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Cytoplasmic Membrane Systems I Lecture 11

Cytoplasmic Membrane Systems I Lecture 11. The Cellular Compartmentalization Problem of Eukaryotic Cells. Cytoplasm: Cytosol plus Organelles Excluding Nucleus. How Do Proteins Get Imported Into Membrane Enclosed Organelles? . Import Requires Input of Energy to Occur!.

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Cytoplasmic Membrane Systems I Lecture 11

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  1. Cytoplasmic Membrane Systems ILecture 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 NucleusWhy 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. Distinctive Features: Nuclear Envelope Nuclear Lamina Chromatin Organization Subcompartments that lack Membranes, including Nucleoli Nuclear Pore Complexes The Eukaryotic Nucleus

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

  8. 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 The Nuclear Lamina Underlies the Nuclear Envelope and Provides Structural Support Nuclear Lamina Chromatin

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

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

  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 Speckles Nucleoli (and Cajal Bodies) Cajal Bodies Bulk Chromatin

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

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

  16. Cytoplasmic side EM view of Xenopus Nuclear Pores 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 Active Transport: Translocation Diffusion ribosomal Proteins snRNPs Small Molecules And Proteins up to 40 kDA nuclear proteins Small uncharged molecules tRNAs mRNPs snRNAs miRNAs ribosomal subunits Nucleus

  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 120 nm 9-10 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 • 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 • 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 IMPORT EXPORT 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 Cytosol 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

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