Nucleus-Cytosol Transport
Membrane Transport

Author: Paolo Pescarmona
Date: 20/11/2008

Description

The nuclear envelope (NE), which contains the DNA and defines the nuclear compartment, consists of two concentric membranes; across them, during interphase there is a continuous bidirectional exchange of molecules, which is necessary to carry out basic biological processes:

  • proteins whose action takes place in the nucleus (e.g. histones, polymerases, transcription factors, proteins belonging to RNP complexes ) are imported
  • RNAs are exported to the cytoplasm for the translation process

1- NUCLEAR PORE COMPLEXES (NPC)

The NE of eukaryotes is pierced by the NPCs, which constitute a selectively permeable barrier to nucleocytoplasmic trafficking.
The NPC is formed by about 30 different proteins dubbed nucleoporins (Nups), present in a copy number of 8 or multiple of 8 and disposed according to an octagonal symmetry; it can be estimated that a mammalian NPC consists of 500-1000 Nup copies. The main mass of the NPC forms a cylindrical structure, composed of a central core consisting of 8 spoke-ring complexes sandwiched between nuclear and cytoplasmic ring structures that are embedded in the NE; attached to the cytoplasmic face are 8 flexible filaments protruding into the cytoplasm, while fibrils emanating on the nuclear side converge at their distal end to form a cagelike structure (the nuclear basket).

This structure contains one or more hydrophilic inner channels through which small soluble molecules such as ions and metabolites can passively diffuse, so that the NE is freely permeable to them. Several cellular proteins and RNA molecules are too large to diffuse rapidly, so they bind specific transport receptors that, by interacting with the NPC, accelerate their passage or allow the passage of macromolecules that would not pass at all because of their size (molecular weight > 30 kDa). The transported macromolecules are called cargoes. Each NPC mediates about 1000 translocation events/s. Macromolecules do not need to be unfolded to pass through NPCs, anyway large RNP complexes likely undergo partial unraveling during transport.

The majority of Nups are located symmetrically on the nuclear and cytoplasmic faces of the NPC with only a few being restricted to one side: the latters are involved in the specific interaction with transport receptors through their protruding fibrils. Although many Nups are common to several trafficking pathways, others are only required for a specific one: different cargo molecules appear to be transported via different routes using particular subsets of Nups, to which the implicated receptors associate in a preferential manner because of a higher affinity. NPC composition in terms of Nups is quite homogeneous and it seems that a single NPC may mediate the transport of different types of macromolecules (different proteins and RNAs), that is it is not specialized for the transport of a specific one; anyway, upon appropriate stimuli and according to the needs of a particular situation the cell can make the transport of certain cargos easier by inducing specific Nups and therefore modyfing NPC composition up to a point.
The NE of a typical mammalian cell contains 3000-4000 NPCs, but their number may vary during different times of interphase. The more active transcription is, the greater the number of the NPCs becomes, so an increase in NPCs occurs during cellular growth and high metabolic activity; this is achieved by enhancing the production of Nups in specific points of the cell cycle.

2- TRANSPORT RECEPTORS

Transport recepors identify their substrate cargoes by recognizing specific signal sequences, that are thought to form a loop on the surface of the cargo molecule:

  • nuclear localization signals (NLS) , present on nuclear proteins; they are recognized by import receptors (importins)
  • nuclear export signals (NES) , present on proteins and RNA molecules that have to be addressed to the cytoplasm; they are recognized by export receptors (exportins).
    Importins and exportins usually bind their cargo molecules directly, but they also may interact with them via adaptor proteins that recognize the signal sequence.

Beta- karyopherin(beta-kap) superfamily
Most transport receptors belong to the Beta-kap superfamily, that mediate the majority of nucleocytoplasmic transport processes in metazoa and yeast; each b-kap can bind a great number of various substrates with structurally similar NLS/NES.
Beta-kap- mediated transport requires energy, which is provided by GTP hydrolysis performed by Ran small GTPase(biology) ; in contrast to RanGDP, RanGTP does bind to b-kap. Conversion between the two Ran conformational states is allowed by two proteins:

  • RanGAP, which triggers rapid GTP hydrolysis and localizes to the cytoplasm so that the cytoplasm contains mainly RanGDP
  • RanGEF, which promotes the exchange of GDP with GTP and localizes to the nucleus, so that the nucleus contains mainly RanGTP

The gradient of the two Ran conformational states confers directionality to the transport; energy is not required directly for the movement of transport complexes through the NPC, but for creating the asymmetrical distribution of RanGTP/RanGDP across the NE.
b-kaps possess binding sites for:

  • Nups: both nuclear and cytoplasmic fibrils and the interior of the central channel have a lot of FG (Phe/Gly-rich) repeats (more than 1000 copies within one NPC) which serve as docking sites for most transport receptors (all b-kaps, but also other unrelated receptors). Transport factors can interact with Nups both in the presence and in the absence of bound cargoes and are therefore able to shuttle through the NPC; importins often have a higher affinity to nuclear Nups, and exportins to cytosolic Nups. The interaction between receptors and FG-repeats is weak, indeed translocation through NPC is thought to involve transient, sequential and stochastic associations/dissociations of transport factors with neighbouring Fg-repeats throughout the central pore.
  • RanGTP
  • cargo NLS/NES

Beta-kap-mediated import: after docking and translocation of the cargo-importin complex to the nuclear side of the NPC, high-affinity binding of RanGTP to the importin determines cargo release because it induces a conformational change incompatible with cargo binding. Then the free importin bound to RanGTP is carried back to the cytosol through NPC, where a Ran-binding protein (Ran-BP) moves RanGTP from the importin so that RanGAP can trigger GTP hydrolysis .

Beta-kap-mediated export: since most exportins have a low affinity for RanGTP and cargo, both bind cooperatively in the nucleus to form a stable export complex. After docking and translocation of the complex to the cytosolic side of the NPC, the Ran-BP moves RanGTP so that binding affinity decreases and destabilization of the cargo-exportin complex takes place; so the cargo is released, the exportin re-enters the nucleus and RanGAP induces GTP hydrolysis.

NB: in both import and export, RanGDP dissociates from Ran-BP and in metazoa is reimported into the nucleus by the importin NTF2 (which is not a b-kap, it belongs to NXF family); in the nucleus RanGEF replaces GDP with GTP, in order to maintain the directionality gradient.

3- NUCLEOCYTOPLASMIC PROTEIN TRANSPORT

Protein import
The main protein importin in metazoa and yeast is importin-beta (Imp-beta), which is a b-kap and binds its cargos via the adaptor importin-alfa (Imp-alfa); in some cases it also binds cargos directly, it depends on specific NLSs. Impb can also form heterodimers with other importins. The main substrates of Impb are ribosomal proteins, histones, transcription factors, snRNP proteins and some viral proteins.

Protein export
The main protein exportin in metazoa and yeast is CRM1 , which is a b-kap and may bind its cargos directly or via adaptor molecules, it depends on specific NESs; its main substrates are RNA-BPs, RNPs, transcription factors, cell cycle regulators, importin adaptor molecules.

Hierarchical regulation of nucleocytoplasmic transport of proteins
The multistage character of translocation, the great number of mediators, the diversity of NES/NLS, the multicomponent structure of NPC imply a possibility of regulation of the nucleocytoplasmic distribution of different proteins in response to stimuli or the cell cycle stage; many proteins, such as transcription factors involved in basic biological processes, contain both a NES and a NLS and their relative localization in a certain situation or cell cycle stage is determined by a fine regulation. The deregulation of many shuttling pathways occurs in several forms of cancer.

  • 1. Regulation at the level of individual cargo : small-scale changes involving only one particular cargo; for instance post-transcriptional modifications in NLS/NES, such as phosphorylation, can alter its conformation and thus influence the accessibility to the transport receptor or its binding affinity
  • 2. Regulation at the level of transport receptors/adaptors : it has an intermediate effect potentially affecting all cargos recognized by that receptor/adaptor; for example variations of the expression level of a transport factor or tissue specific expression of certain factors
  • 3. Regulation at the level of NPC : it is more global since it influences multiple receptors and thus a large number of cargoes simultaneously; for example, changes in Nup composition of the NPC can modulate the efficiency of translocation of some proteins.

4- NUCLEOCYTOPLASMIC TRANSPORT OF RNA: EXPORT OF RNA MOLECULES

tRNA nuclear export
tRNAs are small molecules, so they can diffuse through the NPC but diffusion is very slow.
The main tRNA exportin in metazoa is exportin-t (Exp-t) , a beta-kap; it binds direcly to tRNA without intervention of adaptor proteins. It binds most efficiently to tRNAs with mature 5’-end and 3’-end and after splicing and chemical modifications, although unspliced tRNA can also be exported. Another beta-kap, exportin-5 (Exp5) can export tRNA molecules by direct binding, however its affinity for t RNA is lower and its main role is pre-miRNA export.

pre-miRNA nuclear export
pre-miRNA, deriving from processing by Drosha of the original stem-loop of transcribed or intron-derived pri-miRNA, are exported to the cytosol by Exp5 .
Artificial shRNAs use the same export pathway to produce siRNAs.

snRNAs nuclear export
The exportin for snRNA is CRM1 , which does not interact directly with the cargo, but requires the cap-binding complex (CBC) and a NES-containing adaptor protein called PHAX . Once in the cytoplasm, snRNA associates with specific proteins to form mature snRNPs, that are reimported into the nucleus where they assemble into the functional spliceosome.

mRNA nuclear export
mRNA export is more complicated than the export of other RNAs, since mRNAs are channelled into the specific export pathway coordinately with their processing and assembly into mRNPs, whose composition changes as they proceed from transcription to processing and export.
The main factor responsible for mRNA export in metazoans is the heterodimer TAP-p15 (TAP belongs to NXF family) and its homologue in yeast is Mex67-Mtr2; CRM1 mediates the export of a few specific cellular mRNAs and certain viral mRNAs.
TAP-p15/Mex67-Mtr2 mediates Ran-independent export. TAP can directly bind mRNA sequences called CTE elements, thus behaving as a RNA-BP, but CTE elements are present only in a set of viral pre-mRNAs; TAP-p15 binds cellular mRNA by protein-protein interaction: the bridging factor between TAP-p15 and mRNA is Aly/REF (*Yra1* is its homologue in yeast). mRNA export factors can be recruited on mRNAs by different mechanisms:

  • Splicing-mediated recruitment : this mechanism is mainly found in metazoa, but in may act also in a few yeast mRNAs derived from intron-containing genes. Aly/REF is recruited by UAP56 (Sub2 in yeast), a helicase involved in early spliceosome assembly; Aly/REF and UAP56 are members of the exon junction complex (EJC) , a complex of proteins involved in splicing that binds upstream of exon-exon junctions
  • Cotrascriptional recruitment : this mechanism is mainly found in yeast, where the majority of genes are intronless and therefore do not undergo splicing. Sub2 and Yra1 are recruited to transcribing genes by THO , a complex involved in transcription elongation that associates with Pol II during transcription; the supercomplex consisting of THO, Sub2 and Yra1 is called TREX . When TREX follows the elongating Pol II along the activated gene, members of the THO subcomplex bind to chromatin, whereas Sub2 and Yra1 associate with nascent mRNA via pre-mRNA factors such as CBC. The TREX complex is removed before nuclear export. A TREX complex exists in mammals as well, but it seems to be recruited in a splicing-dependent and cap-dependent manner only to the 5’-end of the mRNA.
  • Recruitment of export factors to 3’-end formation : both 3’-cleavage and polyadelylation are required for efficient mRNA export; perhaps, in a manner akin to THO, as 3’-processing factors travel with Pol II, to which they associate at the beginning of transcription, they recruit export factors to the growing mRNA.
  • Recruitment of export factors by mRNA “identity elements” : the export factors can also bind directly to the mRNA because they seem capable of recognizing introns and unstructured regions; in this case post-transcriptional processing is not required.

The ATP-dependent RNA-helicase Dbp5 , which is primarily cytoplasmic, is positioned to function in late steps of mRNA export, promoting conformational changes in the mRNP necessary for movement into the cytoplasm and discharging of export factors; its local activation could generate directionality, for example by preventing backsliding of the mRNP.

rRNA nuclear export
The ribosomal subunits are assembled in the nucleolus: processed and mature rRNAs associate with ribosomal proteins, which are imported from the cytoplasm, then the subunits are re-exported to the cytosol. The two ribosomal subunits, 40S and 60S, follow separate export routes and associate only in the cytoplasm.

  • 40S subunit export : poorly understood; CRM1 is involved
  • 60S subunit export : 60S subunits belong to the largest particles that have to pass the transport channels of the NPC; their export is mainly mediated by CRM1 and the adaptor NES-containing protein Nmd3, lately recruited in the nucleus, but several different exportins are thought to act, for example Mex67-Mtr2 in yeast, which uses a different interaction surface from the one used for mRNA to bind the pre-60S subunit

Regulation of RNA nuclear export: a quality control
The transport is greatly dependent on correct post-transcriptional modifications of RNA molecules, since usually only mature and fully processed cargos are transported; so transcription, processing and nuclear export proceed in an orderly fashion to avoid the export of aberrant or immature RNAs.

Bibliography

Atlanta Cook, Fulvia Bono, Martin Jinek Elena Conti
Structural Biology of Nucleocytoplasmic Transport
Annu. Rev. Biochem. 2007.76:647-671

H. Fried and U. Kutay
Nucleocytoplasmic transport: taking an inventory
Cell. Mol. Life Sci. 60 (2003) 1659–1688

Laura J. Terry, Eric B. Shows, Susan R. Wente
Crossing the Nuclear Envelope: Hierarchical Regulation of Nucleocytoplasmic Transport
Science 318, 1412 (2007)

A. V. Sorokin, E. R. Kim, and L. P. Ovchinnikov
Nucleocytoplasmic Transport of Proteins
Biochemistry (Moscow), 2007, Vol. 72, No. 13, pp. 1439-1457

Alwin Köhler and Ed Hurt
Exporting RNA from the nucleus to the cytoplasm
Nature Reviews Molecular Cell Biology Vol. 8 October 2007 (761-773)

Richard Bayliss, Anita H. Corbett, and Murray Stewart
The Molecular Mechanism of Transport of Macromolecules Through Nuclear Pore Complexes
Traffic Vol. 1 (2000), Issue 6, pp. 448–456

Bruce Alberts, Alesander Johnson
Molecular Biology of the Cell (IV Edition) Zanichelli

Comments
2008-11-20T14:34:00 - Paolo Pescarmona

Scheda realizzata da Alberto Peretti e Marta Pezzella

prova

Attachments
fileuserdate
Immagine1.bmpppp2008-11-20T00:00:00
Immagine2.bmpppp2008-11-20T00:00:00
Immagine3.bmpppp2008-11-20T00:00:00
Immagine4.bmpppp2008-11-20T00:00:00
Immagine5.bmpppp2008-11-20T00:00:00
Nucleocytoplasmic_Transport.docppp2008-11-20T00:00:00
AddThis Social Bookmark Button