1. Cytoplasmic Membrane Systems I Lecture 11

2. The Cellular Compartmentalization Problem of Eukaryotic Cells Cytoplasm: Cytosol plus Organelles Excluding Nucleus

3. How Do Proteins Get Imported Into Membrane Enclosed Organelles? Import Requires Input of Energy to Occur!

4. Pathways for Protein Trafficking Default Localization (Remain in Cytosol) Signal Mediated Localization –Gated Transport –Transmembrane Transport –Vesicular Transport Secretory Pathway

5. The Eukaryotic Nucleus Why Have One? Separation of mRNA synthesis/processing from translation- Allows for greater regulation of gene expression Separate Fragile Chromosomes from Cytoplasmic Cytoskeletal Filaments Involved in Cell Movement

6. The Eukaryotic Nucleus Distinctive Features: Nuclear Envelope Nuclear Lamina Chromatin Organization Subcompartments that lack Membranes, including Nucleoli Nuclear Pore Complexes

7. The Nucleus is Bound by the Nuclear Envelope Double MembraneLumen Contiguous With ER Lumen Nuclear Pore Complexes The Only Channels for Transport Between Nucleus and Cytoplasm

8. The Nuclear Lamina Underlies the Nuclear Envelope and Provides Structural Support Chromatin Nuclear Lamina Composed of Intermediate Filaments called Lamins Located Beneath the Inner Membrane and Physically Connected by: -Integral Lamina Associated Proteins -Lipid Anchors on Lamins Also Associated with Chromatin via proteins - May be important in organizing Replication and Transcription

9. Chromosomes Occupy Distinct Areas of The Nucleus Called Chromosome Territories Chromosomal Territory Interchromosomal Domain

10. Nucleoli are Sites of RNA synthesis and Processing rRNA tRNA Additional RNA

11. Assembly of Ribosomal Subunits Occurs in the Nucleolus

12. rRNA Synthesis and Processing and Ribosomal Subunit Assembly Occur in Nucleoli

13. Speckles are Sites of snRNP Storage Nucleoli (and Cajal Bodies) Speckles Bulk Chromatin Cajal Bodies

14. Nuclear Pore not simple “holes” in the membrane Selective Gateways for two-way traffic

15. Nucleus Nucleolus Nuclear envelope: Inner membrane Outer membrane Nuclear pore Pore complex Surface of nuclear envelope. Pore complexes (TEM). Nuclear lamina (TEM). Close-up of nuclear envelope Ribosome 1 µm 0.25 µm Nuclear Pore Complexes are Embedded in the Nuclear Membrane

16. EM view of Xenopus Nuclear Pores Cytoplasmic side Nuclear (Basket) Cytoplasmic

17. Large Molecules are Actively Transported Between Nucleus and Cytoplasm Active Transport through the Nuclear Pore Complex has the following Features: Energy Dependent Signal Dependent Temperature Dependent Can Be Saturated These are Features of a Carrier Mediated Process

18. Large Molecules are Actively Transported Between Nucleus and Cytoplasm Nucleus ribosomal Proteins snRNPs ribosomal subunits tRNAs mRNPs snRNAs miRNAs nuclear proteins Small Molecules And Proteins up to 40 kDA Diffusion Active Transport: Translocation Small uncharged molecules

19. Nuclear Pores Are Busy! A Single Human Cell contains 10 million Ribosomes, has ~4000 Nuclear Pores and divides every 24 hours Needs 400, 000 ribosomal proteins imported every minute (~100/pore) Needs 12, 000 Ribosomal Subunits Exported every minute (~3 per pore) If DNA is Synthesized- needs 1 million new Histone Proteins every 3 minutes

20. Nuclear Pores Contain a Open Channel to Allow for Small Molecules to Diffuse 9-10 nm 120 nm

21. Nuclear Pore Complexes are Symmetrical Structures Located Where Inner and Outer Membranes are Fused Composed of 30-50 Different Pore Proteins called Nucleoporins Present in Multiple Copies

22. Proteins Are Selectively Transported through Nuclear Pores 1)Signal Dependent NLS- Nuclear Localization Signal –Basic Amino Acids NES- Nuclear Export Signal - Leucine Rich Sequence 2) Receptor Mediated Process Involves Receptor Transport Proteins that Recognize NLS or NES of Cargo Proteins Nuclear Transport Receptors are often part of the large family of Karyopherin proteins Importins- Involved in Nuclear Import Exportins- Involved in Nuclear Export 3) Regulated by G-protein RAN

23. Nuclear Localization Signals are Necessary and Sufficient for Nuclear Import General Characteristics but No Elaborate Sequence Requirements Classical NLS Sequences 1. Short Stretch of Basic Amino Acids Lysine and Arginine Rich 4-8 residues Best Characterized: NLS of SV 40 T-Antigen PPKKKRKV 2. Bipartite NLS sequences Two stretches of Basic Amino Acids about 10 aa Apart Example: NLS of nucleoplasm protein KRPAATKKAGQAKKKK

24. Mutation of the SV-40 T-Antigen NLS Abolishes Nuclear Import

25. Cytoplasmic Nuclear Transport Receptors Mediate Nuclear Protein Import (Importin)

26. Karyopherins Transiently Bind the Nucleoporins That Line the Route through the Nuclear Pore Nup358 Nup214 Nup62 Nup98 Nup153

27. Movement of Proteins through NPC is regulated by the G-protein RAN

28. Distribution of RAN-GTP Across the Nuclear Envelope GAP: Stimulates RAN GTP Hydrolysis GEF: Stimulates Exchange of GDP for GTP by RAN

29. Nuclear Import: Dissociation of Importin-Cargo Complexes Requires Ran-GTP binding 1.Recognition of NLS of Cargo Protein by Importin (Nuclear Transport Receptor) 2. Importin-Cargo Complex binds specific nucleoporin proteins of cytoplasmic filaments 3. Complex translocated 4. Dissociation of Complex at nuclear side requires RAN-GTP binding

30. Nuclear Export: The Assembly of Exportin-Cargo Complexes Requires RAN-GTP IMPORTEXPORT Cytosol Nucleus 1.Protein with Nuclear Export Signal (NES) Bound by Export Receptor (Exportin) And RAN-GTP 2. Complex Associates with Nucleoporins And Translocates to Cytoplasm 3. Once in Cytosol Exportin-Cargo Complex Dissociates Ran-GTP hydrolysis  RAN-GDP 4. Exportin recycled back to Nucleus

31. Why Aren’t Nuclear Localization Signals Removed? Nuclear Proteins Undergo Repeated Nuclear Entry Some proteins shuttle between nucleus and cytoplasm - Nuclear Proteins are capable of repeated entry into the nucleus When mitosis occurs, the nuclear envelope breaks down- cytosolic and nuclear proteins mix- when it is reformed- nuclear proteins need to be reimported

32. Multiple Classes of RNA are Exported from the Nucleus The Same NPCs used for protein transport are used for RNA export Transport of most RNA is unidirectional from nucleus to cytoplasm Export of RNA is receptor mediated and energy dependent. Different soluble transport factors are required for transport of each class of RNA

33. mRNA Are Exported from the Nucleus as RNA–Protein Complexes Most mRNP require TAP/MEX67 Functions like Exportin

34. Regulated Nuclear Export of Unspliced RNA by the HIV Rev Protein How does HIV Assemble Progeny Virus Containing A Single Stranded Unspliced RNA Genome?

35. Transport of Various RNAs and RNPs Ribosomes –Need to Import Ribosomal Proteins –Need to Export Ribosomal Subunits –Involves Karyopherin family of Receptor proteins –RAN dependent tRNA –Dedicated transport receptor- Exportin-t- only one binds RNA directly –Requires RAN –Aminoacylated and fully processed tRNA are the preferential substrate for Exportin-t microRNA –Dedicated transport receptor Exportin- V –Requires RAN snRNA- –Exported to Cytoplasm to form snRNPS- then imported by Snurportin and karyopherin family members