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. Gated transport Transmembrane transport Vesicular transport Transport is highly regulated

10. Signal sequence and signal patch

13. 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”

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

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

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

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

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

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

21. Nuclear localization of TXN factors control gene expression

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

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

25. The subcompartments of mitochondria and chloroplasts

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

27. Translocases of the outer and inner mito membranes

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

29. Protein import by mitochondria

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

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

32. Proteins destined for various mito space

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

34. Urate oxidase Peroxisomes have one single membrane No DNA or ribosomes Catalase and urate oxidase Oxidative reactions not taken over by mito RH 2 +O 2 ->R+H 2 O 2 H 2 O 2 +R’H 2 ->R’+2H 2 O Catalase Urate oxidate (R=uric acid)  -oxidation biosynthesis of plasmalogens photorespiration glyoxylate cycle

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

36. Summary 1.Cells are highly compartmentalized; proteins are sorted to different compartments; 2.Nuclear transport, nuclear pore, nucleoporins, NLS; 3.Ran GTPase control direction; 4.Nuclear lamina, nuclear lamins; 5.Mitochondria transport, signal sequence, TOM, TIM, energy; 6.Chloroplast transport, thylakoid; 7.Peroxisomes, structure, function, transport, biogenesis.