Nucleo-cytoplasmic transport (5)

Aspect of nuclear pore complexes (NPC)

Baskets that open onto the cytoplasmic side

Size of NPC

125 MDa (huge bc 30x ribosome)

Protein composition

50 proteins in yeast vs 100 in vertebrates

What determines if you need transport and not diffusion throught the NPC?

Size: if bigger than 40-60kDa

FG nucleoporins

Gel meshwork of that result from the interaction of hydrophobic and disordered domains

How do FG nucleoporins allow transport?

Transport depends on reversible hydrophobic interactions

Where are nuclear proteins synthesized?

In the cytoplasm

How are nuclear proteins recognized?

Through NLSs or nuclear localization signals (fluorescence)

Proteins needed for transport (2)

1. Ran

2. Nuclear transport receptors (NTRs)

Conformations of Ran

Ran-GTP and Ran-GDP

Ran-GDP/Ran-GTP cycle

1. Ran-GDP→Ran-GTP through GEF

2. Rand-GTP→Ran-GDP through GAP (hydrolysis)

NTRs

Bind to NLS domains on proteins and facilitate transport by associating with FG repeats

1st step of import

NTRs/Importin binds to NLS marked cargo

2nd step of import

Complex diffuses to the nucleoplasmic side

3rd step of import

Complex interacts with Ran-GTP = Ran-GTP changes Importin conformation = less affinity to the Cargo

Why is Ran-GTP highly concentrated in the nucleus?

Bc the GEF is tied to chromosomes

4th step of import

RanGTP/Importin complex diffuses into the cytoplasm

5th step of import

RanGDP releases Importin

1st step of export

Cargo-Exportin-RanGTP complex diffuses through the gel meshwork

2nd step of export

GAP hydrolyses GTP→GDP= change in conformation of Ran = complex falls apart

3rd step of export

1. GDP→GTP

2. Exportin returns to its initial conformation

Exportin t

Export of tRNAs

Other kind of Exportin

hnRNP for specific mRNAs

Are most mRNAs Ran-independent or Ran-dependent?

Ran-independent

Ran-independent export

NXF1/NXT1 subunits bind to the RNA forming mRNP

Specificity of mRNP export

5’ end must be exported first bc ribosomes associate with the mRNA while it’s still being exported

What is a fully spliced mature mRNA?

mRNA with a 5’ cap and a 3’ poly A tail (determined through cytoplasmic remodelling)

How are NXF1 and NXT1 removed from the mRNA once it’s in the cytoplasm?

By RNA helicase