CBG 34 Flashcards
what are karyophorins?
a group of protein that are involved in transporting molecules between the cytoplasm and nucleus of the cell
Fg nucleoporins
exportins
importins (alpha and beta)
give karyophorin examples
FG nuceloporins
exportins
importins
what aids entry to macromolecules? what aids exit?
importins
exportins
Nuclear pore complex structure?
cytoplasmic fibrils
central framework( FG meshwork core)
nuclear basket
v dynamic,huge,complex
NLS?
nuclear localisation signals. signal peptides
what types of signal sequences are there?
no signal sequence=cytosol
NLS=nuclear
MSS= mitochondriaol
ER SS= plasma membrane,ER and golgi body,endosomes,lysosomes
what was the first experiment to understand signal sequences? what protein did they use? what was the experiment and when?
1 protein- with 5 identical subunits- NUCLEOPLASIN
injected into XENOPUS OOCYTES
-injected in cytosol=went to nucleus
-iinjected in nucleus-stayed in nucleus
Then used PROTEOLYSIS to degrade/partially degrade the tail
-without tail =stayed in cytosol
-number of tails on pentameter correlates with rate of transport
PROVED TAIL LIKELY TO CONTAIN NUCLEAR LOCALISATION SIGNAL
what did the experiment with xenopus oocytes prove?
tail was likely to contain nuclear localisation signal (number of tails on pentamer correlates with rate of transport)
what was used in the second experiment to permeabilize rge plasma membrane whilst keeping the nuclear membrane intact?
DIGITONIN
what was the ihibitor that binds to the NPC and inhibts transport ? (used in 2nd experiment)
Wheat germ agglutin
briefly describe experiment that showed energy and specific associations are needed for nuclear transport?
1) digitonin used to permeabilize the membrane whilst keeping the nuclear membrane intact
2) added cargo with NLS fluorescently tagged
observation was that only when energy had been added to cytosol did the cargo move to nucleus
3)the added WHEAT GERM AGGLUTIN which bound to NPC and inhibited transport
EXPERIMENT SHOWS: energy and specific associations are needed for nuclear transport
explain difference between RANGTP and RANGDP?
RANGTP=active
RANGDP=inactive
what is RAN?
a small G protein involved in nuclear transport tha tbinds to GTP/GDP and is inactive or active depending on what form it is bound to
If active it splits components such as alpha beta importins and the cargo up
what does GAP do? wrt RAN
GTPAse Activating proteins
helps small proteins hydrolyse GTP–> GDP this INACTIVATES RAN
What does GEF do wrt RAN?
Guanine Nucleotide exchange factor
exchanges GDD for GTP –> ACTIVATION
what did louis sullivan say?
form follows function (architect)
what is the structure of alpha importin?
series of repeats and structural folds–> ARMADILLO
-used for scaffold
BINDING SPOT FOR NLS
alpha importin binds and recognises specific TAILS
what is the structure of beta importin?
similar ARMADILLO repeats like alpha importin and scaffolding BUT
BINDS:
FG REPEATS
RAN GTP (location is opposite side of FG)
what is an importin?
a type of karyopherin made up of alpha and beta subunits
what are NTRs?
nuclear transport RECEPTORS- e.g importins or exportins
what 2 things do you need to transport large cargo into the nuclear pore?
chemical energy
specific receptors
what do the FG repeats in the central framework of the nuclear pore complex interact with when cargo is being transported through? Also explain detail of cargo and NLS and importin
FG interacts with B importin side (remember cargo is attached to the Nuclear localisation signal(postcode) and this is attched to importin at alpha binding site for tailswhich is also attached to B importin side which is the part that interacts with the FG repeats)
Explain what happens to the cargo once it is through teh nuclear pore
GEF helps change RANDGDP to RAND GTP
RANDGTP is Active
Beta recognises RANGTP which allows separation of components
alpha importin part breaks off and can return to cytosol freely through passive diffusion
cargo stays inside nucleus
beta and RANGTP returns through pore to cytosol.
once in cytosol GAP hydrolyses GTP so RANGTP–>RANGDP
RANDGDP is inactivated and beta importin and RAN GDP split up
what does F stand for?
phenylalanine (hydrophobic)
what does G stand for?
glycine (hydrophobic)
why do FG repeats not fold?
FG also has lots of K lysine that are hydrophillic to stop globular folding
where are FG repeats?
are abundant in nuclear pore complexes and deleting too many can be dangerous
the conserved hyrdrophic regions represent binding sites for nuclear transport receptors
