Protein Targeting: Nucleus

Question 1

What is the size of the nuclear pore complex?

Answer 1

125 million Da

Question 2

What is the diameter of the nuclear pore complex?

Answer 2

150 - 200 nm

Question 3

What is the Nuclear Pore Complex composed of?

Answer 3

• Octagonal ring structure embedded in the nuclear envelope
• 8 filaments that extend 100nm in nucleoplasm and are joined at their ends to form the nuclear basket
• Cytoplasmic filaments extending into the cytoplasm

Question 4

What size of ions, molecules and some proteins does the nuclear pore complex allow to diffuse freely?

Answer 4

Up to 60 kDa

Question 5

What do GTPase Activating Proteins (GAP) do to the G-protein?

Answer 5

Switches it to a GDP bound state (OFF)

Question 6

What do Guanine Nucleotide Exchange Factors (GEFs) do to the G-protein?

Answer 6

Switches to a GTP bound state (ON)

Question 7

What are the TWO characteristics of Nuclear Localisation Signals (NLS)?

Answer 7

Neccessary & Sufficient

Question 8

Which protein recognises NLS?

Answer 8

Importins

Question 9

What forms the Nuclear Import Receptor?

Answer 9

A heterodimer of Importin α and Importin β

Question 10

What is the role of Importin α?

Answer 10

Binds to basic NLS sequence

Question 11

What is the role of Importin β?

Answer 11

Mediates transport through the nuclear pore complex

Question 12

What part of the nuclear pore complex does Importin β and the cargo (cargo complex) interact with?

Answer 12

FG-nucleoporins
* Phe (F) and Gly (G) repeats

Question 13

Diffusion is non-directional so how are nuclear proteins selectively imported to the nucleus where their concentration is higher than in the cytosol?

Answer 13

• Cargo complex is rapidly and selectively disassembled in nucleus
• Once disassembled from importin, the cargo protein cannot diffuse out of nucleus

Question 14

Which enzyme regulates the assembly and disassembly of the cargo complex?

Answer 14

Ran GTPase

Question 15

What is the role of Ran GTPase in the NUCLEUS?

Answer 15

• GEF keeps Ran in its GTP bound state
• Binding of Ran-GTP to Importin causes dissociation of cargo complex
• Ran-GTP bound to Importin is exported to Cytosol

Question 16

What is the role of Ran GTPase in the CYTOSOL?

Answer 16

• GAP causes Ran to hydolyse GTP and convert to GDP bound state
• Ran-GDP dissociates from Importin
• Importin binds to another cargo protein
• Ran-GDP binds to NTF2 (nuclear transport factor 2) and returns to nucleus

Question 17

Where is Ran-GEF located?

Answer 17

Bound to chromatin so remains in the nucleus

Question 18

Where is Ran-GAP located?

Answer 18

Associated with the cytoplasmic filaments of the nuclear pore

Question 19

Which amino acid are Nuclear Export Signals (NES) rich in?

Answer 19

Leucine

Question 20

What is the Nuclear Export process?

Answer 20

• Exportin-1 forms a complex with Ran-GTP and binds to NES of cargo protein
• All three proteins diffuse through the NPC
• At the exit of the pore, Ran-GTP is converted to Ran-GDP and cargo complex is disassociated
• Cargo protein is released into the cytosol
• Ran and Exportin-1 return separately to the nucleus

Question 21

What is the major difference between nuclear import and nuclear export in terms of Ran?

Answer 21

Ran is part of the cargo complex in nuclear EXPORT

Question 22

What are TWO examples of regulated nuclear transport?

Answer 22

1. T-Cell Activation
2. Steroid Hormone Receptor activity