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. Regulation of gene expression at the level of nucleocytoplasmic transport. Prokaryotes. Eukaryotes.
February 16, 2005
Maria L. Zapp, Ph.D.
Program in Molecular Medicine and
The UMass Center for AIDS Research
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
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.
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
Nuclear Envelope (NE) : Nuclear pore complexes, nuclear lamina, and lipid membranes
Nuclear pore complexes (NPCs)
Possible pathways for molecular movement into the nucleus: lamina, and lipid membranes
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
Micropipette filled with tracer substance lamina, and lipid membranes
Tritiated ( 3H-labeled) dextrans of 3 different sizes
Radii= 12.0 ( ) 23.3 ( ) 35.5 ( )
What is the permeability of the nuclear envelope?
using X. laevis oocytes
Representative 100 mM sections
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
Time course of nuclear permeation, expressed as the average lamina, and lipid membranes
nuclear:cytoplasmic grain density, X n/c as a function of diffusion
time after injection, td (min). Vertical bars = s. e. mean.
Radii= 12.0 ( ) 23.3 ( ) 35.5 ( )
of the data
Paine, et al., 1975. Nature 254: 109-114.
Conclusion: lamina, and lipid membranes
The NE is a molecular sieve that
restricts molecular movement
between the nucleus and the
1. These data demonstrate that the NE is less permeable to larger
dextrans (>23.3 Å) than smaller dextrans (<12.0 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.
Schematic representation of the Nuclear Pore Complex (NPC) lamina, and lipid membranes
Protein constituents of the NPC are known as nucleoporins or “NUPs”.
Selectivity at the nuclear pore lamina, and lipid membranes
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”
Nucleoplasmin injected into the
cytoplasm of frog oocytes enters
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
Conclusion: Nuclear proteins contain nuclear-targeting signals
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.
These oocyte-based approaches help
demonstrate that cellular proteins
and RNA are transported bidirectionally
through the NPC.
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. 99:2216-2222.
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 “prototype”of 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--> Thr ) remains in the cytosol.
Lanford and Butel, 1984. Cell 37:801-813. Kalderon, et al., 1984. Cell 39: 499-509.
1 AA the nucleus
ii. Construction and analysis of chimeric fusion proteins
Mata2 = 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 a2 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
Richardson, et al., 1984. Cell 44: 77-85. Hall, et al., 1984. Cell 36: 1057-1065. Goldfarb, et al., 1986. Nature 322:641-644.
Summary: the nucleus
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
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.
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.
+ NLS Protein
D NLS Protein (lacks an NLS)
+ NLS Protein (contains an NLS)
MT-NLS protein (contains a mutant NLS)
GST-NLS-EGFP the nucleus
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 DNAprecipitation, 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.
iii. the nucleusin 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
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. 5:501-510 ; 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. 123:1649-1659. Rexach and Blobel, 1995. Cell 83: 638-692.
- the nucleusATP
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-
permeabilized 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).
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. 111:807-816.
Molecular events in nucleocytoplasmic transport the nucleus
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
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.
Ran the nucleus
Cargo bearing an NLS
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
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.
GAP/ the nucleus
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 Ran’s 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.
The nucleotide state of Ran determines compartment identity
Summary the nucleus
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.
Selectivity across the nuclear pore the nucleus
Part II. Nucleocytoplasmic transport of RNA
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.
Microinjection assay for RNA export in the nucleusXenopus oocytes
32P-labeled RNA transcript injected
into the nucleus
Incubate at 16oC
view of nuclear-specific
Manually dissect into nuclear (N ) and cytoplasmic (C ) fractions.
Isolate RNA in fractions and analyze RNA species using PAGE and autoradiography.
RNA of interest
T= total RNA injected N= nuclear RNA fraction C = cytoplasmic RNA fraction
Microinjection / RNA titration assay in the nucleusXenopus 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.
Cold rRNA competitor
No RNA Competitor
T= total input RNA
N= nuclear RNA
t = time (min)
1. Similar to nuclear protein import, cellular RNA export is a saturable, carrier-mediated, energy
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.
Yeast cell the nucleus
WT strain at 37oC
WT strain at 25oC
Mutant strain at 25oC
Mutant strain at 37oC
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))
37oC non-permissive temperature
Strains defective in mRNA export accumulate poly A(+) RNA in the nucleus at 37oC, but not at 25oC.
Cole, et al., 2002.MethodsEnzymol. 351:568-587.
Retrovirus Lifecycle the nucleus
9kb the nucleus
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
ARM Domain the nucleus
NLS / RNA binding
Nuclear Export Signal
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
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).
Rev the nucleus
Model of HIV-1 Rev-Mediated RNA Export
RRE Rev Responsive Element
Putative host factor
Rev’s Mechanism of Action:
Rev binds directly to the RRE within incompletely spliced viral RNAs (i.egag-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).
RNA the nucleusin 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).
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. 8:1538-1547; Hope, et al., 1990. J. Virol. 91 :1231-1238.
Summary the nucleus
RNA export can be viewed as a protein process associated with an
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
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.
Rev-mediated export the nucleus
Cellular RNA export
in Xenopus oocytes:
Member of the importin-b
family of transport
Identification of a cellular factor that interacts with the NES:
Discovery of the nuclear export receptor
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.
Cis- the nucleusActing Export Signals on Proteins and RNA
Dreyfuss, et al., 2002. Nat. Rev. Mol. Cell. Biol. 3:195-205 Maniatis and Reed, 2002. Nature 416: 499-506.
TAP / p15 heterodimer the nucleus
CTE Constitutive Transport
Constitutive Transport Element (CTE)-mediated nuclear RNA export
The CTE is a cis-acting RNA element located in the 3’UTR of Mason-Pfizer Monkey Virus RNA (MPMV)
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.
Nascent pre-mRNAs are packaged into hnRNPs. During spliceosome assembly, exons are packaged by non-hnRNP spliceosome components such as SR proteins. After splicing, hnRNP particles remain associated with the introns, which are retained in the nucleus. Partial or mutant pre-mRNAs unable to enter the splicing pathway are also retained in packaged hnRNPs. In contrast, the spliced mRNP is targeted for export by factors recruited during splicing, in particular the export factor Aly/REF. The spliced mRNA is exported by a conserved machinery composed of non-hnRNP factors such as TAP/p15, hGle1, hGle 2, and hDbp5.
Pre-mRNA splicing coupled export model
Adapted from Reed , R. and Magni, K. (2001). Nat Cell Biol. 3 :E201-4.
Adapted from Conti, E. and Izaurralde, E. (2001). splicingCurr. Opin. Cell. Biol. 13: 310-320.
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.
2. splicingProtein 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.
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.
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
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).
P targets for regulation
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.
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 O’Shea (1999.Annu. Rev.Cell.Dev. Biol. 15: 291-339.
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.
Regulation of nuclear import of transcription factors and the cytoplasm
A. The transcription factor NF-B is maintained as an inactive complex with IB, which masks its NLS in the cytoplasm. In response to appropriate extracellular signals, IB 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.