RAN system and Nuclear transport

Question 1

Describe the theory for formation of a eukaryote.

Answer 1

• • An archaea loses its cell wall.
• • This increases horizontal gene transfer, speeding evolution.
• • digestion of other prokaryotes also increases speed of evolution
• • membrane begins to invaginate to allow a more and more protected nucleus.
• • A cell is consumed that is not digested, it is the 1st mitochondria
• • Nucleus also helps to form the ER.

Question 2

G Proteins

Answer 2

Bind GTP, hydrolyze to GDP. GTP is considered generally, the active state.

Question 3

GAP

Answer 3

GTPase Accelerating Proteins. Catalyze GTP–>GDP

Question 4

GEF

Answer 4

Guanine Nucleotide-Exchange Factors. Help with release of GDP from the G protein.

Question 5

What are the two types of G-Proteins?

Answer 5

Heterotrimeric G proteins
Have three subunits, an Alpha (Which has GTPase activity), beta and gamma, form a heterodimer. Heterotrimeric G proteins often couple with G protein-coupled receptors (GPCRs).

Monomeric G proteins

• Evolutionary speaking, related to the alpha protein of the heterotrimeric G proteins.
• Exists in thousand varieties
• Ras was the first monomeric G protein discovered.

Question 6

Where is RAN-GEF located?

Answer 6

Within the Nucleus. So RAN-GTP will exist exclusively in the nucleus.

Question 7

Where is RAN-GAP located?

Answer 7

Within the Cytoplasm. So RAN-GDP will exist exclusively in the cytoplasm.

Question 8

Is RAN monomeric or trimeric?

Answer 8

Monomeric G protein.

Question 9

What is a Nuclear Import Receptor?

What is a Cargo Protein?

Answer 9

A Nuclear Import Receptor is a protein which can bind to RAN-GTP or a Cargo Protein (mutually exclusive). It has poor binding affinity for RAN-GDP.

A cargo protein is any protein carried in or out by import or export receptors.

Question 10

What is a Nuclear Export Receptor?

Answer 10

It is a protein which can only bind it’s cargo protein if RAN-GTP is bound to it. It has low affinity for RAN-GDP (and no affinity for the cargo protein once RAN-GTP leaves).

Question 11

Explain Nuclear Import.

Answer 11

A Nuclear Import Receptor binds a cargo protein. It travels into the nucleus. Inside the nucleus is where RAN-GTP dwells. RAN-GTP binds to the nuclear import receptor, either after it has already released its cargo protein or making it release the cargo protein as it binds. With RAN-GTP bound, the Nuclear Import Receptor cannot bind its cargo protein. Once it leaves the nucleus, RAN-GAP jumps it, and catalyzes GTP–>GDP. GDP has poor affinity for its current binding site, and falls off, leaving the site open for a binding protein.

Question 12

Explain Nuclear Export.

Answer 12

A Nuclear Export Protein must bind RAN-GTP to bind its cargo protein. RAN-GTP is in the nucleus, so we will start our tail with a Nuclear Export Protein who has bound both RAN-GTP and its cargo protein. Once it has bound its cargo protein, the Nuclear Export Protein skedaddles to the cytosol, where RAN-GAP takes RAN-GTP –> RAN-GDP in ~10 ms. Binding of cargo protein is now impossible, so it leaves. The Nuclear Export Protein is returned to the nucleus, ready to undertake its journey once again.

Question 13

Even though the slides didn’t describe one, why must there be a mechanism to move RAN-GDP into the nucleus?

Answer 13

Because RAN-GEF exists only in the nucleus. RAN-GDP rarely releases GDP without RAN-GEF and there is little RAN-GTP in the cytosol (due to RAN-GAP).

Given this, we know the only way to get more RAN-GTP, is to move RAN-GDP to the nucleus, where RAN-GEF can release GDP and GTP can bind to RAN.

Question 14

Is GTP or GDP kept in higher concentrations in the cell? How much higher.

Answer 14

GTP is kept ~20x higher. Therefor by statistics alone it will bind to RAN more often.

Question 15

What are nuclear lamina?

Answer 15

They are an internal structure of the membrane that looks like burlap sacks, and binds to protein pores.

Question 16

What is the lamina made of?

Answer 16

Intermediate fibers named lamins.

Question 17

Phosphorylation of the lamina does what? What phosphorylates it.

Answer 17

Causes the loss of stability and the breakdown of the nuclear envelope. This is phosphorylated by CDK (cyclin dependent kinase).

Question 18

What does dephosphorylation of lamins cause?

Answer 18

The reassemblage of the nuclear envelope.

Question 19

Is it possible to have exclusions of receptors to specific regions or from the whole nuclear envelope.

Answer 19

Yes, this goes down. It changes expression in specific areas.

Question 20

What is calcineurin?

Answer 20

Is a protein phosphatase which is only active when Ca2+ is present in high concentration in the cell.

Question 21

NFAT stands for what?

Answer 21

Nuclear Factor of Activated T-cells.

Question 22

What does NFAT do once inside the nucleus?

Answer 22

It is an activator of transcription.

Question 23

Explain how the NFAT regulatory system works.

Answer 23

NFAT starts in the cytosol. Where it sits, bound to three phosphates, which have shifted its conformation so it shows a cytosolic signal sequence. Increase in intracellular calcium activates calcineurin, a phosphatase, which removes the three phosphates on NFAT. This causes a nucleus signal sequence to be shown. NFAT enters the nucleus, and transcription goes down. Until Ca2+ lowers and a kinase adds 3 phosphates like nobodies business. NFAT has a signal sequence saying pack it all up and move to the cytosol. And NFAT just listens, because that’s the type of Nuclear Factor of Activated T-cell it is.