Macromolecular transport Flashcards
What are the 3 types of nucleoporins?
Structural, membrane, and FG which contain hydrophobic FG repeats
What are the 2 mechanisms of transport?
Passive diffusion and active transport
Describe the mechanism for nuclear import of proteins
- Free importin (nuclear transport receptor) binds to NLS of a cargo protein, forming the importin-cargo complex
- Importin-cargo complex diffuses through NPC by interacting with FG-nucleoporins
- In the nucleus, Ran-GTP binds to the importin, making it release the cargo protein
- Importin-Ran-GTP complex diffuses to cytoplasm
- GAP stimulates Ran to hydrolyze the bound GTP, releasing the importin and resulting in Ran-GDP
- Ran-GDP returns in nucleus where GEF causes the release of GDP, and rebinding of GTP, resulting in Ran-GTP
MEMO: Importin+Cargo, GEF converts Ran-GDP to Ran-GTP in nucleus, GAP converts Ran-GTP to Ran-GDP in cytoplasm
NOTE: Diffusion of complexes are due to concentration gradient
What is the element needed for a protein to be imported?
Nuclear-localization signal (NLS)
Explain the experiment which supports the notion that the import of proteins is directed through interactions with FG nucleoporins.
- Perform antibody depletion to knock out Nup98 FG domain
- Examine whether nuclear import is disrupted using an experimental sample (MBP protein fused with ‘IBB’ NLS) and a control (70kDA dextran)
Perform rescue experiment where Nup98 is restored to confirm Nup98 was the cause of observed phenotype
RESULT:
(undepleted) => Nuclear import is observed as MBP protein is in the nucleus, while dextran remains in cytoplasm.
(-) Nup98 => No nuclear import is observed, while 70kDA dextran can be found everywhere, suggesting that the nuclear pore does not form an effective barrier alone
(+) Nup98 => Nuclear import is restored as MBP protein can be found within the nucleus, while dextran remains in the cytoplasm
- Purify Nup98, forming a gel
- Add MBP-mCherry (control with no NLS) and importing+cargo complex on the buffer side, observing whether they become transported to the gel side containing Nup98
RESULT: Only the importin+cargo complex can be found on the gel side (Fig. c)
What is the mechanism for nuclear export of proteins?
- Exportin binds with NES of cargo protein, and Ran-GTP, forming Cargo-Exportin-RanGTP complex
- Cargo-Exportin-RanGTP complex diffuses through NPC via interactions with FG-repeats to the cytoplasm
- In the cytoplasm, GAP converts Ran-GTP to Ran-GDP, leading to the dissociation of the whole complex
- Cargo protein is released in cytoplasm, whereas Ran-GDP and exportin diffused back into the nucleus
- In the nucleus, GEF converts Ran-GDP to Ran-GTP
MEMO: Cargo+Exportin+RanGTP
Where can Ran-GDP and Ran-GTP be found? Which proteins act on them?
In the nucleus, GEF acts on Ran-GDP, converting it to free Ran-GTP.
In the cytoplasm, GAP, acts on Ran-GTP, converting it to free to Ran-GDP.
Because of the concentration gradient, Ran-GDP diffuses from the cytoplasm from the nucleus.
Ran-GTP diffuses from the nucleus to the cytoplasm.
How and why does a GEF contribute to the process of nuclear import?
GEF acts on Ran-GDP, converting it to Ran-GTP. This conversion is important since the binding of Ran-GTP to the importin is needed for the release of the cargo protein.
What is the main difference in the complex being diffused through the NPC in nuclear import and export?
In nuclear import, the complex is composed of importin and cargo.
In nuclear export, the complex is composed of exportin, Ran-GTP and the cargo.
What is the mechanism for the export of mRNAs?
In the nucleus, after processing, mRNA is associated with mRNP; it is transported by the mRNP exporter, a heterodimeric protein composed of a large subunit (NXF1) and a small subunit (NXT1).
- NXF1/NXT1 dimers bind to mRNPs
- NXF1/NXT1 interacts with FG-repeats through its exterior hydrophobic patches, allowing for the diffusion of the complex to the cytoplasm
- In the cytoplasm, NXF1/NXT1 dissociate from mRNP with the help of RNA helicase Dpb5
- Free NXF1/NXT1 are imported back into the nucleus
NOTE: mRNP cannot interact with FG-repeats so they cannot diffuse back into the nucleus from the cytoplasm + NXF1/NXT1 binding acts as quality control as they only bind to processed mRNA
What role does Dpb5 play in nuclear export of mRNPs?
Dpb5 is a RNA helicase that helps NXF1/NXT1 dissociate from the mRNP in the cytoplasm.
In what way does NXF1/NXT1 help to ensure that only processed mRNAs are exported?
NXF/NXT binds mRNA through REF protein, which only binds to exon splice junctions. Also, NXF/NXT recognizes the RNA PolyA tail.
Explain the mechanism that prevents RNAs with splice site mutations from being exported?
RNAs with splice site mutations still have bound splicing factors, which Mlp1/2 proteins bind to, preventing their export.
HIV mRNAs contain splice sites and thus can be viewed as incompletely spliced pre-RNAs, which would be normally blocked for nuclear export. What is the mechanism that overcomes this?
1) Early in an infection, a few 2kb viral mRNAs are exported, which encodes for Rev, a protein that contains a NES that can interact with exportin 1
2) After translation and nuclear import of Rev, Rev binds to the RRE sequence in unspliced HIV RNAs, and associates with exportin, resulting in the export of HIV mRNAs through the NPC without the use of NXF1/NXT1
Select the false statement:
(A) Concentration gradient is important in mediating nuclear import and export of proteins.
(B) Free NXF1/NXT1 are imported into the nucleus, just as free Ran-GDP.
(C) Ran is a GTPase protein that is regulated by regulatory proteins
(D) When introns are artificially inserted within the lacZ gene, in the Mlp1 -/- variant, we expect to see white colonies.
(E) When introns are artifically inserted within the lacZ gene, in the WT, we expect to see white colonies.
(F) None of the above.
(D) We expect to see blue colonies as mutant genes would still be exported.
