Lecture D

What is the function of an NES (Nuclear Export Signal)?

An NES is a specific amino acid sequence that acts as a 'postal code' to signal that a protein should be exported from the nucleus to the cytoplasm.

What is the most common motif found in an NES?

The most common NES consists of a leucine-rich motif

Which protein mediates the transport of cargo from the nucleus to the cytoplasm?

Exportin, a type of karyopherin, mediates this transport.

What is required for the stable assembly of the exportin-cargo complex in the nucleus?

The binding of Ran-GTP is required for the stable assembly of the exportin-cargo complex.

What happens to the exportin-cargo-Ran-GTP complex once it reaches the cytoplasm?

RanGAP1 triggers GTP hydrolysis, causing the complex to disassemble and release the cargo and Ran-GDP.

How does exportin return to the nucleus after delivering its cargo?

Exportin returns to the nucleus via an exposed NLS (Nuclear Localization Signal) and importin.

Are NES sequences necessary and sufficient for nuclear export?

Yes, similar to NLS sequences for import, NES sequences are both necessary and sufficient for nucleus-to-cytoplasm targeting.

What is the fate of importin α after it is imported into the nucleus?

Importin α binds to exportin 2 and is transported back to the cytoplasm to participate in additional rounds of import.

What does 'piggyback' nuclear protein import refer to?

It refers to the mechanism where proteins are imported into the nucleus by binding to importin subunits.

How does the Ran-GTP gradient facilitate the movement of importin β back to the cytoplasm?

The high concentration of Ran-GTP in the nucleus promotes binding to importin β, and the gradient drives the complex toward the cytoplasm where [Ran-GTP] is low.

How do nuclear proteins lacking an NLS enter the nucleus?

They 'piggyback' by binding to a chaperone protein that contains an NLS.

What role does Importin beta play in the import of proteins lacking an NLS?

It mediates the targeting and import of the protein-protein complex (the chaperone-protein cargo) into the nucleus.

What determines the steady-state localization of a protein that shuttles between the nucleus and cytoplasm?

The relative strength of its NLS (Nuclear Localization Signal) and NES (Nuclear Export Signal).

How can a cell regulate the strength of an NLS or NES?

Through post-translational modifications, such as the phosphorylation of specific amino acid residues adjacent to the targeting signal.

What is the purpose of a Co-Immunoprecipitation (Co-IP) assay in this context?

To determine if a specific nuclear-localized protein physically interacts with importin (alpha/beta subunits).

In a Co-IP assay, what is the 'bait'?

The purified epitope-tagged nuclear protein being tested.

In a Co-IP assay, what is the 'prey'?

The purified importin (alpha/beta subunits).

What is the final step of a Co-IP assay used to visualize the isolated proteins?

SDS-PAGE and Coomassie blue staining.

What is the primary function of the objective lens in a brightfield microscope?

To focus both the light diffracted by the specimen and the undiffracted light to form an image.

What is the benefit of using computational deconvolution in microscopy?

It removes background and out-of-focus light, resulting in better contrast and clarity.

What happens at the release of Importin-a

Exposes nuclear export signal (NES) within Importin-a

How does exportin bind to other proteins exported from the nucleus

Via their NESs

Exportin is the karyopherin that mediates

Nucleus-to-cytoplasm transport

Importin-a (or NES containing protein) binds to ___ due to its high concentration in the nucleus

Ran-GTP

What causes the importin a-exportin-Ran-GTP complex to be transported from the nucleus into the cytoplasm?

Ran-GTP gradient

Once in the cytoplasm, GTP on Ran-GTP is hydrolyzed by

RanGAP1

Once hydrolyzed, Ran-GDP is released from exportin which then

releases importin-a or other NES containing proteins

After release, Ran-GDP moves back into the nucleus due to _ and is converted to ___ by _

Gradient, Ran-GTP, RCC1

After release, exportin moves back

into nucleus via exposed NLS for another round of export

Some nuclear proteins do not have an NLS, how do they get to the nucleus?

Pigggyback via a chaperone with an NLS, binds in the cytoplasm

Targeting and import of protein-protein complexes into the nucleus is mediated by ___ as usual

Importin

If a protein that shuttles between the nucleus and cytoplasm has NLS > NES, where will the majority go

Nucleus

Process of In vitro Co-IP Assay

1. Mix bait and prey in vitro, 2. add agarose beadds coated with anti-epitope tag IgGs, 3. isolate beads along with all associated proteins (centrifugation), 4. SDS-PAGE and Coomassie blue staining

Main components of a standard brightfield microscope

Light source, condenser lens, stage, objective and ocular lenses, detector