Lecture 5 - Exam 3: Protein Import and Export

Question 1

When it comes to protein import (between the nucleus and cytoplasm), what runs the show?

Answer 1

Importin, Ran, and Ran GAP

Question 2

What is the basic mechanism of the protein transport (protein import)?

Answer 2

1. Importins bind to the NLS of a cargo protein and to the nuclear pore. Then, the complexed importin-cargo protein is transported to the nuclear pore.
2. Ran/GTP binds to the importin-cargo complex. The binding of Ran/GTP triggers the release of the cargo protein in the nucleus.
3. The importin-Ran/GTP complex is exported back to the cytoplasm through the nuclear pore.
4. In the cytoplasm, Ran GAP is associated with the cytoplasmic filaments, hydrolyzing the GTP on Ran to GDP, releasing the importin (recycling).
5. The Ran/GDP formed in the cytoplasm is then transported back to the nucleus.

Question 3

What is Ran?

Answer 3

It is a GTP-binding protein called a GTPase.

Question 4

What does GTP stand for? What is it?

Answer 4

Guanosine-5’-triphosphate ; it is a purine nucleoside triphosphate.

Question 5

What role does GTP have?

Answer 5

Has a role as source of energy and as activator of substrates.

Question 6

What is the process of an inactive GTPase to an active GTPase?

Answer 6

GTPase is bound to GDP and GDI to begin with.
GEF (G nucleotide exchange factors) comes and stimulates the release of GDP and binding of GTP, but does NOT convert GDP to GTP.
GTP binds to the GTPase (ACTIVE!!!).
GAP (GTPase activating proteins) stimulates the hydrolysis of GTP, inactivating the GTPase (Ran in most cases) (starts at the beginning now)

Question 7

Signaling via small GTPases = ?

Answer 7

Fast response to stimuli

Question 8

What are the families of small GTPases?

Answer 8

Ran, Ras, Rac, CDC42

Question 9

What is the basic mechanism of protein transport (protein export)?

Answer 9

1. Proteins targeted for export contain a nuclear export signal (NES).
2. NES are recognized by receptors in the nucleus called exportins.
3. Exportins form stable complexes with cargo proteins in association with Ran/GTP in the nucleus.
4. In the cytoplasm, GTP hydrolysis leads to dissociation of the complex Ran/GTP-cargo protein-exportin.
5. Ran/GDP and the exportin are returned to the nucleus with the help of an import receptor NTF2.
6. Ran GEF (bound to chromatin) stimulates the conversion of Ran/GDP to Ran/GTP.

Question 10

Describe the transport of mRNAs.

Answer 10

Following RNA processing, mRNAs are bound by an exporter complex, which mediate their transport through the nuclear pore complex.
A helicase associated with the cytoplasmic face of the nuclear pore complex remodels the mRNA and “releases” the mRNA, providing directionality.

Question 11

What do Karyopherin exportins do?

Answer 11

Transport tRNAs, rRNAs, and miRNAs.

Question 12

Describe nuclear localization signal (NLS) masking.

Answer 12

The transcription factor NF-kB is maintained as an inactive complex with IkB, which masks its nuclear localization sequence (NLS) in the cytoplasm.
In response to appropriate extracellular signals, IkB is phosphorylated and degraded by proteolysis, allowing the import of NF-kB to the nucleus.
This is an important control mechanism for nuclear proteins like transcription factors.

Question 13

Describe regulation (of nuclear import of transcription factors) by phosphorylation.

Answer 13

The yeast transcription factor Pho4 is maintained in the cytoplasm by phosphorylation in the vicinity of its nuclear localization sequence.
Regulated dephosphorylation exposes the NLS and allows Pho4 to be transported (via an importin) to the nucleus.