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What Is Nuclear Transport?

Nuclear transport is, essential, the movement of carge (mRNA, small proteins and select molecules) between the nucleus and the cytoplasm of the cell.

This process involves the movement of cargo through the multiprotien nuclear pore complex shown previously.

It is VERY IMPORTANT that you realise that the nuclear pore complex (NPC) does NOT operate in the same way as a channel protein.

What is the nuclear pore complex?

It is a complex comprised of 30 different proteins called nucleoporins (Nups) which will be discussed later.

Important Bits and What They Do:

Spoke Rings: rings on both the nuclear and cytoplasmic side of the nuclear envelope which may help to stabalise the NPC and may also form a site of attachment of the complex to the cytoskeleton.

Nuclear Basket: protein filaments ("spokes") attached to the spoke ring on the nuclear side of the NPC. Their function is unknown but it has been hypothesised that due to its shape it may be involved in the transport of cargo via vaults.

Cytoplasmic Filaments: protein fibrils that extend into either the cytoplasm of nucleus to "pick up" or guide molecules toward the nuclear pore (imagine a jellyfish eating)

Cargos and Transporters

The main nuclear transport receptor family is known as karyopherins. This includes the proteins Importin and Exportin which are also called transportins.

These proteins are usually very large (90-130 kDa) acidic protein sharing 15 -25% protein sequence identity.

they contain an N-terminal Ran-GDP binding domain, a C-terminal cargo binding domain and capacity to bind the components of the nuclear pore complex (NPC)

molecule must associate with transport receptor (karyopherins)

- recognised by specific amino acid sequence called the nuclear localisation sequence (NLS) on cargo protein

- Importin beta helps transport cargo through NPC

- Importin alpha bings

There are some proteins that are imported into the nucleus without aid of a soluble protein family member. Also there are two families of RNA cargo, mRNA and ribosomal subunits, whose export from nucleus into cytoplasm can be mediated without any help of the protein receptor family members.

Many classes of cargo bind directly to a particular receptor while others bind indirectly via adaptor proteins.

These are proteins with classic basic nuclear localisation signal (NLS) that bind to importin alpha family of adaptors.

Adaptor utilising cargos are imported to the nucleus from the cytoplasm via the importin beta receptor that shares an N-terminal importin beta binding domain.

How Does Cargo Move Through NPC

1) Virtual Gate: NPCs exclude large molecules except those chaperoned by transport factors e.g. transcription/ signalling transduction factors

2) Selective Phase: NPCs function as permeability barrier for inert molecules but become selectively permeable for nuclear transport receptors and receptor cargo complexes

3) Reduction of Dimesionality: NTRs transport proteins by sliding on the surface of FG motifs. Small molecules can pass through narrow pore but binding of specific complex can cause NPC conformation change to make it larger than normal.

*transport receptors can intermittently widen filter pores while other proteins can't*

S. cerevisiae (brewer's yeast) encodes 14 receptors, 9 of these are import receptors, 4 are export receptors, 1 is uncharacterised. Of course in multicellular eukaryotes we have more adaptors and receptors than in S. cerevisiae. At least 6 alpha importin family members exist in higher eukaryotes and there are identical by 45 -85 % but they have different expression patterns and signal ways.

It's important to keep in mind that each receptor is not necessarily dedicated to a particular cargo as well as the cargo is not dedicated to a particular receptor. The overall number of signalling appears to be equal to the number of receptors found in the nucleus membrane.

Extra Info

Simple receptor - import cargo mechanism is mediated by dimeric complex of two receptors - importin beta and importin 7 in which importin 7 acts as a co-receptor. Important to mention can be co-receptor or receptor mode , and as receptor it functions in ribosomal protein import.

A co-receptor is a cell surface receptor that binds a signalling molecule in addition to a primary receptor in order to facilitate ligand recognition and initiate biological processes, such as entry of a pathogen into a host cell

Direction

Ran-GTPase plays a key role in regulating direction of transport. Ran is localised inside the nucleus at steady state like all small GTPases. Talking about Ran, it has very low nucleotide hydrolysis and exchange activities on its own. These are strongly stimulated by a GTPAse-activating protein (RanGAP) and by a guanine nucleotide exchange factor (RanGEF). This illustrates that GTP is found more prominently in the nucleus rather than the cytoplasm and does not dissociate randomly.

Micromolecules cross the nuclear membrane though aqueous channels formed by nuclear pore complexes. These elaborate protein structures of 66MDa (S. cerevisiae) or 125 MDa (higher eukaryotes) are composed of up to about 50 (S. cerevisiae) or 100 (higher eukaryotes) different proteins termed nucleoporins (Nups) .

The diameter of the channel appears to be very flexible from 10 nm - 25 nm it can expand to translocate large cargos.

About half of the Nups contain multiply repeated phenylalanine - glycine (FG) motifs. The FG motif could be predictive of a role in transport by helping to transport macromolecules inside the nucleus and outside the nucleus.

Export: transport from nucleus to cytoplasm

Majority are cellular RNA molecules are synthesised in the nucleus and have to be removed to cytoplasm to carry out their particular functions. These exported RNA's can be grouped into 4 major classes; mRNA, rRNA, tRNA and U snRNA (a small nuclear RNA combined with primer RNA which is essential for the formation of mRNA).

RNA export involves movement from site of transcription to NPC and translocation through the NPC. Each type of RNA blocks its own export when present in excess but not that of the others indicating the existence of class specific export factors. One of the general features of RNP transport is the ability of transport machineries to discriminate between immature and mature RNPs. This ensures that non-functional RNPs do not get into cytoplasm where they would have potenitally negative effects.

*Important to mention that small RNAs are similar to export of protiens and even involves the same of similar karyopherin transport factors. Like mRNP export, the export of both the 40S and 60S has to be very rapid.

Exportin binds to cargo and RanGTP diffuses through pore to cytoplasm. Exportins require GTP to bind to cargo (Importins need it to dissociate). Mature mRNA's and tRNA's are exported by specific exporter proteins i.e. mutated RNA due to inability to bind

Diseases That Affect Nuclear Transport

HIV:

HIV-1MA(matrix) (regulates viral replication) and IN(integrase) carry a conventianal nuclear localisation signal and may use the importin pathway for nuclear import.

HIV-Vpr (HIV-viral protein R) promotes entry of nucleic acids into non-dividing macrophages and it also causes a G2 cell-cycle arrest. Vpr does not carry the nuclear localistaion signal but MA and IN do. Even though it doesn't have a NLS Vpr still shows karyophilicity and governs entry into the nucleus.

Experiments in yeast have shown that overepxression of Vpr or Importin-beta blocks the nuclear transport of mRNA into yeast suggesting the role of HIV-Vpr in hijacking the cell's replication machinery.

Borna Disease:

Borna disease virus (BDV) is a negative-strand RNA virus which infects a variety of warm-blooded animals. Infection can be asymptomatic or cause a variety of behavioral disorders. Unusual features of Borna disease virus biology include nuclear localization of replication and transcription; diverse strategies for regulation of gene expression; and interaction with signaling pathways resulting in subtle neuropathology.

The phosphoproteins of non-segmented negative strand (NNS) RNA viruses are essential cofactors for virus transcription and replication. Their phosphorylation by cellular kinases influences the ability of phosphoproteins to form homomultimers, bind other viral proteins and serve as transcriptional activators. Although there are no direct data concerning the role of phosphoprotein in the BDV lifecycle, it is postulated to have a similar function to other viral phosphoproteins.

Phosphoprotein contains a strong bipartite NLS at its amino-terminus and appears to have weaker NLS motifs toward its carboxyl terminus. BDV also has a similar nuclear export sequences to other viral export proteins such as HIV-1 Rev

http://onlinelibrary.wiley.com/doi/10.1002/rmv.300/pdf

Nuclear Transport

NUPS

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