Protein Targeting Flashcards
What are target sequences?
- a.a sequences that act as address labels
- directs proteins to correct destination in a cell
- e.g. KDEL sequences
What is the KDEL sequence?
Lys-Asp-Glu-Leu
What does the nuclear pore consist of?
Scaffold Nucleoporins Channel nucleoporin Disordered region - line the pore, portions extend into the pore space Nuclear Basket - extends into the nuclear space Cytosolic fibrils - extend to the cytosol
What are nucleoporins?
proteins that make up the nuclear pore
What are importins?
Importins are a type of karyopherin that transports protein molecules into the nucleus by binding to specific recognition sequences (nuclear localisation sequences)
What are the two classes of importins?
Importin-α and importin-β
How can the importins be recognised / carry different cargo proteins?
Each have a slightly different import sequences so will be recognised by different cargo proteins
What are the two structural ways importin-β can function?
- can function as a monomer
- heterodimer with importin-α
- importin-α acts as an adaptor protein
- importin-α is bound to the cargo, and importin-β is bound to importin-α
What is an example of a importin-α cargo?
cellular cargo: BRCA1
viral cargo: HIV-1 integrase
What sort of repeats do nucleoporins contain?
FG (Phe-Gly) repeats
- repeats line the channel of the nuclear pore complex
How are importin-β’s thought to transport their cargo through the nucleus?
They transport through the nuclear pore via weak transient interactions with FG repeats
What is nuclear import and export controlled by?
Ran GTPases
What is the Ran cycle?
Cycle between the two states:
- The switching on of Ran involves the displacement of GDP with GTP
- Conformation change causes the released of GDP
- Hydrolysis of GTP make it into GDP
- Turns it from its ON state into OFF state
What protein and exchange factor is needed during the Ran cycle? Inc locations.
Guanine Nucleotide exchange factor (GEF) needed to replace GDP with GTP - turning it ON
- located in the nucleus, bound to chromatin
- Ran-GEF
GTPase Accessory protein (GAP) helps the process of turning it OFF, converting GTP to GDP
- located in the cytosol
- Ran-GAP
What is the process during the importing of cargo into the nucleus?
- Protein with NLS / cargo is binding to an importin
- Trafficked into the nucleus
- Ran-GTP (active site) binds to the importin
- Will trigger a conformation change - Cargo released into the nucleus
- Importin bound to Ran-GTP is imported back out of the nucleus into cytosol
- Ran-GAP switches off Ran (converts Ran-GTP to Ran-GDP)
- Hydrolysis of GTP
- Ran can no longer take part in importing and releases the importin
What state is Ran when bound to GTP and where in the cell would it be located?
ON state
- nucleus
What is the process during the exporting of cargo out of the nucleus?
- Cargo that needs to be exported has an exporting signal
- Recognised by the Exportin protein
- Bound to Ran-GTP
- Complex transported out of nucleus into the cytosol
- After exiting the complex, Ran-GTP converted into Ran-GDP
- Exportin released as conformational change has occurred
What transcription factor controls expression of the genes involved in T cell activation?
NF-AT
Where is NF-AT found in its inactive state?
NF-AT is found in the cytosol
How is NF-AT expression controlled in T cells?
- Exposure of T cells to antigen results in a rise in intracellular Ca
- When there is high intracellular [Ca], calcineurin binds to NF-AT masking a nuclear export signal
- calcineruin also dephosphorylates NF-AT, exposing a nuclear import signal - NF-AT is then imported into the nucleus
- Activation of gene transcription
- Once T-cell is a resting T-cell this will lead to low Ca conc
- calcineurin no longer binds to NF-AT exposing the nuclear export signal - NF-AT is phosphorylated once more and exported from the nucleus, switching off gene transcription
What are cyclosporin A and FK506?
Cyclosporin A & FK06:
immunosuppressant drugs taken for rheumatoid arthritis, psoriasis, and in organ transplants to prevent rejection.
How does cyclosporin A and FK506 work?
Inhibition of the action of calcineurin. It forms a complex to block the phosphatase activity of calcineurin, resulting in a failure to activate the genes regulated by the NF-AT transcription factor. These genes include those required for B-cell help such as interleukin.
What happens during high cholesterol levels to stop transcription of more cholesterol?
SREBP = transcription factor
- Needs to be moved into the nucleus
High levels of cholesterol:
- SREBP is found in the cytosol associated with SCAP
- SCAP is binds to cholesterol
- This retains SREBP in the ER membrane
- Remains inactive (cannot function as transcription factor)
What happens during low cholesterol levels to encourage transcription of more cholesterol?
Cholesterol wont bind to SCAP
- When cholesterol not bound, SCAP has a conformational change
- Changes into a vesicle and enters the Golgi membrane
- Encounters 2 proteases
- They both cleave SREBP
- releases cytoplasmic domain of SREBP
- also releases a release factor which goes into the nucleus
- Can undergo transcription
How is the sex determined in mammals?
SRY is a transpiration factor that inhibits female anatomical structures in males
promotes formation of tested from undifferentiated gonadal tissue
What is the Swyer Syndrome?
- rare disorder where there is a failure of gonads to develop
- females with syndrome are XY, are externally female but lack ovaries or testes
What is the cause of Swyer Syndrome?
- mutations in the SRY gene on the Y chromosome
- can affect ability of the transcription factor to develop
- mutated SRY cannot interact with importin so cannot be trafficked into the nucleus and interact with the target gene
What other diseases are linked to nuclear transport?
- Tau Interferes with Nuclear Transport in Alzheimer’s Disease
- impaired nucleocytoplasmic trafficking has emerged as a mechanism contributing to ALS
