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Nucleocytoplasmic
Trafficking
Presented by:Ebrahim Eftekhar
Ph.D student of Clinical Biochemistry
Shiraz University of medical science
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Outline
Nuclear pore complex structure (NPC) Required component for nucleocytoplasmic transport
Protein import
RNA export-tRNA export
-SnRNA export
DNA importRegulation of nuclear localization during signaling
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Nuclear pore complex (NPC): Large, elaboratestructure that perforate the nuclear envelopes.
Molecular Mass: 125 million dalton (one of the biggestmacromolecular assemblies = 30 times larger than aribosome)
The nuclear envelope of mammalian cell: contain 3000-4000 NPC.
Proteomic analysis: NPC contain 30different proteinknown as nucleoporins or Nups.
Each NPC can transport up to 500macromoleculespersecond in both direction at the same time.
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NPC structure
Figure 1: An electron micrograph showing a side view
.of two NPC
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Figure 2: A small region of the nuclear envelope
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Figure 3 : NPC complex
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Table 1: Mammalian, S.cerevisiae and C.elegancs nucleoporins
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-Bipartite NLS
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What happens when we use recombinant DNA
techniques to add theNLS
to a protein?
Normal BSA BSA with NLS
Microinjection Pipettes
Cell
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B)Nuclear export signal (NES, Leu rich peptide)
-Leu-Ala-Leu-Lys-Leu-Ala-Leu-Asp-Leu-
2-ReceptorsA) The receptor for the largest class of NLS-
bearing protein is heterodimer ofimportin
and importin .
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Figure 5 : Nuclear import receptors
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B) Exportin 1 (CRM1): transport molecules from
nucleus to cytoplasm.
Importin and exportin receptors are collectively called
Karyopherin.
3-Ran protein: small GTP binding protein that regulate
transport through the pore.
Ran-GTP: predominantly in the nucleoplasm
Ran-GDP: predominantly in the cytoplasm
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4-Ran regulator proteins
Ran-GAP (GTPase activating protein) in cytocol.
Ran-GEF (Guanine nucleotide exchange factor) innucleus.
Ran-BP1 &BP2: Accessory proteins that act
cooperatively with Ran-GAP.
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Step1:Assembly of import complex
Recognition of NLS by importin is crucial
for the formation of import complex.
NLS binding site of importin are formed
from an array of acidic residue.
The N-t of importin bind to importin
through IBB domain.
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Figure 6 : Interaction between importin and
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The IBB domain contain a cluster of basic
residues that is similar to an NLS and can bind
to the NLS binding site.
Therefore in addition to connecting importin to
, IBB domain has an autoinhibitory role.
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Figure 7 : Nuclear protein import
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Step2 : Translocation through NPC
Macromolecules of Mr > 40 kD are excluded
from NPCs, and only those bound to carriers can
move through the channel.
The mechanism by which complexes are
translocated through NPCs remain to beelucidated.
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Phe-Gly (FG) sequence repeats are thought to
be important for mediating movement through
NPCs.
FG nucleoporin repeat form a sieve-like gel
through the interaction between hydrophobiccore.
The diffusion of particles in a cross linked geldepend crucially on the gels pore size.
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hydrogel/sieve model proposes:
interaction with the carrier locally disruptsinteractions between FG-repeat cores that
generate the gel and so transiently opens
adjacent meshes in the gel.
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Step3:Import complex disassemblyStep3:Import complex disassembly
RanGTP dissociates the cargo: carrier import
complex and therefore imposes directionality
on the transport process.
Step 4:Importin recycling
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Figure 7 : Nuclear protein import
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Figure 8 : Structure of the importin: Ran GTP complex
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Figure 9: Microtubules transport cellular proteins to the cellular
.perinuclear region by dynein , facilitating protein nuclear import
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tRNA Export
Exportin t directly bind to the TC and acceptor
arm structure (aminoacylated) of tRNA .
tRNA with immature 5 and 3 end are not effecientlyexported.
Intranuclear aminoacylation of tRNA is a proofreading mechanism to ensure that only correctly
maturated tRNA will be exported.
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Figure 10 : tRNA export from nucleus
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Nuclear Import of DNA
The movement of DNA from cytoplasm tothe nucleus remain one of the major barriertoefficient gene transfer and expression.
Graessman demonstrated that when 1000 to2000 copies of a plasmid were injected into the
cytoplasm, less than3% of the expression wasseen as compared to cells injected in thenucleus.
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Surprisingly, little attention directed toward
discovering the mechanisms used by the cell to
direct DNA to the nucleus.
During mitosis, the nuclear envelope breaks
down, eliminating a major barrier to gene
transfer.
Does DNA ever enter the nuclei ofnon-
dividing cells?
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SV40 DNA was injected in to the cytoplasm.
-Within 2 to 4 hours , DNA was localized in the
perinuclear region, suggesting that the DNA was
accumulating at the NE awaiting import.
-by 6 to 8 hours, DNA was localized to the nucleus.
DNA accumulates in distinct regions of the
nucleus that co-localize with proteins involved intranscription and splicing , indicating that the
DNA is functional for transcription.
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Although SV40 DNA is readily taken up by
nuclei of non-dividing cells, many other
plasmids are not.
SV40 genome contains a sequence that can
mediate nuclear uptake.
when 50 bp of the SV40 enhancer region is
cloned into any of the other bacterial plasmids,
they are targeted to the nucleus.
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Figure 13 : Model for SV40 enhancer mediated sequence-specific
nuclear import
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Incorporation ofNF-B binding sites alonein a plasmid can increase the nuclear
localization of the plasmid in HeLa cells.
In the presence ofNF-B activatorsuch as
TNF- gene expression robustly increase.
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Regulation of nuclear localization
during signaling
NFAT Family of Transcription Factors
NFAT, plays a key role in activating geneexpression in T lymphocytes.
The activity of NFAT is controlled throughits localization.
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In unstimulated cells,NFAT reside in thecytosol.
In stimulated cell , calcineurin dephosphorylatesNFAT and causes its translocation to the nucleus.
If calcineurin inhibited with Cyclosporin ,NFAT is rapidly rephosphorylated and exported
from the nucleus.
When NFAT phosphorylated, NLS is thought tobe inaccessible.
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References:
1-Stwart M.Nature review of molecular
biology,2007:8;195.2-Maximiliano A.Cell,2008:556:1.
3-Potoun CW.Advanced drug delivery, 2007:59;698.
4-Roderick YH.International Review of Cell and
Molecular Biology ,2008:267;343.5-Joshua Z.Advanced drug delivery, 2003:55;703.
6-Andrew E. Cell biology ,2001:155;187.
7-Aitchison JD. Cell biology, 2000:27;23.8-Clarke RP. Trend in cell biology ,2001:11;366.
9-Ohno M.Cell,1998:92;327.
10-Nakielny S. Cell,1999:99;677
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Thank you for Your kind
!attention