4. Oocyte injection assay

5. Protein source : In vitro translation

6. Protein source : Classical protein purification

7. Post-translational Import in oocyte injection assay
Target
In vitro
translation
Classical
protein purification
Nucleus ok
Nuclear localised proteins
do not lose their address tag upon localisation

9. Active and passive transport of proteins in nucleus
39 nm
9nm

10. Proteins move through pores

11. Bidirectional transport ?
tRNA
Nuclear protein

12. Discovery of first nuclear localisation sequence

13. To confirm that a stretch of sequence is
necessary and sufficient to guide a protein
to its desired location

14. Redirecting a cytoplasmic protein to nucleus

15. Conclusions

16. Nuclear localisation sequence (NLS) in nucleoplasmin

18. NLS

20. Digitonin permealisation assay for studying
the mechanism of protein transport to nucleus

23. Ras related nuclear factor Ran
The nuclear import players:
Nuclear import receptor binds the cargo.
NLS is in the amino acid sequence of the cargo protein.
Ran-GTP and Ran-GDP are different forms of Ran bound either to GTP or GDP. Ran-GTP causes the NLS to dissociate from the Nuclear import receptor.
Ran-GAP (in cytoplasm ) is distinct from Ran but causes Ran to hydrolyze GTP. Hence, Ran-GAP promotes the conversion of Ran-GTP to Ran-GDP.
Ran-GEF (in nucleus) is distinct from Ran but causes Ran to release GDP and bind a different molecule of GTP. Hence, Ran-GEF promotes the conversion of Ran-GDP to Ran-GTP.
Ras related nuclear factor Ran

24. Nuclear Import Receptors (IMPORTINS)
bind to Nuclear Localization Signals

25. interact with importins
FG nucleoporins
interact with importins

28. Ras related nuclear factor Ran
The nuclear import players:
Nuclear import receptor binds the cargo.
NLS is in the amino acid sequence of the cargo protein.
Ran-GTP and Ran-GDP are different forms of Ran bound either to GTP or GDP. Ran-GTP causes the NLS to dissociate from the Nuclear import receptor.
Ran-GAP (in cytoplasm ) is distinct from Ran but causes Ran to hydrolyze GTP. Hence, Ran-GAP promotes the conversion of Ran-GTP to Ran-GDP.
Ran-GEF (in nucleus) is distinct from Ran but causes Ran to release GDP and bind a different molecule of GTP. Hence, Ran-GEF promotes the conversion of Ran-GDP to Ran-GTP.
Ras related nuclear factor Ran

29. Monomeric
GTPases
In the cell, this cycle only runs counter-clockwise because the cell maintains a high GTP/GDP ratio. The rate of the cycle is controlled by GAP and GEF because the intrinsic GTPase activity and rate of nucleotide exchange for the monomeric GTPase are slow. GAP and GEF lower the activation energies of these processes.
Modified from Fig. 3-72
Monomeric GTPases are a family of proteins that exist in a GDP-bound state or a GTP-bound state. These two states have different activities.
The transition from the GTP state to the GDP state involves hydrolysis of the GTP and the rate of hydrolysis is accelerated by a GTPase-activating protein (GAP).
The transition from the GDP state to the GTP state is accelerated by a Guanine nucleotide exchange factor (GEF).
Note that one complete cycle of changes hydrolyzes GTP. This hydrolysis of GTP is used to power cellular functions.

30. Ras related nuclear factor Ran
The nuclear import players:
Nuclear import receptor binds the cargo.
NLS is in the amino acid sequence of the cargo protein.
Ran-GTP and Ran-GDP are different forms of Ran bound either to GTP or GDP. Ran-GTP causes the NLS to dissociate from the Nuclear import receptor.
Ran-GAP (in cytoplasm ) is distinct from Ran but causes Ran to hydrolyze GTP. Hence, Ran-GAP promotes the conversion of Ran-GTP to Ran-GDP.
Ran-GEF (in nucleus) is distinct from Ran but causes Ran to release GDP and bind a different molecule of GTP. Hence, Ran-GEF promotes the conversion of Ran-GDP to Ran-GTP.
Ras related nuclear factor Ran

31. The GTP binding protein Ran regulates Nuclear Transport
The Ran-GTP/GDP cycle

32. Import process
1. Nuclear import receptor associates with cargo and brings the cargo to the nuclear pore.
2. Somehow the receptor/cargo complex moves through the pore.
3. Once in the nucleus, Ran-GTP displaces the cargo from the receptor. Ran-GTP is present in the nucleus because of the Ran-GEF.
4. Receptor/Ran-GTP exits the nucleus through the pore.
5. Once the receptor returns to the cytosol, Ran-GAP induces Ran to hydrolyze its bound GTP.
6. Ran-GDP dissociates from the receptor.

33. PROTEIN EXPORT
from nucleus

34. Protein export from nucleus Principle of assay

35. The GTP binding protein Ran regulates Nuclear Transport
EXPORT
IMPORT

36. The activity of many transcription factors is regulated by
control of Nuclear Import and Export
Stimulation of
the T-Cell
The Transcription factor
NF-AT and
T-Cell Activation

37. The activity of many transcription factors is regulated by
control of Nuclear Import and Export
Immunosuppressant
Drugs, eg FK506
The Transcription factor
NF-AT and
T-Cell Activation