Nucleocytoplasmic Trafficking Susan Wente Department of Cell & Developmental Biology U-3209 Medical Research Building III

Lecture Readings: 1) Text: Molecular Biology of the Cell Alberts et al., 4th Edition pg Molecular Cell Biology Lodish et al., 4th Edition pg ) “Recent” Review: Suntharalingam and Wente “Peering through the Pore: Nuclear Pore Complex Structure, Assembly, and Function” Dev. Cell (2003) 4: ) Assigned Paper: Takano, et al “tRNA actively shuttles between the nucleus and cytosol in yeast” Science (2005) 309: Main paper plus Supplemental Information

Segregation of protein function and genomic material -specialized machinery to maintain protein composition and communication between compartments Why are there membranes in the first place?

Nucleocytoplasmic Trafficking Nucleus Nuclear Envelope -two double lipid bilayer membrane structure -pores formed by the fusion of the inner and outer membranes molecules smaller than kD freely diffusable larger molecules traverse by active mechanism

Nucleocytoplasmic Trafficking Plasma membrane

Bidirectional Transport In and Out of the Nucleus N C N C Protein Import N C RNA Export Shuttling: Import & Export

Estimates of Transport Flux through Nuclear Pore Complexes HeLa Cell: -10 million ribosomes generated for each cell cycle (24 hrs) 10 X 10 6 / 24 / 60 = 7000 ribosomes/min Per minute: 560,000 ribosomal proteins imported 14,000 ribosomal subunits exported Through each NPC per minute: 100 ribosomal proteins imported 3 ribosomal subunits exported In vitro Import Measurements: (Ribbeck & Gorlich, 2001) 10 3 translocation events per NPC per second

Impacts on Disease and Development Kua et al., “Nuclear transport and cancer: from mechanism to intervention” Nat. Rev. Cancer (2004) 4: Cronshaw & Matunis “The nuclear pore complex: disease associations and functional correlations” Trends Endocrinol. Metab. (2004) 15:34-39 Cancer: -regulation of p53 tumor suppressor import/export -FG Nups genes linked to translocations in hematological malignancies, myeloid leukemias -overexpression of Nups in ovarian cancer tumors -mutation of a import factor in breast cancer cell line Autoimmune diseases -primary biliary cirrhosis Viral pathogenesis

Drugs that target transport pathways -Cyclosporin A -Leptomycin B

Nuclear Pore Complexes from Xenopus laevis Landmarks in Cell Biology: Unwin and Milligan J. Cell Bio. 1982

NPC proteins = Nucleoporins (Nups) ~ 30 Nups ~ 60 MDa cytoplasm nucleus Ribosome = ~ 80 proteins ~ 4 MDa

Higher Resolution Structural Analysis -Cryo-electron tomography -X-ray structure analysis of individual Nups -Mapping nearest neighbor protein-protein interactions

Movie of NPC Structure Modeling from MICHAEL ROUT, Rockefeller University - Have large amount of data on who’s next to who, and who forms complexes with whom - Using MODELLER, protein modeling software - Model proteins as soft spheres - Radius of sphere corresponds to mass of protein - Define spatial restraints between the proteins – taken from our lists of NUP-NUP interactions and isolated subcomplex compositions, include any other info we have on position etc. of NUPs - Randomize protein positions - Then, allow model to reform, fulfilling all of the restraints, if possible - Do this 1000s of times - Look for models which best fulfill the restraints (i.e., agree with all your original interaction data) - Here’s MODELLER generating one Model - Starts with Formation of the Model - Then, “Jiggling” is Optimization Process

Structural Homology Between Nups and Vesicle Coat Proteins

Model for Common Ancestorial Link between Coated Vesicles and NPCs

-How do macromolecules move through? -How is the NPC assembled? -How are transport and assembly regulated? General Strategy: Attack at the Site of Entry and Exit

Cytoplasm + General Mechanisms for Protein Import into Organelles Smith & Schnell, 2001 ER Mitochondria Chloroplasts Peroxisomes Thylakoid,  pH Bacterial Export, Tat Nucleus

Cytoplasm + General Mechanisms for Protein Import into Organelles Smith & Schnell, 2001 ER Mitochondria Chloroplasts Peroxisomes Thylakoid,  pH Bacterial Export, Tat Nucleus -Signal hypothesis: “addresses” Specific “addresses” recognized by organelle specific receptors

-Nuclear Localization and the Signal Hypothesis -Translocation Models -RanGTPase cycle -Examples of Experimental Strategies Key Points of the Nucleocytoplasmic Transport Mechanism

Localization Signals: Import and Export -amino acid sequences that are both necessary and sufficient for the import or export of a protein -uncleaved and at variable points in each individual protein -”folded” substrates -piggy-backing by a carrier Multiple different signals = Multiple distinct receptors

Pyruvate Kinase Alone Experimental Strategy for Demonstrating an NLS is Sufficient for Import: Ectopic Expression in Cultured Cells and Localization by Indirect Immunofluorescence Microscopy PK fused to the NLS SV40 Large T Antigen.

Localization Signals: Import and Export -amino acid sequences that are both necessary and sufficient for the import or export of a protein -uncleaved and at variable points in each individual protein -”folded” substrates -piggy-backing by a carrier Multiple different signals = Multiple distinct receptors

Family of Importin/Karyopherin  Transport Factors Ohno et al., in S. cerevisiae >21 in humans -each specific for a given cargo NC RanGTP Binding Domains: Adapter/cargo with NLS/NES Nucleoporins

Prototypical Protein Import Pathway SIGNALS DOCKING TRANSLOCATE nucleus cytoplasm Nuclear Localization Signal RANGTP CARGO RELEASE  RANGTP RANGDP RANGTP Shuttling Transporter Cargo RECYCLING

Leucine-rich NES Protein Export Pathway SIGNALS KAP  -DOCKING TRANSLOCATE nucleuscytoplasmRANGTP CARGO RELEASE RANGTP RANGDP RANGTP Leucine rich NES Rev “LPPLERLTL”NES NES NES NESNES Crm1 RECYCLING

RanGTP Differentially Modulates Transport Factor-Cargo Interaction nucleuscytoplasmRANGTP RANGDP NES NESRANGTP  Import cargo release Export cargo binding NLS

The Ran GTPase is required for: -modulating cargo binding to karyopherins -modulating karyopherin interactions with FG nucleoporins -GTP hydrolysis is not required for the actual translocation step = directionality Steady-state Ran localization = nuclear N C

Experimental Strategy for Testing the Nuclear Import Activity of A Transport Factor: Permeabilized Cells

mRNA TAP/Mex67 Adapted from Cullen, 2003

Novel Shuttling Factors in the mRNA Export PathwayGle1 Gle2

Wild type Mutants polyA+RNADAPI Experimental Strategy for Testing the Role of a Factor in mRNA export: In situ Hybridization to Detect polyA+ RNA Localization in Yeast Mutant Cells -Digoxigenin-Oligo (dT) 30 -Anti-Dig FITC FAB

-The Nups: Translocon -The Shuttling Transport Factors -Karyopherins -mRNA export Nuclear Entry and Exit: The Rules and the Players

NPC Proteins = Nucleoporins = Nups 1/3rd of NPC mass from FG Nups FG FXFG GLFG -non-tandem repeats, polar spacer sequences -localized in all substructures -docking sites for transport factors

Yeast FG Nucleoporins Nup159 Nsp1 Nup100 Nup145N Nup116 Nup60 Nup2 Nup1 Nup42 Nup49 Nup57 FG GLFG FxFG Modified from Allen et al., 2001

FG nups reside throughout the NPC Nup159-FG Nup42-FG Nup2-FXFG Nup60-FXF Nup1-FXFG Nup100-GLFG Nup145N-GLFG Nup116-FG,GLFG Nup49-GLFG Nsp1-FG,FXFG Nup57-GLFG exclusively cytoplasmic exclusively nucleoplasmic cytoplasmic & nucleoplasmic

FG nups are binding sites for movement through the NPC nucleus cytoplasm FG 1. Complex formation 2. Docking at NPC 3. Translocation 4. Release Mechanism?

Affinity Brownian Selective Oily-Spaghetti Gradient (Virtual) Phase Gating Partitioning FG Nups in Models for NPC Translocation Modified from Weis, 2003

Nup42-FG Nup159-FG Nup116-FG,GLFG Nup49-GLFG Nsp1-FG,FxFG Nup57-GLFG Nup145N-GLFG Nup2-FxFG Nup60-FxF Nup1-FxFG and 2 ofand 3 of OR Nup100-GLFG Nup49-GLFG Nsp1-FG,FxFG Nup57-GLFG Nup145N-GLFG FG Experimental Strategy for Testing Role of FG Domains in Nuclear Transport: Generating Minimal FG NPCs in Yeast Strawn et al., 2004 Nature Cell Biol. 6:

HeLa cells were fused with Xenopus laevis cells, treated with actinomyosin D and stained for human hnRNP A1(red) and hnRNP C (green). Unfused HeLa cells, arrowhead. Unfused X.l. cells, dotted arrow. Heterokaryon, solid arrow. Experimental Strategy for Analyzing Shuttling (Import & Export Cycles): Heterokaryon Transport Studies

-How do macromolecules move through? -How is the NPC assembled? -How are transport and assembly regulated? General Strategy: Attack at the Site of Entry and Exit

Supplemental Lecture Information

Formation of a pore and NPC in an intact nuclear envelope Intact double membranes Hemi-fusion of inner leaflets Full fusion to form pore

Mitotic NPC Assembly Burke and Ellenberg, 2002, Nat. Rev. INTERPHASE PROPHASE METAPHASE ANAPHASE CYTOKINESIS

Structural Changes in the NPC and the hnRNP are Observed During Export of Balbiani Ring Particles Kiseleva et al, 1996 What mediates or signals these changes?

Survivor’s Guide to Nuclear Transport Nomenclature NUP = nucleoporin NLS = nuclear localization sequence NES = nuclear export sequence RAN = small GTPase (Gsp1) RanGEF = RCC1, Prp20 RanGAP = Rna1 Import and Export factors Importin/Karyopherin/Transportin (cargo) -  and  /Kap95 - Kap60 (cNLS) -transportin 1,Kap  2 (M9-NLS hnRNP A1) Exportin/Karyopherin (example cargo) -exportin/Crm1/Xpo1 (leucine rich-NES) -CAS/Cse1 (alpha) -exportin-t/Los1 (tRNA) -exportin-5/Msn5 (tRNA-eEF1A complex, pre-miRNAs) mRNA export factors (vertebrate/S.cerevisiae) TAP/Mex67 p15, NXT1/Mtr2 RAE1/Gle2 Gle1 Dbp5

Strategies to Reveal Transport Mechanisms -Tissue Culture Cell Microinjection* -Ectopic Expression in Cells and Localization* -Heterokaryons to Analyze Shuttling* Assigned Reading -In vitro Transport Assays Coupled with Biochemical Fractionation -digitonin-permeabilized cells -Two-hybrid Approach -Genomic Approach -searching for RanGTP binding proteins -Genetic Strategies* -Screens for yeast mutants with defective transport