oocyte injection assay protein source : in vitro translation
TRANSCRIPT
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Oocyte injection assay
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Protein source : In vitro translation
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Protein source : Classical protein purification
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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
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Active and passive transport of proteins in nucleus
39 nm
9nm
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Proteins move through pores
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Bidirectional transport ?tRNA
Nuclear protein
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Discovery of first nuclear localisation sequence
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To confirm that a stretch of sequence is necessary and sufficient to guide a protein
to its desired location
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Redirecting a cytoplasmic protein to nucleus
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Conclusions
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Nuclear localisation sequence (NLS) in nucleoplasmin
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NLS
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Digitonin permealisation assay for studying the mechanism of protein transport to nucleus
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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
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Nuclear Import Receptors (IMPORTINS) bind to Nuclear Localization Signals
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FG nucleoporins interact with importins
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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
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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.
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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
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The Ran-GTP/GDP cycle
The GTP binding protein Ran regulates Nuclear Transport
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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.
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PROTEIN EXPORT from nucleus
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Protein export from nucleus Principle of assay
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The GTP binding protein Ran regulates Nuclear Transport
IMPORT EXPORT
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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
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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