1. Lectures 5 & 6 Nuclear Import, Export and Targeting

2. 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.

3. 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 2 0 antibody Cell membrane Nuclear membrane T antigen Infected mammalian cells

4. 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 constructPyruvate kinase - NLS construct

5. 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 proteinsImport detection Synthetic/ real nuclei + Xenopus extract or permeabilized cells + cytoplasmic extract RadiolabeledBiochemical quantitation Fluorescent labeledFluorescence microscopy Colloidal gold labeledElectron microscopy NPC Chromatin Outer nuclear membrane Inner nuclear membrane

6. 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

7. Xenopus Ext + real nuclei  fluor/c.g. probe  import  Fluor 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]

8. 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 NPC ATP Dependent Step: Translocation Through the NPC

9. 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) NUP

10. 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 experiment and “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]

11. Finley and Forbes experiment contd… Reconstitution Exp : sperm chromatin + depleted ext. + NPC-GP’s  synthetic nuclei (reconst) + 7-mer HSA-probe  NPC 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.

12. Mechanisms of Nuclear Import and Export Gene Expression in Prokaryotes: DNA  RNA  Protein Gene Expression in Eukaryotes: DNA  RNA N -----  RNA C  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  --------- 

13. 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

14. 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

15. 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-mRNA RNA protein RNP particle

16. 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

17. 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 restrictedShuttling protein Heterokaryon 1. Construct heterokaryons Human cell Mouse cell + Cell Fusion Polyethylene glycol (PEG)

18. 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 HeLa Xenopus HeLa HK H Xen HK H Xen

19. 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

20. 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. MtaseFusion p 1-455 ** 1-385 207-511** 245-511 ** Leonhardt et al. Cell (1992) 71:865-873 [207- 455 REQUIRED FOR TARGETING TO RS]

21. 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-MTaseAnti-β-galMerged