MBB 267 Week 10: Mitchel 9

Question 1

How are proteins and RNAs transported across the nuclear membrane?

Answer 1

Via nuclear pore complexes (NPCs)

Question 2

What is the structure of NPCs?

Answer 2

Nuclear pore complexes are large structures (~ 60 MDa; that’s 14 times the molecular weight of a ribosome) but contain approx. 30 different proteins called nucleoporins. The nuclear , pore complexes consist of an annular ring structure that constitutes the pore, from which extends a nuclear basket structure on the nuclear side and extended cytoplasmic filaments on the cytoplasmic side. Both structures allow molecular interactions with proteins on either side of the pore. The nuclear side of the membrane is in close contact with the nuclear lamina, a network of fibrillar proteins that supports the nuclear membrane and anchors the nuclear pore complexes in the membrane

Question 3

What are FG-repeat nucelporins?

Answer 3

The channel of the nuclear pore is lined with proteins called FG-repeat nucleoporins, which form a hydrophobic gel that constricts the pore diameter.

Question 4

Are proteins transported in their folded or non folded state?

Answer 4

Proteins are selectively transported and in a folded state

Question 5

What are nuclear localisation signals?

Answer 5

Import of reporter proteins were dependent upon the presence of a nuclear localization signal (NLS), a peptide sequence that allows recognition by the carrier protein, and microinjection of cytoplasmic cell extract

Question 6

What is Ran?

-How is it used to transport molecules across the nuclear membrane?

Answer 6

Ran is a key factor in nuclear transport. Ran belongs to the Ras family of small GTPases: molecular switches that are controlled by GTPase activating proteins(GAPs) and guanine nucleotide exchange factors (GEFs).
-Ran/GAP is cytoplasmic, while Ran/GEF is nuclear. Hence, nuclear Ran is bound to GTP and cytoplasmic Ran is bound to GDP. Molecules can tag along the Ran while it is moving along the membrane.

Question 7

What are karyopherins?

• How do karyopherins operate?
• Why are karyopherins able to move through nuclear pores?

Answer 7

Proteins that directly mediate nuclear transport by interacting with the cargo molecules and facilitating their diffusion through the nuclear pores are called karyopherins (importins or exportins)

• Karyopherins (whether an importin or an exportin) interact tightly with Ran when it is bound to GTP (in the nucleus) and dissociate from Ran upon GTP hydrolysis (in the cytoplasm). Thus, karyopherins interact with Ran in the nucleus, can pass through the nuclear pore and then dissociate from Ran upon GTP hydrolysis in the cytoplasm.
• Karyopherins can diffuse through the nuclear pores because they can interact with the hydrophobic FG-repeat nucleoporins

Question 8

What is the difference between importins and exportins?

Answer 8

Importins release cargo upon binding to Ran-GTP in the nucleus. Exportins release cargo upon Ran-GTP hydrolysis. The key difference between importins and exportins that allows correct directionality of transport is that their affinity for their respective cargo molecules is affected by Ran in distinct ways. Importins can bind cargo in the absence of Ran and dissociate from their cargoes upon interaction with Ran (in the nucleus), while exportins bind cargo only when bound to Ran (in the nucleus) and dissociate from their cargoes upon loss of Ran binding (in the cytoplasm).

Question 9

What is the mechanism of importins?

Answer 9

Importins bind to their cargo proteins via peptide sequences called nuclear localisation signals (NLS) or nuclear export signals (NES). Importin/cargo complex moves with Ran/GDP to the nucleus. When they are in the nucleus, Ran-GEF releases the cargo protein and the Ran/GTP exits the nucleus.

Question 10

What is the mechanism of exportins?

Answer 10

Exportins bind their cargo via nuclear export signals. Cargo is bound to exportins when Ran is bound to GTP (importins bind Ran to GDP)

Question 11

How is directionality ensured between exportins and importins?

Answer 11

the directionality of transport is ensured by (i) the differential localisation of Ran GEF and Ran GAP, (ii) the affinity of karyopherins for their cargoes and for GTP-bound Ran, and (iii) the differential affinity of karyopherins for their cargoes upon interaction/loss of Ran binding

Question 12

What does NXF1 do?

-How does it do that?

Answer 12

Mediates mRNA export from nucleus.
-NXF1 binds to RNA and to FG repeat nucleoporins. mRNA export doesnt require Ran. During the process of pre-mRNA splicing, the exon junction complex (EJC) is deposited on the mRNA at the site of splicing. One component of this complex is an RNA-binding protein called Ref (RNA export factor). Ref also interacts with NXF1. Binding of Ref to NXF1 destabilises its interaction with RNA and stimulates RNA-binding activity of NXF1, causing the RNA to dissociate from Ref and bind to NXF1 (a molecular “hand- over” event). Since only spliced pre-mRNA contains the EJC, only spliced mRNPs are exported to the cytoplasm. Upon export, the NXF1/mRNP complex is disassembled through the chaperone activity of the RNA helicase Dbp5. Dbp5 is localised to the cytoplasmic fibrils of the nuclear pore complex. Upon export, mRNP particles are extensively remodelled to remove nuclear associated proteins and allow the binding of cytoplasmic factors that promote mRNA translation. Notably, the EJC remains bound until the initial round of translation