oocyte injection assay protein source : in vitro translation

Oocyte injection assay

Protein source : In vitro translation

Protein source : Classical protein purification

Target In vitro translation

Classical protein

purification

Nucleus ok ok

Post-translational Import in oocyte injection assay

Nuclear localised proteins do not lose their address tag upon localisation

Active and passive transport of proteins in nucleus

39 nm

9nm

Proteins move through pores

Bidirectional transport ?tRNA

Nuclear protein

Discovery of first nuclear localisation sequence

To confirm that a stretch of sequence is necessary and sufficient to guide a protein

to its desired location

Redirecting a cytoplasmic protein to nucleus

Conclusions

Nuclear localisation sequence (NLS) in nucleoplasmin

NLS

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

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

Nuclear Import Receptors (IMPORTINS) bind to Nuclear Localization Signals

FG nucleoporins interact with importins

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

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.

MonomericGTPases

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.

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

The Ran-GTP/GDP cycle

The GTP binding protein Ran regulates Nuclear Transport

Import process1. 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.

PROTEIN EXPORT from nucleus

Protein export from nucleus Principle of assay

The GTP binding protein Ran regulates Nuclear Transport

IMPORT EXPORT

The activity of many transcription factors is regulated by control of Nuclear Import and Export

Stimulation of the T-Cell

The Transcription factorNF-AT and T-Cell Activation

The activity of many transcription factors is regulated by control of Nuclear Import and Export

The Transcription factorNF-AT and T-Cell Activation

ImmunosuppressantDrugs, eg FK506