1. Targeting of Proteins to the Organelles

2. Targeting of Proteins: Nucleus and Mitochondria

3. The Nucleus
Central Dogma
Functions

4. Proteins are made in cytoplasm-transported to other locations
Central dogma:
DNA--> RNA--> Protein
Nucleus
Cytoplasm
nucleus
mitochondria
Rough endoplasmic reticulum (ER)
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
Here is an electron micrograph of the NPC.
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. How are proteins targeted to the nucleus?
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

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
K—Lysine
R--Arginine

36. 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

51. Juxtaposition of Inner and Outer Membranes at Transport Sites

52. Mitochondrial Protein Transport
Cytosolic Hsc70 keeps precursor Protein in partially unfolded state
Tom20/22 Receptor recognized matrix-targeting sequence
Transfer of Precursor through Tom40 channel
Passes through Tim23/17 channel
Matrix Hsc70 helps pull precursor through channel
Matrix protease cleaves off signal sequence

53. Mitochondrial Transport Movie

54. Transport of Mitochondrial Membrane Proteins

55. Stop-Transfer Players Tom20/22 Tom40 Tim23/17 Tim44 Hsc70
Matrix Protease

56. Stop-Transfer Process same as with Matrix-targeted protein
Have Matrix targeting sequence
BUT
Internal Stop-Transfer sequence recognized by Tim23/17 channel
Sequence is hydrophobic
Precursor is transferred into the inner membrane bilayer

57. Path B
Proteins contain a matrix targeting sequence and internal hydrophobic domains
Oxa1
Tom40, Tim23/17 are involved in transport

58. Path C Proteins do not contain a matrix targeting sequence
Recognized by the Tom70/22 complex

59. Intermembrane Space Localization

60. Path A Major Pathway Proteins contain a matrix targeting sequence
Translocation to the matrix begins

61. Path B
Some proteins can just move through Tom40

62. Outermembrane proteins
Like Tom40 itself
Proteins interact with Tom40
Then transferred to the SAM complex (sorting and assembly machinery)

63. Section assignments for next week
Undergraduate
Read article on Ran in Nuclear Transport
Graduate
Read article on Tubular ER network

64. Problem Set 1 Undergraduates Problem Set 1 will be posted on Friday
Due Feb 28th by