Lectures 5 & 6

Nuclear Import, Export and Targeting

Nuclear Import and the Role of Nuclear Pore Complex Proteins

• Nuclear Localization sequences (NLS’s) are necessary & sufficient signals for a protein to be imported into the nucleus from the cytoplasm.

• Simple SV-40 type: PKKKRKV 7-mer necessary and sufficient for nuclear import.

• Bipartite or split NLS’s: KR-[PATKKAGQA]- KKK: Spacers can be mutated by SDM and still have import but not NLS.

Discovery of the SV-40 7-mer NLS • SV-40 is a DNA virus that infects mammalian cells and takes

over the genomic machinery of the cell nucleus.

• SV-40 (T antigen) antigen is a viral protein essential for the regulation of viral DNA replication and transcription.

• In normal cells the antigen is made in the cytoplasm and is imported to the nucleus where it is concentrated 10 fold more than in the cytoplasm.

• During a mutagenesis study of the SV-40 virus, a class of mutants were isolated that had a higher concentration of T-antigen in the cytoplasm than in the nucleus.

• It was then shown that a single amino acid substitution (K-128) was sufficient enough to block nuclear import.

• In vitro site directed mutagenesis defined a 7-mer sequence around K-128 that was critical for nuclear import.

Normal T Mutant T

Immunofluorescence using anti-T-antigen & FITC conjugated 20 antibody

Cell membrane

Nuclear membrane

T antigen

Infected mammalian cells

The 7-mer NLS is Necessay and Sufficient for Nuclear Import

• A DNA construct was engineered in which the 7- mer peptide was ligated to the 3’ end of the pyruvate kinase gene.

• Pyruvate kinase is a cytoplasmic protein not found in the nucleus.

• Transfection into cells tested whether the 7-mer is both necessary and sufficient for nuclear import.

• Results along with the control experiment are shown below.

Pyruvate kinase control construct Pyruvate kinase - NLS construct

In vitro Systems for Nuclear Import• DNA oocytes in vivo mininuclei

DNA or sperm chromatin + Xenopus oocyte extract synthetic nuclei

or permeablized cells + cytosol NUCLEAR IMPORT • real nuclei + Xenopus oocyte extract NUCLEAR IMPORT

Nuclear Source Nuclear import systems

Labeled proteins Import detection

Synthetic/ real nuclei + Xenopus extract or permeabilized cells + cytoplasmic extract

Radiolabeled Biochemical quantitation

Fluorescent labeled Fluorescence microscopy

Colloidal gold labeled Electron microscopy

NPC

ChromatinOuter nuclear membrane

Inner nuclear membrane

Demonstration of ATP and Cytosol Dependent Transport in Permeabilized Mammalian Cells

• Digitonin was used to permeabilize cells

• The transport of a normally cytoplasmic protein into the nucleus was observed when it was conjugated at the N-T to a 7-mer NLS.

- Lysate

• Xenopus Ext + real nuclei fluor/c.g. probeimportFluor Mic or EM• 3 major findings: (1) 7-mer NLS is sufficient & necessary; (2) Role of ATP; (3) Effect of WGA• Protein Probes: 7-mer(norm or mutant)-HSA-FITC (Green) [FM]• HSA= human serum albumin“ “ “ -HSA-colloidal gold [EM]• Import Assay: 7-mer-HSA (FITC or CG) + Xenopus ext + rat liver nuclei

examine• Exp. Results: normal 7-mer: (1) Binds to NPC (2) Accumulates inside nucleus Mutant 7-mer: (1) No binding to NPC (2) No accumul. inside the nucleus

Newmeyer and Forbes Experiment

Normal 7-mer Mutant 7-mer

[Newmeyer & Forbes, Cell 52 (1988) 641-653]

ATP Requirement

• Xenopus ext + apyrase ATP depleted extract + 7-mer-HSA + nuclei Binds to NPC but no import

Identifies Two Steps for Nuclear Import

ATP Independent Step: Binding to the NPCATP Dependent Step: Translocation Through

the NPC

Wheat Germ Agglutinin (WGA) Effect

• WGA Effect: WGA (Wheat germ agglutinin) – plant lectin that binds N-actylglucosamine (GlcNc): Many NPC proteins have 0-linked GlcNc

• Exp: 7-mer probe + extract + WGA + nuclei 7-mer binds to NPC but no import. Expect that WGA sterically blocks the GlcNcs of NUPs.

+

NUP

WGA

WGA sterically blocks GlcNc’s of NUP’s

Further Distinguishes Nuclear Import into Two Steps: (1) Binding to NPC (ATP independent and WGA resistant) ; (2) Translocation through the NPC (ATP dependent and WGA sensitive)

NUPNUP

Finley and Forbes Experiment

Depletion experiment to determine the role of NPC glycoproteins (NPC-GPs) in nuclear

import Xen ext WGA sepharose CENTRIFUGE beads ext GlcNc NPC-GPs

beads supernatant

(Xenopus extract depleted of NPC-GPs)

Depletion Exp: sperm chromatin + depleted extract synthetic nuclei (depleted)

Synthetic nuclei (depleted) + 7-mer HSA-probe No NPC binding or import

Remarkably, the NPCs reassemble in this depletion experimentand “look the same” as “normal” NPCs

The absence of binding to the NPC indicates that the NPC-GP’s are required for NPC binding but that the GlcNc residues on the GPs are not required.

[Finley & Forbes, Cell 60 (1990) 17-29]

Finley and Forbes experiment contd…

Reconstitution Exp: sperm chromatin + depleted ext. + NPC-GP’s synthetic nuclei (reconst) + 7-mer HSA-probeNPC binding and import

CONCLUSION: Glycosylated (GlucNc) NPC proteins are required for both steps of nuclear import in vitro [NPC binding and translocation] but are not essential for the overall architecture of the NPC.

Mechanisms of Nuclear Import and Export

Gene Expression in Prokaryotes:

DNA RNA Protein

Gene Expression in Eukaryotes:

DNA RNAN ----- RNAC Protein export

CYTOPLASM --------- Nuclear Proteins

Shuttling Proteins

NUCLEAR IMPORT/EXPORT ARE ESSENTIAL PROCESSES FOR GENE REGULATION IN EUKARYOTES AND ARE LIKELY HIGHLY

REGULATED PROCESSES

NUCLEUS

N E

Import Export

import ---------

Nuclear Import Mechanisms• Proteins to be imported into the nucleus have Nuclear Localization Signals (NLS’s) that enable nuclear import.

• NLS’s bind to importin α subunit of an importin α-β complex.

• Transport through the NPC is mediated by interaction of degenerative sequences in the NPC proteins with the importin β subunit.

• Key to function and regulation are RAN GTP [high in nucleus by RCC1 (Ran nucleotide exchange factor)] & RAN GDP [high in cytoplasm by RAN GAP (RAN GTP activating protein)].

• The asymmetric distribution of RCC1 in the nucleus and RAN GAP in the cytoplasm drives the nuclear import process.

Ran

• The exporting proteins have special sequences called Nuclear Export Signals (NES’s) that mediate export through binding to a class of proteins that function in export called exportins.

• Exportins are typically monomeric and function in a reverse manner to importin under the control of RAN.

• Thus the cargo complex requires RAN-GTP which is found only in the nucleus.

• Disassociation of the ‘cargo” from the exportin requires RAN-GDP which occurs only in the cytoplasm.

NUCLEAR EXPORT

Machinery of nuclear import/export contd…RNA Export:

• In the current model, RNA export occurs by the export of multiple RNP proteins that cover the mRNA during transport.

•So a more precise term is: “RNP (ribonucleoprotein) export”.

• In the cell nucleus pre-mRNA is packaged into particles called pre-mRNP or hnRNP particles.

pre-mRNARNA

protein

RNP particle

Machinery of Nuclear Import/Export contd…

• During RNA splicing changes occur in the proteins associated with the hnRNP particles.

• Most dramatic change occurs at the moment of nuclear export where some of the RNP proteins are “nuclear restricted” and are therefore released from the RNP particles while others stay associated with the RNA.

• CBC (cap binding protein complex) initiates the export of RNA from the 5’ end.

• In the cytoplasm the remaining nuclear RNP proteins are removed and the RNA gets associated with cytoplasmic specific proteins which enable the mRNA to associate with ribosomes and carry out protein synthesis.

• The disassociated RNP proteins are imported back into the nucleus where they associate with other pre m-RNA’s.

• The RNP proteins imported back into the nucleus “shuttle” between nucleus and the cytoplasm through the NPC.

• Shuttling proteins have both a NES and NLS sequences.

Shuttling protein

NLS NES

HETEROKARON ANALYSIS TO DETERMINE IF A NUCLEAR PROTEIN SHUTTLES BETWEEN NUCLEUS AND CYTOPLASM

2. Do IF with Ab to protein of interest – Ab must recognize only the species of interest, e.g., human specific Ab to protein X and see if X is found only in the human cell nucleus of heterokaryon [nuclear restricted protein] or in both the human and mouse nucleus [a shuttling protein].

Nuclear restricted Shuttling protein

Heterokaryon

1. Construct heterokaryons

Human cell

Mouse cell

+Cell Fusion

Polyethylene glycol (PEG)

Demonstration of a nuclear restricted (hnRNP C, green) versus a shuttling (hnRNA A, red) protein by heterokaryon analysis after cell fusion of human (HeLa) and Xenopus laevis (frog) cells using antibodies specific for the human proteins

Heterokaryon - solid arrow

HeLa cell – arrowhead

Xenopus cell – dotted arrow

Xenopus

HeLaHeLa

Xenopus

HeLa

HK HK

H

XenHK

H

Xen

Nuclear Targeting

• Aside from NLS’s and NES’s there is growing evidence that many nuclear proteins contain Nuclear Targeting Sequences (NTS’s) that target individual proteins to the sites of genomic function/organization.

• A classic example is the DNA methyl transferase (MTase) which is an enzyme associated with replication sites in cells and is responsible for maintaining the methylation patterns of the DNA from cell generation to generation.

• This is important for regulation of transcription ( highly methylated genes are generally not transcribed).

Co-localization of MTase (red) with BrdU labeled (green) RS.

Leonhardt et al. Cell (1992) 71:865-873

Question: How is MTase targeted to RS? Is there a specific region of the MTase protein that is responsible for targeting the MTase to RS??

• Construct a series of deletion mutants of MTase• Transfect mammalian cells with MTase constructs fused to the beta- galactosidase (β-gal) gene.

• Use anti-β-gal antibodies to detect localization of the fusion protein in the nucleus and with RS labeled with BrdU method.

Mtase Fusion p

1-455**

1-385

207-511**

245-511

**

**

Leonhardt et al. Cell (1992) 71:865-873

[207- 455 REQUIRED FOR TARGETING TO RS]

Nuclear Targeting contd… Results: A region of the N-terminal MTase is necessary and

sufficient to target β-gal to RS. The targeting sequence is a 248 aa track from aa 207-455 of the 1,502 aa sequence of the whole protein.

1-1490

1-109;

308-1490

Anti-MTase Anti-β-gal

Merged