Nucleocytoplasmic Trafficking

8/14/2019 Nucleocytoplasmic Trafficking

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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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Nucleocytoplasmic Trafficking

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