Nuclear Flashcards
Describe the structure of nuclear pores
What are the characteristics of nuclear localisation signals?
1-2 stretches of basic residues.
How did they work out what was required for nuclear import?
A receptor: Was saturatable, when they attached an NLS to albumin, other nuclear localised things couldnt get in cuz was busy w the albumin
Cytosolic factors: They removed they cytosol and replaced with buffer and it didn’t work, replace the buffer with cytosol and it did work
They then fractionated the cytosol and found the components which allow for successful NLS.
Requires 4 soluble factors:
* Imp alpha
* Imp beta
* Ran
* NTF2
What are the two steps to nuclear import?
1) Docking
* Docking, requires energy
* No protein going into nuclei yet
2) Translocation,
* energy, Ran and NTF2 dependent
* Energy in form of GTP
Describe the role of RCC1 and RanGAP1 in the Ran GTPase cycle.
Ran is a GTPase. RCC1 and RanGAP1 control whether RAN is GDP or GTP
- As usual, exists in two confirmations, GTP and GDP
- Not great at hydrolysing GTP on their own
- Needs assistance from another protein
- Also pretty bad at exchanging GTP to GDP
- Needs help from another protein to release the Pi
RCC1 (GEF)
* Allows GDP to be release
* More likely to come back as GTP, as is much higher concentration in Ran
RanGAP1
* Release the phosphate
How is a RanGTP gradietn created across the membrane?
- The GEF and GAPS controlling if RanGTP/GDP are localised
- RCC1 bound to chromatin in the nucleus
- RanGAP1 is cytosolic
- Means the nucleus is gonna be high concentration of Ran GTP
- And high concentration of Ran GDP in the cytosol
Explain the process of nuclear import
- Cytosol:
- Impbeta allows the cargo complex to interact with the pore. It wont fully move through the pore until it can interact with RanGTP
- When it moves through nuclear pore, comes into contact with Ran GTP, this binds to ImpB, causing it to disociate.
- The Imp alpha then disscoiates from the cargo because of this
- Imp beta remains bound to the RanGTP, taking it back to the cytosol
- In the cytosol, RanGAP causes hydroloysis of Ran GTP, and this RanGDP then dissociates from Impbeta.
- Impbeta then binds to imp alpha and the cargo
How does nuclear export work?
- Exportin binds only to RanGTP
- Bound exportin is required to load up cargo, so binds cargo and rangtp
- In the cytoplasm, where the RanGTP hydrolyses into Ran GDP, exportin dissociates and therefore cargo does
How to does CAS enable nuclear export of impalpha? (which allows the cycle to continue)
CAS binds to RanGTP which allows imp alpha to bind to CAS. CAS then can take the complex through the pore.
Ran GTP is then hydrolysed into GDP causing the complex to dissociate
Describe a nucleaser export signal.
Nuclear Export Signal
* Can regulate amount of import and export at any one time
* Rich in Leucine residues, small and hydrophobic
How do exon junction complex prevent exported of non spliced RNA
- When an intron is removed
- Exon junction complex is left at site where intron was removed
- Marker that the sequence has had its intron removed
- Mex67 / Tap able to bind the complex
- Provides a method to prevent export of non splice RNA
How does RanGDP return to nucleus?
Binds to NTF2 which gets it back in.
- NTF2 can interact with nuclea pore complex, so can bring RanGDP back
- RCC1 stimulates exchange
Generating Ran GTP
how is RAN used in mitotic spindle formation
- Localised enrichment of Ran GTP around chromatin as that is where the rcc1 is found.
- This localised ran gtp drives spindle formation
- Ran GDP means TPX2 is bound and inactive, when it turns to GTP around chromatin cuz of rcc1, TPX2 is release
- TPX2 causes microtubule formation
Why do mRNA localised themselves?
- Can localise mRNA to one side of the cell to create an asymmetric devision, whereby one has the mRNA or transcription factor and one doesnt, diverging the two cells along different differentiation pathways
- Generate cell polarity, basal and apical membrane differences
- Co tranlsational interactions, for when it is needed at the place it is made
- Localised responses
- Synthesising proteins from mRNA at the location, the protein is needed
- To specifically localise themselves (viruses) to places in the cell
How is mRNA localised?
How is it localised
3 major ways:
* Made in the nucleus and then exported: 1) Cytoskeleton as tramlines to deliver to the site via motor proteins 2) Random diffusion around cell, then trapping at the required localisation 3) Random diffusion/generation, then all mRNA begins to degrade apart fromt he mRNA attached to the desired location
