Targeting of Proteins to the Organelles

1. Targeting of Proteins to the Organelles

2. Targeting of Proteins: Nucleus and Mitochondria

3. The Nucleus • Central Dogma • Functions

4. Nucleus Cytoplasm nucleus mitochondria Rough endoplasmic reticulum (ER) Proteins are made in cytoplasm-transported to other locations Central dogma: DNA--> RNA--> Protein Fig 13.1 animation

5. The Nucleus Structures Nuclear Envelope Nuclear Lamina Nuclear Matrix Chromosomal Domains Nuclear Pore Complex Houses DNA Organized Regulated movements

6. The Nucleus • Nucleus holds DNA • Keeps DNA organized throughout the cell life cycle • The nucleus is organized similar to a mini-cell within a cell

7. The Nucleus: DNA organization • Chromatin • Discrete DNA localization

8. The Nucleus: Nuclear Lamina • Supportive mesh on the inside of the nuclear envelope • Nuclear Matrix • Provides a structured space

9. The Nucleus • The Nuclear Envelope is a continuous membrane that forms a double bilayer • Outer: • Inner:

10. The Nucleus: Nuclear Pore Complex • How do molecules get into the nucleus? • Nuclear Pore complex

11. Nuclear Pore Complex Cytoplasmic Nucleoplasm Regulates movement of proteins between cytoplasm and nucleus

12. The Nucleus: Nuclear Pore Complex • The NPC is one of the largest protein complexes in the cell • Composed of: • Limited diffusion: • Membrane bound:

13. The Nucleus: Nuclear Pore Complex • Basket portion on the nucleoplasm side is joined by filaments

14. The Nucleus: Nuclear Pore Complex • Center of the nuclear pore is aqueous • Imported proteins move through the NPC via a brownian gate model

15. The Nucleus: Nuclear Pore Complex • Some channel and basket nucleoporins form hydrophobic stretches • Structural Nups:

16. NPC Structure Cytoplasmic Nups Symmetric Nups Nuclear Nups

17. Nuclear Import and Export • Many proteins are too large to pass through the NPC alone • How does this occur?

18. Nuclear Import PLAYERS Cargo Proteins Import Proteins Ran Ran-GEF Ran-GAP

19. Nuclear Import • Importins • Exportins • FG repeats

20. Nuclear Import • Importins can form a heterodimeric nuclear import receptor • Ran

21. Nuclear Import • Free Importin binds to NLS in the cytoplasm

22. Nuclear Import • Upon entering the nucleus, importin interacts with Ran-GTP

23. Nuclear Import • Ran-GTP/Importin complex then diffuses out of the nucleus • Ran-GAP

24. Nuclear Import • Ran GDP returns to nucleus through NPC • Ran-GEF

25. Ran: GTPase in Nuclear Transport G protein switches Two confirmations GTP-bound GDP-bound GTPase hydrolyzes GTP to GDP; slow enzyme Modifying Proteins Guanine nucleotide exchange factors (GEF) GDP GTP Nuclear GTPase Activating Protein (GAP) ***this protein is not a GTPase!! GTP GDP Cytoplasmic

26. Nuclear Import • Diffusion through the pores is random, but transport is directional • How is this achieved?

27. Nuclear Export • Exportin binds to cargo

28. Nuclear Export • Ran-GTP makes contact with Ran-GAP in cytoplasm • Complex dissociates

29. Nuclear Import and Export • Both use Ran-GTP • Import: • Export:

30. Nuclear Import

31. Nuclear Transport Proteins Imported Proteins DNA replication DNA repair Transcription Unassembled ribosome Exported Proteins mRNA (bound to proteins) tRNA (bound to proteins) Assembled ribosome How are proteins targeted to the nucleus?

32. Signal Sequence: Cellular Address Barack Obama 1600 Pennsylvania Avenue Washington, D.C. 20003

33. Signal Sequences NLS

34. Signal Sequences • Signal sequences direct the final protein destination • Chemical properties of the amino acids in the sequence direct interactions

35. Signal Sequences • These sequences are read by specific transport receptors

36. Nuclear Localization Signal • Nuclear localization signal • Pro-Lys-Lys-Lys-Arg-Lys-Val • Digitonin

37. Nuclear Export Signal • Used to transport proteins, tRNA, ribosomal subunits out of the nucleus • Exportins bind the NES to start the process

38. Mitochondria Power house of cell • Outer membrane • simple bilayer • permeable to ions and small molecules • Inner membrane • IMPERMEABLE to all material except through carrier channels • Complex in conformation • Contains all electron transport chain machinery • Contains all ATP synthesis machinery

39. Mitochondria Power house of cell • Intermembrane space • Complex shape (follows contours of inner membrane • pH ~7 • Matrix • all enzymes required for Krebs cycle • Hold mitochondrial genome, ribosomes, enzymes for mitochondrial division • pH ~8

40. Mitochondria • Mitochondria harness energy • Glycolysis (pyruvate) • Generate ATP

41. Mitochondria • cellular respiration • “Breathing” on a cellular level • Occurs in the inner membrane and the inner membrane space • membrane surface area

42. Electron Transport Chain

43. Mitochondria • Matrix contains all necessary components for mitochondrial replication • Mix of mitochondrial encoded proteins and those from the nuclear genome

44. Signal Sequences

45. Mitochondrial targeting sequences • The signal for matrix mitochondrial proteins is part sequence, but also part structure • Amphipathic • Receptors that bind the sequences can bind to more than one specific sequence

46. Mitochondrial Protein Transport • Transport of proteins in to the mitochondria requires: • Transport occurs:

47. Mitochondrial Protein Transport Players Hsc70 Chaperones Outer membrane Translocon Outer membrane receptors- Tom 20/22 or Tom 70/22 Outer membrane channel- Tom 40 Inner membrane Translocon Inner membrane channel- Tim 23/17 Matrix Proteases

48. Mitochondrial Protein Transport • Chaperones (HSC 70) keep proteins unfolded

49. Mitochondrial Protein Transport • Precursor binds to the import receptor on the outer membrane • Import receptors: Tom 20/22

50. Mitochondrial Protein Transport • Transport to the matrix occurs simultaneously • Tim23/17 mediate transport to the matrix