CBG 34

Question 1

what are karyophorins?

Answer 1

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)

Question 2

give karyophorin examples

Answer 2

FG nuceloporins
exportins
importins

Question 3

what aids entry to macromolecules? what aids exit?

Answer 3

importins

exportins

Question 4

Nuclear pore complex structure?

Answer 4

cytoplasmic fibrils
central framework( FG meshwork core)
nuclear basket

v dynamic,huge,complex

Question 5

NLS?

Answer 5

nuclear localisation signals. signal peptides

Question 6

what types of signal sequences are there?

Answer 6

no signal sequence=cytosol
NLS=nuclear
MSS= mitochondriaol
ER SS= plasma membrane,ER and golgi body,endosomes,lysosomes

Question 7

what was the first experiment to understand signal sequences? what protein did they use? what was the experiment and when?

Answer 7

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

Question 8

what did the experiment with xenopus oocytes prove?

Answer 8

tail was likely to contain nuclear localisation signal (number of tails on pentamer correlates with rate of transport)

Question 9

what was used in the second experiment to permeabilize rge plasma membrane whilst keeping the nuclear membrane intact?

Answer 9

DIGITONIN

Question 10

what was the ihibitor that binds to the NPC and inhibts transport ? (used in 2nd experiment)

Answer 10

Wheat germ agglutin

Question 11

briefly describe experiment that showed energy and specific associations are needed for nuclear transport?

Answer 11

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

Question 12

explain difference between RANGTP and RANGDP?

Answer 12

RANGTP=active

RANGDP=inactive

Question 13

what is RAN?

Answer 13

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

Question 14

what does GAP do? wrt RAN

Answer 14

GTPAse Activating proteins

helps small proteins hydrolyse GTP–> GDP this INACTIVATES RAN

Question 15

What does GEF do wrt RAN?

Answer 15

Guanine Nucleotide exchange factor

exchanges GDD for GTP –> ACTIVATION

Question 16

what did louis sullivan say?

Answer 16

form follows function (architect)

Question 17

what is the structure of alpha importin?

Answer 17

series of repeats and structural folds–> ARMADILLO
-used for scaffold
BINDING SPOT FOR NLS
alpha importin binds and recognises specific TAILS

Question 18

what is the structure of beta importin?

Answer 18

similar ARMADILLO repeats like alpha importin and scaffolding BUT
BINDS:
FG REPEATS
RAN GTP (location is opposite side of FG)

Question 19

what is an importin?

Answer 19

a type of karyopherin made up of alpha and beta subunits

Question 20

what are NTRs?

Answer 20

nuclear transport RECEPTORS- e.g importins or exportins

Question 21

what 2 things do you need to transport large cargo into the nuclear pore?

Answer 21

chemical energy

specific receptors

Question 22

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

Answer 22

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)

Question 23

Explain what happens to the cargo once it is through teh nuclear pore

Answer 23

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

Question 24

what does F stand for?

Answer 24

phenylalanine (hydrophobic)

Question 25

what does G stand for?

Answer 25

glycine (hydrophobic)

Question 26

why do FG repeats not fold?

Answer 26

FG also has lots of K lysine that are hydrophillic to stop globular folding

Question 27

where are FG repeats?

Answer 27

are abundant in nuclear pore complexes and deleting too many can be dangerous
the conserved hyrdrophic regions represent binding sites for nuclear transport receptors