ultra-low temperature autoradiography. Count the
grain densities.
7
Schematic representation of the data
Time (minutes)
Paine, et al., 1975. Nature 254 109-114.
8
Conclusion The NE is a molecular sieve that
restricts molecular movement between the nucleus
and the cytoplasm.
Summary 1. These data demonstrate that the NE is
less permeable to larger dextrans (gt23.3 Å)
than smaller dextrans (lt12.0 Å). 2. The
permeability of the NE plays a major role in
limiting the rate of nuclear entry. 3. These
classical studies suggested that the NE is a
diffusion- restrictive barrier. The data
are consistent with nuclear entry kinetics
expected for passage through an envelope with
pores.
9
Schematic representation of the Nuclear Pore
Complex (NPC)
Protein constituents of the NPC are known as
nucleoporins or NUPs.
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Selectivity at the nuclear pore Part I
Nuclear-cytoplasmic transport of proteins
Key observations Large proteins can enter the
nucleus and remain there. Cytoplasmic proteins
do not enter the nucleus, and remain localized in
the cytoplasm. Some proteins re-equilibrate
between the nucleus and the cytoplasm.
Approach and Results Nucleoplasmin is a
pentameric nuclear protein that contains
a protease-resistant core domain and a
protease-sensitive tail domain. Nucleoplasmin
injected into the cytoplasm of frog oocytes
enters the nucleus. When the tail domain is
removed by digestion, the residual core
domain remains a pentamer and is UNABLE to enter
the nucleus. The detached tail domains rapidly
accumulate in the nucleus, suggesting the tail
domain contains a signal for nuclear
accumulation.
Conclusion Nuclear proteins contain
nuclear-targeting signals
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Nuclear protein transport occurs through the NPCs
and requires ATP
Key observations Direct visualization of
intracellular migration of nucleoplasmin- coated
colloidal-gold particles through oocyte NPCs
using EM. Particle movement is altered
dramatically by ATP depletion and low
temperature. Additional EM work visualized an
RNA-coated gold particle moving through the NPC
to the cytoplasm.
Significance These oocyte-based approaches
help demonstrate that cellular proteins and RNA
are transported bidirectionally through the NPC.
Conclusions 1. The steady-state distribution of
cellular proteins between the nucleus and the
cytoplasm is governed by an intrinsic
property of the polypeptides. 2. Nuclear proteins
contain specific Nuclear Localization Signals
(NLS) that promote nuclear uptake. 3. Nuclear
protein uptake occurs via NPCs.
Bonner, et al., 1975. J.Cell Biol. 64 431-437.
Dingwall, et al., 1982. Cell 30 449-458.
Feldherr, et al., 1984. J. Cell Biol.
992216-2222.
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The NLS of a protein selectively promotes its
import into the nucleus
Approaches to identify sequences which mediate
nuclear localization of proteins
i. Deletion analysis of SV40 virus large
T-antigen
Construction and characterization of viral
protein mutants defective in nuclear import.
The first NLS was identified in SV40 large
T-antigen and consists of numerous charged amino
acid residues. The SV40 T-antigen sequence is
the prototypeof classical NLSs.
Immunofluorescence (IF) micrographs showing the
intracellular distribution of the SV40 virus
T-antigen containing or lacking a short peptide
that serves as an NLS. (Left panel) The wild
type T-antigen protein contains the lysine-rich
sequence indicated and it is imported to its site
of action in the nucleus, as shown by IF staining
with an antibody against the T-antigen. (Right
panel) An SV40 T- antigen protein with a mutant
NLS peptide (Lys--gt Thr ) remains in the cytosol.
Lanford and Butel, 1984. Cell 37801-813.
Kalderon, et al., 1984. Cell 39 499-509.
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ii. Construction and analysis of chimeric fusion
proteins
Mata?2 A yeast protein involved in mating. The
protein is nuclear localized.
b-galactosidase (b-gal ) A bacterial enzyme
involved in metabolism. The protein is localized
in the cytoplasm of yeast cells.
Generate a yeast expression vector Sequences
that encode Mat a?2 were cloned in frame with
sequences that encode b-gal. Transform plasmid
into yeast cells and analyze the intracellular
distribution of the fusion protein.
Analysis of Protein Localization
yMATa2
yMATa2-b-galactosidase
b-galactosidase
Richardson, et al., 1984. Cell 44 77-85. Hall,
et al., 1984. Cell 36 1057-1065. Goldfarb, et
al., 1986. Nature 322641-644.
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Summary

• 1. The addition of an NLS can facilitate nuclear
  entry of a protein that is too large to
• enter by diffusion.
• 2. Nuclear proteins contain specific amino acid
  sequences that selectively promote
• nuclear localization.
• 3. Additional NLS peptide competition studies in
  frog oocytes indicated that nuclear
• protein localization or nuclear import is a
  saturable process. The saturation
• kinetics and competition effects observed
  suggested nuclear protein import
• is a carrier-mediated process.
• 4. Nuclear import of proteins is a
  receptor-mediated process. The NLS may interact
• with a component of the nuclear transport
  machinery.
• 5. Large proteins may interact with cellular
  receptors for nuclear import. Specific
• interactions would result in a selective
  distribution of proteins between the nucleus
• and the cytoplasm.

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Development of novel assays for nuclear protein
import
To determine whether the protein of interest
contained an NLS. To identify the molecular steps
required for nuclear protein import. To identify
cellular factors that mediate nuclear protein
import.
i. Mammalian cell microinjection assay
Inject a fluorescently-labeled protein into the
cytoplasm of a mammalian cell, then
determine its intracellular localization using
fluorescence microscopy.
Cytoplasm
Nucleus
NLS Protein
MT- NLS protein
?D?NLS Protein
Injection substrates
D NLS Protein (lacks an NLS)
NLS Protein (contains an NLS)
MT- NLS protein (contains a mutant NLS)
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ii. Mammalian cell transient transfection
assay Glutathione-S-Transferase (GST) is an
enzyme from S. japonicum. GST 26 kDa. Green
Fluorescent Protein (EGFP) is a light-converting
protein from A. victoria. GST 27kDa. Enhanced
GFP (EGFP) is a variant of wild type GFP protein,
which has been optimized for brighter
fluorescence and high expression in mammalian
cells. Construct plasmids for transient
expression of a GST- EGFP fusion protein that
contains an NLS (GFP-NLS-EGFP) or lacks an NLS
(GST-DNLS-EGFP) in mammalian tissue culture
cells. Introduce DNA into cells using standard
methods (i. e. CaPO4-mediated DNA precipitation,
cationic liposomes, DEAE-dextran or
Electroporation). Analyze the intracellular
distribution of the protein using indirect
fluorescence microscopy.
hRIP Control or Marker protein. hRIP is an
endogenous protein that is
localized at the nuclear periphery.
17

• iii. in vitro reconstituted nuclei.
• Assemble an assay mix containing isolated
  intact nuclei from mammalian cells,
• frog egg extract, and a fluorescently labeled
  protein.
• Results Isolated mammalian cell nuclei import
  nuclear proteins efficiently when
• incubated in this mix, but
  exclude non-nuclear proteins. Nuclear import
• of the protein substrate displays
  the same characteristics for an active
• protein import system a
  requirement for an NLS, ATP, an intact NE, and
• temperature dependence.

Summary
1. These three assay systems provided evidence
that nuclear protein import occurs in two
distinct steps rapid binding or docking at the
NE, followed by trans- location through the
NPC. 2. The binding and translocation steps can
be uncoupled by incubating cells at low
temperature or by treating them with inhibitors
of ATP production. Translocation through
the NPC is energy-dependent. 3. The NPC contains
multiple docking sites that guide the movement of
NLS- containing proteins from the cytoplasm
to the nucleoplasmic face of the NPC. 4. Docking
of the NLS-containing protein to the NPC, as well
as its subsequent movement through the NPC
requires cellular transport factors.
Newmeyer, et al., 1986. EMBO J. 5501-510 J.
Cell Biol. 103 2091-2103. Richardson, et al.,
1988. Cell 52 655-664. Adams, et al., 1990. J.
Cell Biol.111 807-816. Adams and Gerace,
1991. Cell 66 837-847. Moore and Blobel, 1993.
Nature 365 661-663 PNAS 91 10212-10216.
Melchior, et al., 1993. J. Cell Biol.
1231649-1659. Rexach and Blobel, 1995. Cell
83 638-692.
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Cellular factors which selectively interact with
the NLS Identification of nuclear protein import
receptors
i. Development of an in vitro reconstitution
assay for protein import using digitonin- permeabi
lized mammalian cell nuclei. This unique assay
system offers several technical advantages for
identifying mediators of protein import
Fluorescently labeled (FITC) or epitope- tagged
import substrate can be introduced into cells
and nuclear uptake monitored microscopically.
Cells are depleted of their soluble cytoplasmic
components thus re-import requires re-addition
of a cytosolic fraction(s). RESULTS Cytosolic
fractions were added to digitonin- permeabilized
cells to restore nuclear import of an
FITC-labeled or epitope-tagged NLS- containing
protein. Fractions demonstrated to support
protein import into nuclei were subfractionated
to identify components of the protein import
machinery. Ultimately, cytosolic fractions were
replaced with purified recombinant factors for
functional analysis.
ii. Chemical crosslinking of cellular proteins
that bind to an NLS-containing protein.
Adams, et al., 1990. J. Cell Biol. 111807-816.
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Molecular events in nucleocytoplasmic transport
Overview
Nucleocytoplasmic transport is largely mediated
by a superfamily of transport receptors that
interact directly with the NPC. These transport
receptors are related, albeit often distantly,
to the cellular protein importin-b (Imp b), and
share an N-terminal GTPase binding motif. Based
on the direction these transport receptors carry
their cargo, they are called importins or
exportins. These transport receptors are
sometimes referred to as karyopherins, a more
historical nomenclature. Transport receptors
bind their cargo on one side of the NE,
translocate to the other side, release the cargo,
and return to their original cellular compartment
to mediate the next round of transport.
Specifically, importins bind cargo in the
cytoplasm and release it in the nucleus
conversely, exportins bind their cargo in the
nucleus and release it in the cytoplasm. In the
simplest case, the cargo is recognized directly
by its cognate transport receptor. In others,
cargo recognition is more complicated and
requires additional adapter molecules. In the
most complex cases, the same receptor binds one
cargo for nuclear import and a different cargo
for nuclear export.
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The nuclear protein import cycle
Key adapter molecules 1. Importin-a (Imp a)
or the NLS receptor mediates NLS recognition.
2. Importin-b (Imp b) mediates interactions with
the NPC to drive translocation of cargo. 3. A
nuclear GTPase system- Ran, RCC, a Ran GAP,
binding proteins 1 and 2, NTF-2


1. Imp a directly binds to the NLS of the
cargo, then interacts with Imp b. 2. Imp b docks
the trimeric complex to the NPC and mediates
translocation. 3. Translocation is terminated by
direct binding of Ran-GTP to Imp b, which
releases the complex from the NPC, and
dissociates Imp a from Imp b. 4. Imp a and b are
recycled to the cytoplasm separately. Imp b /
Ran-GTP complexes leave the nucleus
directly. Imp a requires a specialized
exportin (CAS 1), thus helping to explain how
NLS-containing proteins remain in the
nucleus. 5. Proteins with an M9-like NLS bind
directly to Transportin, and do not require
an adapter or a-like protein. Ran also
regulates these interactions.
nucleus
cytoplasm
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Ran GTPase system Regulation of cargo loading
onto transport receptors
Ran is a small nuclear GTPase that switches
between a GDP- and a GTP-bound form. This
switch can only be accomplished by the aid of
regulators of Rans nucleotide bound state.
These regulatory proteins are localized on
opposite sides of the NE the Ran
GTPase-nucleotide Exchange Factor (GEF) is
nuclear, whereas the Ran GTPase Activating
Protein (GAP) is cytoplasmic. Ran binding
proteins are also cytoplasmic.
The intrinsic GTPase activity of Ran is activated
by the concerted action of the GAP and RanBP1.
Because both proteins are in the cytoplasm, Ran
is in the GDP-bound form in this compartment.
Conversion of Ran-GDP to Ran-GTP requires the
GEF. Because the GEF is bound to chromatin,
nuclear Ran is in the GTP-bound form. The
overall result of this nuclear GTPase cycle is a
Ran-GTP gradient across the NE with a high
concentration of Ran-GTP in the nucleus, and a
low concentration in the cytoplasm.
GTP
GDP
Ran GTP
Ran GDP
GEF/Rcc1
nucleus
NE
cytoplasm
RanBP1
The nucleotide state of Ran determines
compartment identity
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Summary
The existence of a Ran-GTP gradient provides a
plausible explanation as to how functional
asymmetry can be imposed on the transport cycle.
Importins bind their cargo in the cytoplasm, and
release them upon binding Ran-GTP in the nucleus.
Importins then return to the cytoplasm as
Ran-GTP complexes minus cargo. Ran-GTP must then
be removed from the Importins to allow binding
of another cargo molecule.
Exportins bind their cargo in the nucleus forming
a trimeric complex with Ran-GTP. This
cargo-exportin-Ran-GTP complex is then
transferred to the cytoplasm, where it
disassembles following GTP hydrolysis. The cargo
free, Ran-GTP free exportin can then re-enter the
nucleus and bind another cargo molecule. The
release of the one cargo molecule requires energy
in the form of one molecule of GTP hydrolyzed
per transport cycle.
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Selectivity across the nuclear pore Part II.
Nucleocytoplasmic transport of RNA
RNA Cargo 1. Messenger RNA (mRNA)
transcripts must exit the nucleus to engage the
protein translation machinery. 2.
Ribosomal (rRNA) and transfer (tRNA) RNAs must
exit the nucleus to participate in
protein translation. 3. Small nuclear RNAs
required for pre-mRNA splicing must exit the
nucleus to undergo maturation to small
ribonucleoprotein particles (snRNPs)
within the cytoplasm. 4. Certain viral RNAs
must exit the nucleus for viral replication.
Advances in the nuclear protein import field
contributed significantly to our current
understanding of nucleocytoplasmic RNA
transport. Identification of cellular
factors that mediate nuclear protein import
(soluble importins, insoluble NPC components).
Establishment of novel assay systems to
directly analyze the movement of
biomolecules between the nucleus and the
cytoplasm.
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Microinjection assay for RNA export in Xenopus
oocytes
32P-labeled RNA transcript injected into the
nucleus
Nucleus

Incubate at 16oC
Longitudinal cross-sectional view of
nuclear-specific microinjection
Manually dissect into nuclear (N ) and
cytoplasmic (C ) fractions.
N
C
Isolate RNA in fractions and analyze RNA species
using PAGE and autoradiography.
N
C
T
N
C
RNA of interest
Control RNA
t0
t0
t0
t2hr
t2hr
Time (t)
T total RNA injected N nuclear RNA fraction
C cytoplasmic RNA fraction
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Microinjection / RNA titration assay in Xenopus
oocytes.
Purpose To determine whether different classes
of RNAs use the same or different export pathways.
Approach Test whether export of a specific class
of RNA is affected by the presence of increasing
amounts of an RNA competitor.
Results
Cold rRNA competitor
(pmol)
No RNA Competitor
0. 5
2. 5
5.0
N C

T N C
N C
N C
N C
32P-rRNA
T total input RNA Ccytoplasmic RNA N nuclear
RNA t time (min)
T N C
N C
N C
N C
N C
32P-U1snRNA

T N C
N C
N C
N C
N C
32P-mRNA
t0
t45
t45
t45
t45
Time (minutes)
Summary
1. Similar to nuclear protein import, cellular
RNA export is a saturable, carrier-mediated,
energy dependent process. 2. Competition
studies using this assay system indicate that
specific factors are required for export of
an individual class of cellular RNAs, and that
such factors may be limiting.
Conversely, nuclear export of the different
classes of cellular RNAs may require common or
shared factors which are not limiting.
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Genetic analysis of nuclear RNA export in budding
yeast
Yeast genetic approaches facilitated the
identification and functional characterization of
cellular factors that mediate nuclear RNA export.
Approaches i. Development of temperature
sensitive (ts ) mutant strains
ii. Synthetic lethality screens for
transport-defective strains.
Example approach i. Incubate yeast cells with a
chemical mutagen, and screen for mutants
defective in mRNA export at the non-permissive
temperature (37oC) using fluorescent RNA in situ
hybridization (FISH).
FISH analysis of poly A() RNA localization in
wild type or temperature sensitive (ts) yeast
cells
poly A () RNA visualized using a FITC-conjugated
oligo probe complementary to the poly A tail
(i.e. FITC-oligo dT (52))
25oC permissive temperature
37oC non-permissive temperature
Result
Strains defective in mRNA export accumulate poly
A() RNA in the nucleus at 37oC, but not at 25oC.
Cole, et al., 2002. Methods Enzymol. 351568-587.
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Retrovirus Lifecycle
28
HIV-1 Rev-mediated nuclear export as a model
system to study RNA export
The Rev protein facilitates the cytoplasmic
accumulation of unspliced or incompletely
spliced HIV RNAs, which encode the viral
structural proteins. In the absence of Rev,
these RNAs are retained in the nucleus. Thus, Rev
function is essential for viral replication.
Northern blot of cytoplasmic HIV RNAs
mock
WT HIV
Rev mutant HIV
2kb class
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Functional domains of the HIV-1 Rev protein
LE Amino acids 78 and 79 of Rev. Note The
mutant Rev M10 protein contains amino acid
substitutions in these residues
i. in vitro binding assays demonstrated that Rev
contains an arginine-rich motif (ARM) which
binds, in a sequence-specific manner, to a
cis-acting RNA sequence known as the Rev
Responsive Element (RRE). The RRE is located in
the second intron of unspliced (i.e. gag-pol) or
incompletely spliced (i.e. env) viral RNAs.
ii. Genetic analysis in mammalian cells
identified a second functional domain, a
leucine-rich Effector domain. Point mutations
within its coding sequences abolish Rev function
(L78, 79E to D78, A79). This particular Rev
mutant, Rev M10, is a trans-dominant negative
inhibitor of Rev function. These key
observations suggested the Rev Effector domain
interacts with a cellular cofactor (s).
30
Model of HIV-1 Rev-Mediated RNA Export
Nucleus
RRE
Rev
AAAAA
cytoplasm
AAAAA
Rev
RRE
AAAAA
Rev
RRE Rev Responsive Element
Putative host factor
Revs Mechanism of Action
Rev binds directly to the RRE within incompletely
spliced viral RNAs (i.e gag-pol and env ). The
Rev effector domain interacts with cellular
factors which mediate RNA export.
Rev M10 does not support viral replication and
does not promote the cytoplasmic accumulation of
RRE-containing viral RNAs. The inability of Rev
M10 to exit the nucleus was shown to correlate
with its inability to support Rev function.
Thus, the Rev effector domain contains a Nuclear
Export Signal (NES).
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RNA in situ hybridization assay for studying
Rev-mediated RNA export
Approach Mammalian cells are transiently
transfected with a plasmid that expresses an
RRE-containing HIV RNA (gag-pol) in the absence
or presence of a Rev expression plasmid
(Rev). The intracellular distribution of these
RNAs is analyzed by fluorescent RNA in situ
hybridization(FISH) using a Cy3-conjugated oligo
probe that is complementary to the RRE RNA.
Note Cy3 is an orange fluorescing cyanine dye
that produces an intense red signal easy detected
using a rhodamine filter (660nm).
Rev
RevM10
HIV gag-pol
HIV gag-pol
No DNA
HIV gag-pol
probe
probe
probe
probe
Additional experimental approaches that have been
developed for analyzing Rev function 1. HIV-1
or chimeric HIV-based genetic analysis. 2.
Transfection assays using an Rev-dependent
reporter construct. 3. Oocyte microinjection
using recombinant Rev protein or peptides. 4.
Yeast-based colorimetric assays using a
Rev-dependent reporter construct.
Sanchez-Velar et al., 2004. Genes Devel. 18
23-34 Meyers and Malim, 1994. Gene s Devel.
81538-1547 Hope, et al., 1990. J. Virol. 91
1231-1238.
32
Summary
RNA export can be viewed as a protein process
associated with an RNA cargo. HIV-1
Rev-mediated and certain classes of cellular RNAs
require NES- containing proteins as RNA transport
cofactors. HIV-1 Rev-mediated and cellular RNA
pathways share one or more dedicated
components. Several cellular proteins contain
leucine-rich NESs TFIIIA, IkB, PKI Unique NES
in the hnRNP A1 protein, the M9 domain, acts as
an NLS and an NES.
33
Identification of a cellular factor that
interacts with the NES
Discovery of the nuclear export receptor
CRM 1

• Evidence
• 1. Leptomycin B (LMB), a lipophilic antibiotic,
  was shown to block Rev or Rev-
• dependent RNA export in HeLa cells.
• 2. LMB had been previously shown to be toxic to
  fission yeast. The molecular
• target of LMB is the CRM1 gene mutants
  resistant to LMB map to that gene.
• 3. Immunoprecipitation studies revealed that
  human CRM1, a member of the
• importin-b protein family, interacts directly
  with NUP 214/CAN.
• Collective data from mammalian cell-based assays,
  oocyte microinjection studies,
• and genetic screens in yeast demonstrated CRM1
  is the nuclear export receptor (NER)
• for Rev. Additional studies showed CRM1 is the
  NER for cellular and viral proteins
• that contain a leucine-rich NES nuclear export
  of these proteins is inhibited by LMB.

34
Cis-Acting Export Signals on Proteins and RNA
Dreyfuss, et al., 2002. Nat. Rev. Mol. Cell.
Biol. 3195-205 Maniatis and Reed, 2002. Nature
416 499-506.
35
Constitutive Transport Element (CTE)-mediated
nuclear RNA export
CTE
The CTE is a cis-acting RNA element located in
the 3UTR of Mason-Pfizer Monkey Virus RNA (MPMV)
TAP
p15
AAAAA
Nucleus
cytoplasm
CTE
p15
AAAAA
CTE
TAP
p15
AAAAA
Mechanism of Action TAP/ p15 binds directly to
the CTE to promote nuclear export of MPMV RNAs.
TAP /p15 function requires an interaction with
components of the cellular export machinery.
Hammarskjold, M.L. (2001). Curr. Top. Microbiol.
Immunol. 259 77-93.
36
Pre-mRNA splicing coupled export model
Adapted from Reed , R. and Magni, K. (2001). Nat
Cell Biol. 3 E201-4.



37
Adapted from Conti, E. and Izaurralde, E.
(2001). Curr. Opin. Cell. Biol. 13 310-320.
38
Nucleocytoplasmic Transport Regulation
Eukaryotic cells control many biological
processes by regulating the movement of
macromolecules in and out of the nucleus.
Similar to other steps in gene expression,
nucleocytoplasmic transport may be subject to
positive or negative regulation.
1. To regulate a given response 2. To
communicate cytoplasmic and nuclear events
allowing cells to respond to environmental
changes or cell cycle position 3. To generate a
more robust molecular switch or affect its
nature (i.e on / off)
Two important issues concerning regulated nuclear
translocation
1. Steady-State Localization of a Cellular
Protein. The steady-state distribution of a
protein is determined by its relative rate of
nuclear import and export. Changes in the rate
of import or export can lead to a shift in the
steady-state localization of the protein. Since
both import and export can be regulated, it is
essential to experimentally observe import in the
absence of export (or vice versa ) to determine
which rate is subject to regulation.
39
2. Protein Shuttling Shuttling proteins move
continuously between the nucleus and the
cytoplasm. The steady-state localization of a
shuttling protein reflects a dynamic process of
nuclear entry and exit. To date, two classes of
shuttling proteins have been identified Carrier
proteins - Proteins associated with hnRNP
particles, presumably are exported to the
cytoplasm bound to RNA and then re-imported into
the nucleus for another round of transport.
HIV-1 Rev is an example. Non-Carrier proteins-
Proteins that use shuttling as a way of
regulating their activity. These proteins would
be localized in the cytoplasm at steady-state
because their nuclear export is more efficient
than nuclear import. Their nuclear export is
blocked under conditions in which
their activities are required in the
nucleus. Thus, protein shuttling as a mode of
regulation may be important for coordinating
nuclear and cytoplasmic events. Additionally, it
offers a simple, reversible, and rapid mechanism
for regulating nuclear activity.
40
How do you determine whether a protein shuttles
between the nucleus and the cytoplasm A
heterokaryon assay
Schematic representation of approaches for
detecting nucleoplasmic shuttling of proteins.
(A) Migration of fluorescently labeled (FITC)
or epitope-tagged nuclear proteins in
interspecies heterokaryons. (B)
Antigen-mediated nuclear accumulation of
antibodies injected into the cytosol. In both
types of experiments, cyclohexamide (CX) was used
to distinguish the migration of pre-existing
proteins from the contribution of newly
synthesized proteins. Nuclear protein export in
this assay is sensitive to LMB treatment.
41
Possible steps in nuclear translocation that
could be targets for regulation
1. The binding of the cargo to an import or
export receptor. 2. The activity of the soluble
transport machinery. 3. The NPC can be modified
to affect its transport properties. 4. The
cargo-receptor complex can be tethered to an
insoluble component, thereby preventing it
from binding to the NPC.
Regulation of Cargo-Receptor Complex
Formation i. Phosphorylation Regulate the
affinity of a cargo for its transport receptor,
thus regulating the sub-cellular localization
of the cargo. ii. Intermolecular Association
Regulate cargo interactions with accessory
adapter proteins. Note These modes of
regulation are not mutually exclusive because
they can be used sequentially to regulate nuclear
localization. These mechanisms can enhance or
decrease the affinity of a cargo for its receptor
(i.e. have a positive or negative effect).
42
Nuclear Factor of Activated T-Cells (NF-AT) A
Cellular Factor Whose Function is Regulated at
the Level of Nucleocytoplasmic Transport
Mode of Regulation Phosphorylation and molecular
associations affect its sub-cellular localization
by modulating its rate of nuclear import and
export.
Stimulation of T-cell receptors leads to
activation of signal transduction pathways which
induce cytokines and cell surface molecule gene
express- ion. T-cell receptor stimulation also
causes an elevation in cytosolic Ca2 levels,
which activates the phosphatase Calcineurin .
Active calcineurin leads to dephosphorylation of
NF-AT.
NF-AT
P
NLS
NES
N
C
Dephosphorylation of NF-AT results in formation
of a dephosphorylated NF-AT/ calcineurin complex.
Once formed, the complex translocates into the
nucleus and facilitates transcription of
genes required for T-cell specific
activation. Phosphorylation of NF-AT inhibits its
nuclear import rate by inducing an
intra-molecular conformational change that makes
the NLS inaccessible for receptor binding.
Calcineurin maintains NF-AT in its
unphosphorylated form, leading to a decrease in
its rate of nuclear export.
Direct binding and masking of the NF-AT NES by
calcineurin inhibits its association with
export receptors, leading to nuclear accumulation
of NF-AT. This model provides a simple
explanation for the observation that
NF-AT/calcineurin is imported to the nucleus as a
complex.
Kaffman and OShea (1999.Annu. Rev.Cell.Dev.
Biol. 15 291-339.
43
Transport of small nuclear RNAs (snRNAs) between
the nucleus and the cytoplasm
Regulation by localization
snRNAs (U1, U2, etc.) are transcribed in the
nucleus and exported to the cytoplasm in a
CRM1-dependent fashion. In the cytoplasm,
they associate with SM proteins to form small
nuclear ribonucleoprotein particles (snRNPs).
The assembled snRNPs are then imported back into
the nucleus, the site of their function.
44
Regulation of nuclear import of transcription
factors
A
B
A. The transcription factor NF-?B is maintained
as an inactive complex with I?B, which
masks its NLS in the cytoplasm. In response to
appropriate extracellular signals, I?B is
phosphorylated and degraded by proteolysis,
allowing the import of NF-?B to the nucleus.
B. In contrast, the yeast transcription factor
SW15 is maintained in the cytoplasm by
phosphorylation in the vicinity of its NLS.
Regulated dephosphorylation exposes
the NLS and allows SW15 to be transported into
the nucleus at the appropriate stage of the cell
cycle.