Import and Export from the Nucleus

Question 1

NPC structure

Answer 1

8 fold rotational symmetry (spoke) Contains:
membrane embedded scaffold around a central transport channel
Cytoplasmic ring
Nuclear ring
8 filaments attached to each ring. Nuclear filaments are connected to distal nuclear ring (nuclear basket of the NPC).

Question 2

NPC molecular structure

Answer 2

NPCs are made of multiple copies of about 30 diff. NUPs (nucleoporins).

Question 3

FG repeats

Answer 3

Mediate active protein transport through NPC. Domain in NUPs. Have hydrophobic domains.
Carry 4–48 FG repeats to fill the central channel of the NPC (this meshwork is responsible for controlling transport and determines pore permeability limit).

Question 4

NUP structure.

Answer 4

limited set of domains (incl TM domains)
Alpha helices, beta propellers, WD domains, FG repeats at N-terminal end. C-terminal is coiled coils.
Often associate in stable sub-complexes (usually in 8 or multiple of 8 copies b/c of NPC 8-fold symmetry)

Question 5

Are nuclear pores long lasting?

Answer 5

No they are dynamic/plastic. Respond to cell changes. 1/3 of components in constant exchange. Scaffolds last longer (don’t turn over post-mitotically).

Question 6

Karyopherin

Answer 6

Transport receptor that mediates nuclear transport (except mRNA and Ran-GDP). Importins/exportins, transport cargo.
Bind to receptors via nuclear localization signal (NLS) or NES.
Interaction b/w NPC/karyopherin facilitates transport.
They require Ran binding. Interact directly with FG Nups.

Question 7

Types of transport

Answer 7

Size filtering diffusion: NP's don't close, physical barrier to hydrophobic mlcs. small things go more easily. traffic down the middle. 
Spontaneous migration: amphiphilic molecules. 
Facilitated transport (NLS NES etc.)

Question 8

Import and cargo release

Answer 8

Release cargo from interaction with RAN-GTP in the nucleus

Question 9

Export and cargo release

Answer 9

RAN-GTP helps assemble receptor/cargo, release happens with GTP hydrolysis.

Question 10

GAP

Answer 10

GTPase activating protein. CYTOPLASMIC. amplifies GTPase activity.

Question 11

GEF

Answer 11

Stimulates removal of old GDP and replacement with GTP. NUCLEAR.

Question 12

Import overview

Answer 12

1. ) Cargo with NLS binds to nuclear import receptor (NIR) and enters nucleus. (Ran-GDP loads cargo in import).
2. ) Ran-GTP in nucleus allows release of cargo. Also binds/recycles the nuclear import receptor back to cytoplasm.
3. ) In cytoplasm, RAN-GTP is hydrolyzed by GAP, released from NIR
4. ) NIR ready for import into nucleus again.

Question 13

Why is Ran-GTP necessary in import?

Answer 13

Ran-GTP is not necessary for entry in import. But it is required for returning NIR to the cytoplasm.

Question 14

Export overview

Answer 14

1. ) Ran-GTP in the nucleus promotes cargo binding to the NER
2. ) Cargo + NER + Ran-GTP –> cytosol
3. ) GAP hydrolyzes RAN-GTP to RAN-GDP, cargo is released, NER is released
4. ) NER dislikes being naked. It returns by itself to the nucleus.

Question 15

Regulation?

Answer 15

By GEF and GAP, can regulate NPC, transport, receptor, or cargo (modification via masking or increased affinity).
Regulated by entropy barrier from nups.
Ran-gdp/gtp gradient,
cargo transporters interacting with nucleoporins.
cargo modifications/assocations that affect interaction with receptors.

Question 16

Cleavage of NLS/NES

Answer 16

does NOT happen. So they can be recycled.

Question 17

General transport picture

Answer 17

Starts when import/export receptor binds to its cargo, the complexes move through diffusion barrier of the pore by interacting with FG repeats of NUPs to reach final destination where cargo is released.

Question 18

Np and disease

Answer 18

Fusions in cancer.
Can get nuclear export of tumor suppressors.
In AML 2 NPC’s fuse & interact with hox proteins, turn trx of bad genes on.

Question 19

NP and aging disease

Answer 19

post-mitotic cells can be prone to oxidative damage, get leaky. Wrong proteins accumulate in nucleus.
No new NPC’s made. get leaky nuclear pore.

Question 20

Basic structure of the nuclear pore

Answer 20

3 simple layers: membrane, scaffold, barrier.

Question 21

What does facilitated transport depend upon?

Answer 21

Energy, temp, signal dependent.
Saturable.
nuclear localization/export signals on cargo, interacts with carrier.

Question 22

NLS

Answer 22

has lysine/arginine residues. separated by spacer.

Question 23

Beta karyopherin

Answer 23

Receptor family. Directly interact with FG repeats, may bind cargo

Question 24

Alpha karyopherins

Answer 24

Adaptor family. Facilitate cargo selective binding.

Question 25

Ran concentration

Answer 25

is higher in the nucleus. NTF2 brings it from cytoplasm to the nucleus.

Question 26

RanGAP RanBp

Answer 26

facilitate GTPase activity of ran

Question 27

RCC1

Answer 27

changes RanGDP–> RanGTP ( a gef)

Question 28

Directionality of transport?

Answer 28

Ran-GTP determines this.