Nucleocytoplasmic Trafficking

Nucleocytoplasmic Trafficking Presented by:Ebrahim Eftekhar Ph.D student of Clinical Biochemistry Shiraz University of medical science

Outline ♠ Nuclear pore complex structure (NPC) ♠ Required component for nucleocytoplasmic transport ♠ Protein import ♠ RNA export -tRNA export -SnRNA export

♠ DNA import ♠ Regulation of nuclear localization during signaling

♠ Nuclear pore complex (NPC): Large, elaborate structure that perforate the nuclear envelopes.

♠ Molecular Mass: 125 million dalton (one of the biggest macromolecular assemblies = 30 times larger than a ribosome)

♠ The nuclear envelope of mammalian cell: contain 30004000 NPC. ♠ Proteomic analysis: NPC contain 30 different protein known as nucleoporins or Nups. ♠ Each NPC can transport up to 500 macromolecules per second in both direction at the same time.

♠ Small water soluble molecules (40 -60 KD) can diffuse passively through NPC.

♠ Macromolecules larger than 60 KD can only pass the NPC by active transport.

NPC structure

Figure 1: An electron micrograph showing a side view .of two NPC

Figure 2: A small region of the nuclear envelope

Figure 3 : NPC complex

Figure 44 : Localization of NPC component

Table 1: Mammalian, S.cerevisiae and C.elegancs nucleoporins

Required component for nucleocytopladsmic transport 1- Signal peptide A) Nuclear localizing signal (NLS, Lys rich peptide)

-Monopartite NLS

-Bipartite NLS

♣♣ What happens when we use recombinant DNA techniques to add the NLS to a protein?

Microinjection Pipettes

BSA with NLS

Normal BSA

Cell

B)Nuclear export signal (NES, Leu rich peptide) -Leu-Ala-Leu-Lys-Leu-Ala-Leu-Asp-Leu❀❀❀❀❀❀❀❀

2-Receptors A) The receptor for the largest class of NLSbearing protein is heterodimer of importin α and importin β.

Figure 5 : Nuclear import receptors

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

4-Ran regulator proteins ❖ Ran-GAP (GTPase activating protein) in cytocol. ❖ Ran-GEF (Guanine nucleotide exchange factor) in nucleus. ❖ Ran-BP1 &BP2: Accessory proteins that act cooperatively with Ran-GAP.

Four steps for Nuclear protein import ❋Assembly of cargo:carreir import complex in the cytoplasm ❋Translocation through NPC ❋Import-complex disassembly in the nucleus ❋Importin recycling

Step1:Assembly of import complex ♣ Recognition of NLS by importin α is crucial for the formation of import complex. ♣ NLS binding site of importin α are formed from an array of acidic residue. ♣ The N-t of importin α bind to importin β through IBB domain.

Figure 6 : Interaction between importinα andβ

♣ 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 α to β, IBB domain has an autoinhibitory role.

Figure 7 : Nuclear protein import

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 be elucidated.

♣ 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 hydrophobic core. ♣ The diffusion of particles in a cross linked gel depend crucially on the gels pore size.

hydrogel/sieve model proposes: ❀❀ interaction with the carrier locally disrupts interactions between FG-repeat cores that generate the gel and so transiently opens adjacent meshes in the gel.

Step3:Import complex disassembly RanGTP dissociates the cargo: carrier import complex and therefore imposes directionality on the transport process.

Step 4:Importin recycling

Figure 7 : Nuclear protein import

Figure 8 : Structure of the importinβ: Ran GTP complex

Figure 9: Microtubules transport cellular proteins to the cellular .perinuclear region by dynein , facilitating protein nuclear import

tRNA Export ♠ ♠ Exportin t directly bind to the TΨC and acceptor arm structure (aminoacylated) of tRNA . ♠ ♠ tRNA with immature 5 and 3 end are not effeciently exported. ♠ ♠ Intranuclear aminoacylation of tRNA is a proof reading mechanism to ensure that only correctly maturated tRNA will be exported.

Figure 10 : tRNA export from nucleus

SnRNA Export

Figure 11 : Export and import of SnRNA

Nuclear Import of DNA ♣ ♣ The movement of DNA from cytoplasm to the nucleus remain one of the major barrier to efficient gene transfer and expression. ♣ ♣ Graessman demonstrated that when 1000 to 2000 copies of a plasmid were injected into the cytoplasm, less than 3% of the expression was seen as compared to cells injected in the nucleus.

♣ ♣ 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 of nondividing cells?

♣ 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 in transcription and splicing , indicating that the DNA is functional for transcription.

♣ 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.

♣ The SV40 enhancer contains binding sites for a number of transcription factors (TF). ♣ In cytoplam TF attach to its binding site on exogenous DNA to form a protein-DNA complex. ♣ One or more TF may have an NLS that is exposed at the surface of the complex such that it can interact with the importin machinery .

Figure 12: SV40 origin region

Figure 13 : Model for SV40 enhancer mediated sequence-specific nuclear import

♣ ♣ Incorporation of NF-κB binding sites alone in a plasmid can increase the nuclear localization of the plasmid in HeLa cells. ♣ ♣ In the presence of NF-κB activator such as TNF-α gene expression robustly increase.

Regulation of nuclear localization during signaling NFAT Family of Transcription Factors ✿ ✿ NFAT, plays a key role in activating gene expression in T lymphocytes. ✿ ✿ The activity of NFAT is controlled through its localization.

✿ In unstimulated cells,NFAT reside in the cytosol. ✿ In stimulated cell , calcineurin dephosphorylates NFAT 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 to be inaccessible.

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