Lectures 5 & 6

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-mernecessary 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. Cell membrane Nuclear membrane T antigen Mutant T Normal T 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 mutagenesisdefined a 7-mer sequence around K-128 that was critical for nuclear import. Infected mammalian cells Immunofluorescence using anti-T-antigen & FITC conjugated 20 antibody

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 construct Pyruvate kinase - NLS construct

5. In vitro Systems for Nuclear Import •  DNA oocytes in vivo mininuclei NPC Inner nuclear membrane Chromatin Outer nuclear membrane •  DNA or sperm chromatin + Xenopus oocyteextract synthetic nuclei • or permeablized cells + cytosol NUCLEAR IMPORT • real nuclei + Xenopus oocyte extractNUCLEAR IMPORT

6. Demonstration of ATP and Cytosol Dependent Transport in Permeabilized Mammalian Cells - Lysate • Digitonin was used to permeabilize cells • The transport of a normally cytoplasmic protein into the nucleus was observedwhen it was conjugated at the N-T to a 7-mer NLS.

7. Newmeyer and Forbes Experiment [Newmeyer & Forbes, Cell 52 (1988) 641-653] • 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 Mutant 7-mer Normal 7-mer

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 NUP WGA NUP 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)

10. Finley and Forbes Experiment [Finley & Forbes, Cell 60 (1990) 17-29] 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 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. Remarkably, the NPCs reassemble in this depletion experiment and “look the same” as “normal” NPCs

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 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 N E NUCLEUS import --------- Import Export

13. Proteins to be imported into the nucleus haveNuclear Localization Signals (NLS’s)that enable nuclear import. • NLS’sbind toimportin αsubunit of an importin α-β complex. • Transport through the NPC is mediated by interaction of degenerative sequences in the NPC proteins with theimportin β subunit. • Key to function and regulation areRAN GTP[high in nucleus byRCC1 (Ran nucleotide exchange factor)]&RAN GDP[high in cytoplasm byRAN GAP(RAN GTP activating protein)]. • The asymmetric distribution of RCC1 in the nucleus and RAN GAP in the cytoplasm drives the nuclear import process. Nuclear Import Mechanisms 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. protein RNA pre-mRNA RNP particle

16. Machinery of Nuclear Import/Export contd… NLS NES Shuttling protein • 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.

17. 1. Construct heterokaryons Cell Fusion + Polyethylene glycol (PEG) Human cell Mouse cell Heterokaryon 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 HETEROKARON ANALYSIS TO DETERMINE IF A NUCLEAR PROTEIN SHUTTLES BETWEEN NUCLEUS AND CYTOPLASM

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 HeLa H H HK HK HK Xenopus Xen Xen HeLa HeLa Xenopus Heterokaryon - solid arrow HeLa cell – arrowhead Xenopus cell – dotted arrow

19. Nuclear Targeting • Aside from NLS’s and NES’s there is growing evidence that many nuclear proteins containNuclear Targeting Sequences (NTS’s)that target individual proteins to the sites of genomic function/organization. • A classic example is theDNA 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). Leonhardt et al. Cell (1992) 71:865-873 Co-localization ofMTase (red)withBrdU labeled (green) RS.

20. 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 [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-MTase Anti-β-gal Merged