Nuclear Transport

Question 1

3 Types of trafficking via sorting signals

Answer 1

1. gated transport (movement via NPC)
2. transmembrane transport (protein translocation from cytosol into a topologically distinct space)
3. vesicular transport (vesicles carry proteins between topologically equivalent compartments)

Question 2

Nuclear Pore Complexes

Answer 2

Made of disordered mesh, scaffold nucleoporins, and channel nucleoporins
Major site of protein import is via NPC, with 500 macromolecules transporting through per second.
The mechanism regulating this is unknown.
NPCs have basket like structure going down and horn like structures going up.
The pore is aqueos and flexible so folded proteins can enter and exit
Proteins need a signal to be carried through (NLS)

Question 3

Nuclear Localisation Signal Experiments

Answer 3

Key experiment - John Gurdon.
- used frog oocytes to show that nuclear proteins accumulate in the nucleus if injected in the cytosol
Key experiment - Dingwall, Laskey, Sharnick
- found that nuclear protein has a domain acting a signal for nuclear entry

Question 4

NLS

Answer 4

• the ‘tail’ of the signal is the rate determining step, as they are key to moving proteins into the nucleus
• direct proteins to the correct location
• each signal specifies a specific cellular destination, the NLS specifies the nucleus
• complementary sorting receptors guide proteins to their appropriate destination

Question 5

Nuclear Import/Export

Answer 5

Based on a choreographed set of interactions between cargo proteins and import/export chaperones
Recognition between molecules is key
The process is orchestrated by Ran - when RAN changes its GTP state is changes its conformation when changes its interaction with proteins

Question 6

Import/Export Cycle

Answer 6

1. Cargo binds to importin a/B via the NLS signal
2. Complex passes through the NPC + Ran GDP enters nucleus as well
3. RAN gef converts RAN gdp to RAN gtp
4. RAN gtp binds to the complex (higher affinity than the cargo does) and disrupts the cargo binding
   - cargo and importin a knocked off
5. importin is taken up by the CAS nuclear export factor catalysed by Nup50
6. importin a/b and bound RAN gtp taken back through the NPC to the cytosol
7. ran GAP hydrolyzes GTP and dissociates complex

Question 7

Karyopherins (importins/exportins)

Answer 7

• Large proteins which need importation have NLS

- cargo protein NLS binds to importins/exportins (products of the karyopherin gene family)

Question 8

NLS and NPC interaction

Answer 8

Importins/exportins bind F-G repeats in the unstructured domains of the NPC nucleoporins in the meshwork core
Random interactions between the repeats and karyopherins have low affinity, but at high concentrations move the complex through the pore
Receptor-cargo complexes bind, dissociate, then rebind to adjacent repeats
This process only occurs going in, creating directionality of movement

Question 9

Export Cycle

Answer 9

Relies on NES (nuclear export signal) and exportins that bind to the signal and NPC proteins to guide cargo out

1. During import, the RAN gtp binds to the cargo complex to promote disassembly or ‘unloading’
2. Exportin binds to NES of cargo and the complex exits via the NPC
3. During export, RAN gtp promotes cargo binding to the export receptor and binds to complex
4. As the exportin moves into the cytosol, RAN gap causes GTP hydrolysis and cargo/exportin release
5. Exportin and RAN gdp moves back into the nucleus

Question 10

Extra

Answer 10

Some proteins have both NLS and NES signals, and can be moved in and out cyclicly
The rate of movement in both ways determines the steady state localisations of shuttling proteins
Protein movement can also be turned on or off by phosphorylation of amino acids close to the signal sequences

Question 11

Importin/Exportin Cycle Proteins

Answer 11

1. RAN
   - RAN gdp (cyotsol)
   - RAN gtp (nucleus)
2. karyopherins (importins and exportins)
3. cargo (contains NLS)
4. Nucleotide exchange factors (RAN gef and gap)