Nuclear Transport

Question 1

Describe gated transport and the nuclear pore complex

Answer 1

Gated transport is between cytosol and nucleus
Nuclear Pore Complex - 1000s per nucleus, each comprised of nucleoporins, mediate import and export of proteins
Traffic into nucleus = histone, RNA polymerase, Lamin, transcription factors
Traffic out of nucleus = ribosomes, mRNA
Less than 5 kDa = molecules diffuse freely
More than 5 kDa = molecules kept out by disordered mesh
- proteins require a signal to be carried across

Question 2

Describe the architecture of NPC

Answer 2

• pore is aqueous so folded proteins with hydrophobic cores not exposed to surface can travel through
  Very large
  Flexible and dynamic

Question 3

What is nuclear localisation signal and nuclear export signal?

Answer 3

Nuclear Localisation Signal (NLS) is found on cargo protein transported INTO nucleus
- sequence dependent - usually basic amino acids
- conserved - can search genome to find NLS
- can change to control traffic into nucleus
MORE NLS means FASTER diffusion into nucleus
Nuclear Export Signal (NES) on cargo protein transported OUT of nucleus

Question 4

How was the receptors for import signals discovered?

Answer 4

1. Treat cells with digitonin (permeabilise plasma membrane, nuclear envelopes intact)
2. Add fluorescent protein substrate with NLS + energy + cytosol
   Saw that protein was trafficked and translocated into nucleus
3. Use wheat germ agglutinin which binds to NPC (and inhibits transport)
   No longer see fluorescent tag in nucleus

Therefore nuclear import requires NLS and specific receptors that recognise signal

Question 5

What are the roles of GEF, GAP and Ran in nuclear transport

Answer 5

Ran (RAs-related Nuclear Protein) = GTPase
GEF = Guanine nucleotide exchange factor
- recharges GDP for GTP
- nuclear, binds to chromatin
GAP = GTPase activating protein
- stimulates GTP hydrolysis
- binds outside nucleus - cytoplasmic

Ran GDP forms Ran GTP via Ran GEF
Ran GTP forms Ran GDP via Ran GAP

Question 6

What are karyopherins (importing/exportins) and how do alpha and beta importins inform how cargo is transported?

Answer 6

Karyopherins (importins/exportins) are chaperone proteins that bind to NLS of cargo proteins
Importins have 2 Subunits: α-importing and β-importins
α-importins - bind to NLS of cargo protein
β-importins - bind to α-importins chain (IBB) with cargo binded from NLS
inner surface binds to a RanGTP and outer surface binds to FG repeats on NPC
* cannot bind to Ran GTP and α-importin at the same time

FG repeats = phenylalanine (aromatic, hydrophobic, none polar) and glycine (no side chain)
Acts as docking sites for importing on NPC
Needs to be flexible to allow cargo to pass
specific, highly conserved

Question 7

How does the Ran cycle work to allow cargo to enter nucleus?

Answer 7

1. Cargo with NLS binds to α of αβ-importin which pass through nuclear pore complex into nucleus
2. Ran GTP binds to β-importin causing it to dissociate from α-importing and cargo to be released
3. α-importin taken your by CAS nuclear export factor, catalysed by Nup50
4. α or β importin taken back through NPC to cytosol taking Ran GTP
5. In cytosol, Ran GAP stimulates hydrolysis of Ran GTP to Ran GDP
6. Ran GDP binds to nuclear transport factor 2 (NTF2) which shuttles it back to nucleus
7. In nucleus, Ran GEF catalysts conversion of Ran GDP to Ran GTP
8. Ran GTP gets taken back to cytoplasm by transporting proteins like β-importing

Net result:
Ran GTP concentrated in nucleus because of Ran GEF
NLS cargo gets taken into nucleus
Ran GTP and importins get recycled for future import
Ran GDP/GTP gradient drives import