As described in the earlier post, the Nuclear Pore Complex (NPC) serves two key purposes:
Once transported, the NPC must also ensure that the molecules are retained in their respective cytoplasmic and nuclear compartments. This calls for regulation of nuclear transport at multiple stages.
At any given moment, there occurs a rapid movement of thousands of proteins and RNAs into and out of the nucleus. How is this deed accomplished?
Well, for starters, it requires transport signals namely Nuclear Localization Signal (NLS) and Nuclear Export Signal (NES) for import and export respectively.
Transport Signals
The minimal requirements for any sort of aided translocation of proteins or other molecules are:
As mentioned in the earlier post, the NLS consists of one or two short amino acid sequences that are rich in positively charged amino acids lysine and arginine.
NES
Studies conducted so far have shown that the NES is a loosely conserved motif containing three to four hydrophobic residues and recognized by nuclear export receptors.
So what exactly are these receptors?
Most of the soluble transport receptors come from a family of proteins known as the karyopherins. They are also referred to as importins, exportins, and tranportins) and were the first family of shuttling transport factors to be discovered.
A salient feature of karyopherins are the tandem HEAT-repeats which form a super helix creating arches at the N- and C-terminals that provide extensive interaction surfaces.
While most karyopherins directly bind their cargo molecules, some require an adaptor protein as an additional element in order to recognize the signal sequences. Karyopherin-α known also as importin-α is the most-studied adapter protein.
Karyopherins have three basic functional domains:
So how do these shuttling receptors carry out their fuctions?
The third domain of shuttling receptors are known to have an affinity to bind a particular GTPase called the Ran GTPase.
In vitro binding studies have revealed that dissociation of import complexes are dissociated by RanGTP binding. Conversely, export complexes are formed through RanGTP association. This forms the basis of transport mechanism.
It has been found that the Ran GTPase activating protein (RanGAP) in localized in the cytoplasm whereas the Ran guanine nucleotide exchange factor (RanGEF) in localized in the nucleus. Based on this finding, it has been proposed that cytoplasmic Ran is primarily in the GDP-bound state (RanGDP) while nucleoplasmic Ran is in the GTP-bound state (RanGTP) and that this RanGTP gradient across the two sides of the NPC is essential for directional receptor-mediated transport.
So, does that mean RanGTPase plays a vital role in nucle-cytoplasmic transport of proteins?
Indeed. It had also been hypothesized intitially that Ran plays a vital role in the nuclear import of proteins. This hypothesis was first based on two facts:
Scheme of nuclear import/export of NLS/NES-containing proteins
The general scheme of nuclear import of NLS-containing proteins is as follows:
The general scheme of nuclear export of NES-containing proteins is not much different except that it is in reverse and is as follows:
What role does the NPC play?
Some nucleoporins on the NPC (often referred to as Nups) contain FG repeat regions consisting of multiple small hydrophobic clusters containing FG (Phe-Gly) dipeptides (usually in the form of FG, GLFG or FXFG) separated by hydrophilic linkers.
These FG repeat regions do not posses a secondary structure and instead persist in the form of a dense meshwork of filaments in the central channels thus facilitating the exclusion of macromolecules from the area and at the same time allowing small molecules to pass through. This is the Brownian affinity gating model.
Alternatively, two more models have been proposed so far as to how the selective transport process occurs:
Although poorly understood as compared to those of soluble molecules, the mechanism for nucleo-cytoplasmic transport of integral membrane proteins also requires multiple FG sites on the integral protein in addition to the ones found on a particular Nup called Nup35.
The diversity of NLS/NES, receptors and numerous mediators in the transport process all imply that the transport of proteins and other material into and out of the nucleus be regulated in terms of growth of the cell, its proliferation and differentiation or its stage in the cell cycle.
Some of the various means of regulation is as follows:
More information about transport through the nuclear pore complexes can be found here
- to form a barrier of selective permeability within the pore (preventing the passage of nonspecific macromolecules and at the same time allowing the free diffusion of water molecules, sugars and ions) and
- to facilitate transport of selected macromolecules across it
Once transported, the NPC must also ensure that the molecules are retained in their respective cytoplasmic and nuclear compartments. This calls for regulation of nuclear transport at multiple stages.
At any given moment, there occurs a rapid movement of thousands of proteins and RNAs into and out of the nucleus. How is this deed accomplished?
Well, for starters, it requires transport signals namely Nuclear Localization Signal (NLS) and Nuclear Export Signal (NES) for import and export respectively.
Transport Signals
The minimal requirements for any sort of aided translocation of proteins or other molecules are:
- a transport signal (specific amino acid sequences containing all the information required to target a protein into or out of the nucleus) and
- an import/export receptor or shuttling receptor for that transport signal (soluble cytoplasmic molecules that bring cargo to the NPC directly or through means of an adaptor protein)
As mentioned in the earlier post, the NLS consists of one or two short amino acid sequences that are rich in positively charged amino acids lysine and arginine.
NES
Studies conducted so far have shown that the NES is a loosely conserved motif containing three to four hydrophobic residues and recognized by nuclear export receptors.
So what exactly are these receptors?
The Karyopherin Family of Transport Receptors
Most of the soluble transport receptors come from a family of proteins known as the karyopherins. They are also referred to as importins, exportins, and tranportins) and were the first family of shuttling transport factors to be discovered.
A salient feature of karyopherins are the tandem HEAT-repeats which form a super helix creating arches at the N- and C-terminals that provide extensive interaction surfaces.
While most karyopherins directly bind their cargo molecules, some require an adaptor protein as an additional element in order to recognize the signal sequences. Karyopherin-α known also as importin-α is the most-studied adapter protein.
Karyopherins have three basic functional domains:
- a cargo-binding domain
- NPC-binding domain(s)
- Ran GTPase binding domain at the amino-terminus (Ran GTPase is a small monomeric Ras-like GTPase)
So how do these shuttling receptors carry out their fuctions?
Ran-Dependent nucleocytoplasmic transport of proteins and other molecules
The third domain of shuttling receptors are known to have an affinity to bind a particular GTPase called the Ran GTPase.
In vitro binding studies have revealed that dissociation of import complexes are dissociated by RanGTP binding. Conversely, export complexes are formed through RanGTP association. This forms the basis of transport mechanism.
It has been found that the Ran GTPase activating protein (RanGAP) in localized in the cytoplasm whereas the Ran guanine nucleotide exchange factor (RanGEF) in localized in the nucleus. Based on this finding, it has been proposed that cytoplasmic Ran is primarily in the GDP-bound state (RanGDP) while nucleoplasmic Ran is in the GTP-bound state (RanGTP) and that this RanGTP gradient across the two sides of the NPC is essential for directional receptor-mediated transport.
So, does that mean RanGTPase plays a vital role in nucle-cytoplasmic transport of proteins?
Indeed. It had also been hypothesized intitially that Ran plays a vital role in the nuclear import of proteins. This hypothesis was first based on two facts:
- induced mutations in Ran resulted in impairment of nuclear import as observed in vivo
- Ran is an essential cytoplasmic component required for transport of NLS-containing cargo molecules as observed in vitro.
Scheme of nuclear import/export of NLS/NES-containing proteins
 |
| Taken from Cold Spring Harb Perspect Biol 010;2:a000562 |
- Formation of the nuclear import receptor:adaptor protein:NLS-containing protein complex (import complex) in the presence of RanGAP and low concentrations of RanGTP in the cytoplasm
- Docking of the import complex to cytoplasmic fibrils
- Transition across the central channel or pore
- RanGTP-stimulated release of adaptor protein:NLS-containing protein into the nucleus
- Retention of nuclear import receptor in the NPC and its eventual transport back into the cytoplasm in complex with RanGTP
- Transport of adaptor protein also in complex with RanGTP back to the cytoplasm after its dissociation from NLS-containing protein
The general scheme of nuclear export of NES-containing proteins is not much different except that it is in reverse and is as follows:
- NES-containing proteins are exported from the nucleus in complex with RanGTP
- On the way out of the NPC, all exported complexes dissociate due to GTP hydrolysis in Ran stimulated by collaboration of RanBP and RanGAP (RanBP displaces RanGTP from export receptor allowing RanGAP to trigger RanGTP hydrolysis to RanGDP)
- Import of RanGDP back to the nucleus (mediated by factors that are beyond the scope of this post)
- Once in the nucleus, RanGEF converts RanGDP back into RanGTP
What role does the NPC play?
Some nucleoporins on the NPC (often referred to as Nups) contain FG repeat regions consisting of multiple small hydrophobic clusters containing FG (Phe-Gly) dipeptides (usually in the form of FG, GLFG or FXFG) separated by hydrophilic linkers.
These FG repeat regions do not posses a secondary structure and instead persist in the form of a dense meshwork of filaments in the central channels thus facilitating the exclusion of macromolecules from the area and at the same time allowing small molecules to pass through. This is the Brownian affinity gating model.
Alternatively, two more models have been proposed so far as to how the selective transport process occurs:
- Selective phase hypothesis - similar to the Brownian gating model, explains that the electron-dense core channel of the NPC acts as a network of hydrophobic interactions among FG-repeats that allow the passage of small molecules and restricting larger ones.
- Affinity gradient mechanism - suggests that the transfer of receptor:cargo complexes between nucleoporins is preferred in the direction of the nucleoplasmic side of the NPC as a result of the increasing receptor-nucleoporin affinities in that direction. This model implies that a receptor:cargo complex is released from one nucleoporin binding site only upon binding to a second nucleoporin site.
Mechanism of nucleo-cytoplasmic transport of integral membrane proteins
Ran-Independent nucleo-cytoplasmic transport of proteins and other molecules
Alternative pathways of translocation for the classical Ran-dependent transport have been described. These are:
- β-catenin-mediated pathway
- Ca2+ binding protein Calmodulin (CaM-mediated pathway)
Regulation of nucleo-cytoplasmic transport
The diversity of NLS/NES, receptors and numerous mediators in the transport process all imply that the transport of proteins and other material into and out of the nucleus be regulated in terms of growth of the cell, its proliferation and differentiation or its stage in the cell cycle.
Some of the various means of regulation is as follows:
- amplified/diminished importin/exportin interations with the NLS/NES-containing cargo
- masking of NLS/NES-containing cargo molecules from recognition by importin/exportin
- retention of proteins in cytoplasm or nucleus until required
- co-transport and changing the cargo-binding properties of importins/exportins
- changing the variety of nucleoporins
- changing the variety of importins/exportins
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