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Lecture 6 Intracellular Compartments and Protein Sorting

Lecture 6 Intracellular Compartments and Protein Sorting. Membrane-enclosed compartments. Proteins: enzymes, transporter and surface markers. 10,000-20,000 proteins delivered to different compartments. Major intracellular compartments.

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Lecture 6 Intracellular Compartments and Protein Sorting

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  1. Lecture 6 • Intracellular Compartments • and Protein Sorting

  2. Membrane-enclosed compartments Proteins: enzymes, transporter and surface markers 10,000-20,000 proteins delivered to different compartments

  3. Major intracellular compartments Vital chemical reactions take place in or on membrane surface Compartments increase surface and isolate reactions

  4. Microtubules help the localization of the ER and the Golgi apparatus Bacteria have no Internal membranes Eukaryotic cells are 1000-10,000 times greater--need internal membranes

  5. Development of plastids

  6. topology

  7. Topologically equivalent spaces are shown in red

  8. Transport is highly regulated Gated transport Transmembrane transport Vesicular transport

  9. Signal sequence and signal patch

  10. 50 nucleoporins Octagonal Variable numbers of pores (3000-4000) depending on TXN 100 histone molecules per minute per pore 6 large and small ribosomal subunits per minute per pore SEM “Basket”

  11. Ribosome 30 nm DNA, RNA polymerases 100-200 Kd subunits Results from injection: <5000 Daltons: fast diffusion 17 Kd: 2 minutes >60 Kd: cannot enter Channel is 9 nm in diameter 15 nm long

  12. An experiment using recombinant DNA technique One or two short sequences Rich in positively charged aa Lys, Arg Immunofluorescence micrographs showing T-antigen localization

  13. Gold particles coated with nuclear localization signals Not through lipid bilayer Folded confomration Pore dilates to 26 nm Nuclear import Receptors!!! Bind to nucleoporins FG-repeats

  14. Nuclear export signals Nuclear export receptors Nuclear transport receptors (karyopherins) A single pore complex conducts traffic in both directions

  15. The Ran GTPase drives directional transport Ran is required for both import and export Asymmetical Localizatin of GAP And GEF! GTPases are molecular switches GTPase-activating Protein (GAP) Guanine exchange Factor (GEF)

  16. Ran-GTP causes cargo binding of export receptor Ran-GTP causes cargo release of import receptor Free export receptors return to the nucleus GTP-bound import receptors return to the cytosol

  17. Nuclear localization of TXN factors control gene expression

  18. The nuclear lamina Meshwork of interconnected protein subunits, nuclear lamins Intermediate filament proteins, interact with nuclear pore complexes and integral membrane proteins, chromatin

  19. NLS is not cleaved off after transport--repeated import

  20. The subcompartments of mitochondria and chloroplasts

  21. Mitochondrial proteins are first fully synthesized: different from proteins transported into ER Signal sequence: Amphipathic a helix Signal peptidase

  22. Translocases of the outer and inner mito membranes

  23. Proteins transiently spanning the inner and outer membranes during their translocation into the matrix Precursor proteins remain unfolded before transport

  24. Protein import by mitochondria

  25. ATP hydrolyses at two sites plus a H+ gradient across inner membrane Release from cytosolic hsp 70 Further translocation through TIM requires H+ gradient Release from mito hsp70 Signal peptide is positively charged

  26. Two models of how mito hsp70 could drive protein transport

  27. Proteins destined for various mito space

  28. Two signal sequences are required for proteins directed to the thylakoid membrane in chloroplasts GTP and ATP Signal sequences for mito and chloroplasts are different Four routes into the thylakoid space

  29. Peroxisomes have one single membrane No DNA or ribosomes Catalase and urate oxidase Urate oxidase Oxidative reactions not taken over by mito RH2+O2->R+H2O2 Urate oxidate (R=uric acid) H2O2+R’H2->R’+2H2O Catalase b-oxidation biosynthesis of plasmalogens photorespiration glyoxylate cycle

  30. A model of how new peroxisomes are produced From preexisting peroxisomes Transport mechanisms unknown: no unfolding necessary growth 23 peroxins Similar to nuclear transport fission

  31. Summary Cells are highly compartmentalized; proteins are sorted to different compartments; Nuclear transport, nuclear pore, nucleoporins, NLS; Ran GTPase control direction; Nuclear lamina, nuclear lamins; Mitochondria transport, signal sequence, TOM, TIM, energy; Chloroplast transport, thylakoid; Peroxisomes, structure, function, transport, biogenesis.

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