lecture 22 (RR10): Nucleo-Cytoplasmic Transport

Question 1

What are nuclear pore complexes?

Answer 1

Nuclear pore complexes resemble baskets, and are the sites of nucleocytoplasmic transport
* Have to move from the nucleus into the cytoplasm in order for the machinery, ribosomes and associated proteins, to translate those mRNAs into those gene products.
* Need to get mRNA into cytoplasm so it can be translated to make proteins.
* Need to transport it through the nuclear pore
* Spherical figures with many pores
* Regions that allow for movement through the inner and outer membrane seen in photo
- Cytoplasmic face looking down
- Nucleoplasmic face looking up

Question 2

Nuclear Pore Complex Structure

Answer 2

The nuclear pore complex is a highly ordered structure
* Nuclear membrane is full of “baskets” that ensure movement between the cytoplasm and the nucleoplasm
* On the plasmic side, you have extended filaments that extend off the basket and a pore that goes through both membranes. And then a basket like structure.
* They act as gate-keepers
* Small molecules can freely diffuse through the nuclear pore
* Larger proteins and protein complexes cannot get through the pore. They have to go through an assisted manner.
* Large spliceosome complexes can’t get into the cytoplasm

Question 3

Nuclear Pore Complex (NPC) Size?

Answer 3

• Ensure movement in nuclear membrane
• It’s huge: 125 megadaltons or 30x bigger than a ribosome
• Composed of ~50 (yeast) to ~100 (vertebrates) different proteins
• Molecules up to ~60 kDa can freely diffuse through the NPC. Bigger molecules and multimolecular complexes (RNPs) must be actively transported.

Question 4

Transport through the NPC

Answer 4

Transport through the NPC depends on reversible hydrophobic interactions
* Nucleoporins are proteins that make up the nuclear pore
* FG nucleoporins are hydrophobic
- They have FG repeats that separate the hydrophilic regions
- FG repeats end up forming a hydrophobic place in a mesh of hydrophilic regions within the nucleoporin
- Because FG repeats are hydrophobic, they will be used by hydrophobic proteins as they make their way through the nuclear pore
- A protein travelling through the pore will interact with the FG repeats
- Because they have lots of FG repeats they are disordered.
→ This gives rise to a gel-like substance within the nuclear pore that blocks the diffusion of the larger molecules in and out through the pore and therefore it requires specific transporters to take large molecules in and out.
→ Transporters interact with the FG repeats and allow it to go through the nuclear pore carrying specific cargoes.

Question 5

Protein import through the NPC (mechanism relevant to RNA export)

Answer 5

• All nuclear proteins are synthesized in the cytoplasm and imported through NPCs
  → Protein has to be made in the cytoplasm and it makes its way to the nucleus in order to carry out appropriate functions
• These proteins contain a nuclear localization signal (NLS)
• These sequences can be just a few residues on the polypeptide which can be recognized telling the protein what has to go into the nucleus

Question 6

Addition of an NLS to a cytoplasmic protein

Answer 6

Addition of an NLS to a cytoplasmic protein targets it to the nucleus
* Used DNA viruses (SV40) to understand nuclear processes. A mutation affected the region of T-antigen that corresponds to a stretch of amino acids that was responsible for telling the cell that T-antigen has to go into the nucleus (NLS).
* Pyruvate kinase is usually present in the cytoplasm
* When you put an NLS on it – it goes into the nucleus
* It’s sufficient to put a target protein into the nucleus
* Usually has a lot of lysine and arginine.

Question 7

Proteins required for nuclear import

Answer 7

• It requires two groups of proteins: Ran and importins
• Ran: a monomeric G protein that exists in two conformations, one when bound to GTP and a different one when bound to GDP.
  → In its GDP-bound state, it will interact with the cargo (importins)
  → GAP = positive regulator
  → GEF = negative regulator
• Nuclear Transport Receptors (importins): these proteins bind to NLS domains present on cargo proteins to facilitate transport through the pore by association with FG-nucleoporins. Brings the proteins to the cytoplasm where it can carry out important functions.
  → It interacts with Ran
• In the cytoplasm, Ran GDP will interact with the cargo to form a ternary complex and will make its way into the nuclear pore

Question 8

Mechanism for nuclear import of NLS-containing cargo proteins
https://achieve.macmillanlearning.com/courses/h5wo7t/e-book good video explanation in 13.6

Answer 8

• There’s a concentration gradient between Ran GDP and its GTP bound state
• A driving force will move proteins translated in the cytoplasm through the nuclear pore and to its nuclear destination through a concentration gradient. There is a high concentration of import complex in the cytoplasm and a low concentration in the nucleoplasm.
• Once in the nucleus, Ran GDP is confronted by a GEF (GEF always present in nucleosome bound to the chromosomes)
• A GEF (Guanine Nucleotide Exchange Factor) will kick out the GDP and put a GTP – the complex changes and lets go
• Cargo complex made up of target protein and the importin. Ran GP will interact with the cargo complex, which will lead to a change in confirmation and it will let go of the cargo.
• Ran GTP will bind to importin and because of the concentration gradient, it will take the importin back into the cytoplasm.
• In the nucleus, there is a high concentration of Ran GTP/importin complex and a low concentration of Ran GDP and that’s what drives the concentration gradient
  cytoplasm through the nuclear pore and via interactions with FG repeats on FG nucleoporins
• Once the complex returns to the cytoplasm, Ran GTP is met by a GAP (GTPase Activating Protein) – this will hydrolyze GDP to GTP
• Ran GTP is high in the nucleus and low in the cytoplasm – GDP is high in the cytoplasm and low in the nucleus
• When Ran GTP is hydrolyzed, it lets go of the importin so it can interact with another protein cargo

1) Importin binds to the NLS of the cargo in the cytoplasm 2) it passes through the nuclear pore as the importin interacts with the FG repeats in the nuclear pore to allow for it to pass though (carrying the cargo) 3) Once the importin + cargo complex enters the nucleus a Ran GTP binds to the importin and changes the importin conformation which displaces the NLS and so the cargo is released in nucleoplasm. 4) The Ran GTP + importin complex is transported through the nuclear pore and into the cytoplasm. 5) A GAP factor changes the GTP to GDP on Ran by hydrolysis. This causes a conformational change to the importin and releases it so that it can start all over again. 6) Ran GDP is transported back into the nucleus where GEF causes the Ran to release the GDP and exchange it by a GTP. *The direction of the transport depends on the localization of RanGEF, predominantly in the nucleoplasm, and Ran GAP, predominantly in the cytoplasm.

Question 9

Export of proteins from nucleus into the cytoplasm

Answer 9

• Form a trimolecular cargo complex (trimeric complex) in the nucleus which will include a Ran-GTP and an exportin which will bind to a cargo
• It will then go through the nuclear pore using the FG repeats in the FG nucleoporins (exportin will interact with the FG)
• Again, this is due to the concentration gradient.
• Eventually will get into the cytoplasm
• Once in the cytoplasm, GAP recognizes GTP and will hydrolyze it to GDP
• A conformational change will take place and all the contents will be released into the cytoplasm.

Ran-dependent mechanism for nuclear export of cargo proteins containing a leucine-rich nuclear-export signal (NES). In the nucleoplasm (bottom), the protein exportin 1 binds cooperatively to the NES of the cargo protein to be transported and to Ran GTP . After the resulting cargo complex diffuses through an NPC via transient interactions with FG-repeats in FG-nucleoporins, the GAP associated with the NPC cytoplasmic filaments stimulates GTP hydrolysis, converting Ran GTP to Ran GDP. The accompanying conformational change in Ran leads to dissociation of the complex. The NES-containing cargo protein is released into the cytosol, whereas exportin 1 and Ran GDP are transported back into the nucleus through an NPC. Ran-GEF in the nucleoplasm then stimulates conversion of Ran GDP to Ran GTP.

Question 10

How are the 3 different RNAs (tRNA, rRNA and mRNAs) exported?

Answer 10

Some RNAs are exported through associating with Ran:
tRNAs:
* Exportin t functions to export tRNAs. It binds fully processed tRNAs and Ran-GTP and passes through NPCs.
* The complex dissociates in the cytosol when it interacts with Ran-GAP (carries out the process exactly like exportin 1).
**rRNAs: **
* Export of ribosomal subunits requires Ran.
mRNAs:
* Some specific mRNAs that associate with particular hnRNP(host of different RNAs) proteins (HIV Rev for example) can be exported through association with Ran.
* Most mRNAs are exported in a Ran-independent process using an mRNA exporter

Question 11

The mRNA exporters

Answer 11

• Consists of two subunits (Nxf1/Nxt1)
  → Together they bind RNA cooperatively with specific mRNP proteins including the SR proteins
  → They form a domain that interacts with FG repeats in FG-nucleoporins
• Transport of mRNP through the NPC is Ran- independent
• They are critical for moving the mRNAs that have been matured (gone through transcription, processing and polyadenylation) to move on to export.

Ran-independent nuclear export of mRNAs. 1) The heterodimeric NXF1/NXT1 complex binds to mRNA-protein complexes (mRNPs) in the nucleus. 2) NXF1/NXT1 acts as a nuclear transport receptor and directs the associated mRNP to the central channel of the NPC by transiently interacting with FG-nucleoporins. 3) An RNA helicase (Dbp5) located on the cytoplasmic side of the NPC removes NXF1 and NXT1 from the mRNA in a reaction that is powered by ATP hydrolysis. Free NXF1 and NXT1 proteins are recycled back into the nucleus by the Ran-dependent import process depicted above.

Question 12

mRNP packaging in Balbiani Rings

Answer 12

• Insect polytene chromosomes (Balbiani Rings) provide a system where transcription and mRNP export can be microscopically imaged.
  → Polytene = multiple copies of DNA (they make lots)
• They form sticky proteins that allow them to stick onto larvae.
• The little things coming of the DNA are little mRNAs that are being wound up into heterogeneous nuclear RNPs (hnRNPs) that will give rise to an mRNA ribonucleoprotein (mRNP).
• These mRNP make their way to the nuclear basket where there is a specific polarity of these structures that allows for the mRNA to go through the pore and immediately is greeted by the ribosomes.
• Since ribosomes associate with the mRNA while it is still being exported, the 5’ end must be exported first (5’ end is immediately accessible to ribosomes so that it can start to translate).

a) The gradual increase in size of the hnRNPs reflects the increasing length of RNA transcripts at greater distances from the transcription start site. T (b) Model of the morphology of the BR hnRNP as it grows in length during its transcription. Following processing and cleavage/polyadenylation of the pre-mRNA, the resulting ribonucleoprotein particle, referred to as an mRNP, appeared like a microscopic croissant by EM. (c) Model for the transport of BR mRNPs through the nuclear pore complex (NPC) based on electron microscopic studies. Note that the curved mRNPs appear to uncoil as they pass through nuclear pores. As the mRNA enters the cytoplasm, it rapidly associates with ribosomes, indicating that the 5' end passes through the NPC first.

Question 13

Model of mRNP export

Answer 13

* mRNA exporter immediately interacts with proteins on the mRNA once it has been matured. On nucleoplasmic side, RNA is decorated with all the important proteins.
* mRNA exporter with the two subunits are bound to the SR proteins on the mature mRNA and they follow the arrow into the cytoplasm
* It must go in a specific manner. It goes through the NPC thanks to the interactions of exporter (NXF1, NTX1) with the FG repeats.
* RNA helicase will rip everything off the RNA molecule so that it will liberate the exporter, all the SR proteins, all proteins bound to the mRNA in the nucleus, will get ripped off and will make their way back into the nucleus to get recycled
* This takes place through normal nuclear import
* Once in the cytoplasm, mRNAs take on a completely different group of proteins that will bind to them. Replaced by cytoplasmic proteins which are required to protect the RNA and direct the next steps for protein synthesis. Called cytoplasmic remodelling → changing the proteins that were present in nucleus and exchanging them for proteins that very often carry out the same kinds of functions, but on the cytoplasmic side.
- mRNAs are completely remodelled
- PABPII bound to the poly A tail goes back and stays in the nucleus
- Another PABP will take over in the cytoplasm – PABPCI which binds to the poly A tail
- A number of other factors associate with the mRNA on the cytoplasmic side that would not associate with it on the nuclear side
* mRNP goes through remodelling stage where proteins are replaced with cytoplasmic proteins that may bind to similar structure but carry out very different functions in the cytoplasm
* Since ribosomes associate with the mRNA while it is still being exported, the 5’ end must be exported first.

Question 14

Pre-mRNAs in the export system?

Answer 14

Pre-mRNAs are excluded from the export system
* Only fully spliced mature mRNAs get exported; mechanisms of this restriction are not fully understood
* This phenomenon may be associated with the activity of a protein bound to a nucleoporin that actively blocks pre-mRNAs from leaving the nucleus