Nuclear pore
Encyclopedia
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
complexes that cross the nuclear envelope
Nuclear envelope
A nuclear envelope is a double lipid bilayer that encloses the genetic material in eukaryotic cells. The nuclear envelope also serves as the physical barrier, separating the contents of the nucleus from the cytosol...
, which is the double membrane
Endomembrane system
The endomembrane system is composed of the different membranes that are suspended in the cytoplasm within a eukaryotic cell. These membranes divide the cell into functional and structural compartments, or organelles...
surrounding the eukaryotic
Eukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...
cell
Cell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
nucleus
Cell nucleus
In cell biology, the nucleus is a membrane-enclosed organelle found in eukaryotic cells. It contains most of the cell's genetic material, organized as multiple long linear DNA molecules in complex with a large variety of proteins, such as histones, to form chromosomes. The genes within these...
. There are about on average 2000 nuclear pore complexes in the nuclear envelope
Nuclear envelope
A nuclear envelope is a double lipid bilayer that encloses the genetic material in eukaryotic cells. The nuclear envelope also serves as the physical barrier, separating the contents of the nucleus from the cytosol...
of a vertebrate cell, but it varies depending on cell type and the stage in the life cycle. The proteins that make up the nuclear pore complex are known as nucleoporins. About half of the nucleoporins typically contain either an alpha solenoid
Alpha solenoid
An alpha solenoid is a protein fold found in the protein subunits of light-harvesting complexes, particularly in the peridinin chlorophyll proteins of dinoflagellates, and as domains of larger eukaryotic proteins that make up the nuclear pore complex. The fold consists of alpha helices arranged in...
or a beta-propeller fold, or in some cases both as separate structural domains. Each NPC contains at least 456 individual protein molecules and is composed of 30 distinct proteins (nucleoporins). The other half show structural characteristics typical of "natively unfolded" proteins, i.e. they are highly flexible proteins that lack ordered secondary structure. These disordered proteins are the FG nucleoporins, so called because their amino-acid sequence contains many repeats of the peptide phenylalanine
Phenylalanine
Phenylalanine is an α-amino acid with the formula C6H5CH2CHCOOH. This essential amino acid is classified as nonpolar because of the hydrophobic nature of the benzyl side chain. L-Phenylalanine is an electrically neutral amino acid, one of the twenty common amino acids used to biochemically form...
—glycine
Glycine
Glycine is an organic compound with the formula NH2CH2COOH. Having a hydrogen substituent as its 'side chain', glycine is the smallest of the 20 amino acids commonly found in proteins. Its codons are GGU, GGC, GGA, GGG cf. the genetic code.Glycine is a colourless, sweet-tasting crystalline solid...
.
Nuclear pore complexes allow the transport of water-soluble molecules across the nuclear envelope. This transport includes RNA
RNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
and ribosomes moving from nucleus to the cytoplasm and protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s (such as DNA polymerase
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....
and lamin
Lamin
Nuclear lamins, also known as Class V intermediate filaments, are fibrous proteins providing structural function and transcriptional regulation in the cell nucleus. Nuclear lamins interact with membrane-associated proteins to form the nuclear lamina on the interior of the nuclear envelope...
s), carbohydrates, signal molecules
Signaling molecule
A signaling molecule is a chemical involved in transmitting information between cells. Such molecules are released from the cell sending the signal, cross over the gap between cells by diffusion, and interact with specific receptors in another cell, triggering a response in that cell by activating...
and lipids moving into the nucleus. It is notable that the nuclear pore complex (NPC) can actively conduct 1000 translocations per complex per second. Although smaller molecules simply diffuse
Diffusion
Molecular diffusion, often called simply diffusion, is the thermal motion of all particles at temperatures above absolute zero. The rate of this movement is a function of temperature, viscosity of the fluid and the size of the particles...
through the pores, larger molecules may be recognized by specific signal sequences and then be diffused with the help of nucleoporins into or out of the nucleus. This is known as the RAN cycle
Ran (biology)
Ran is a small 25Kda protein that is involved in transport into and out of the cell nucleus during interphase and also involved in mitosis. It is a member of the Ras superfamily....
. Each of the eight protein subunits surrounding the actual pore (the outer ring) projects a spoke-shaped protein into the pore channel. The center of the pore often appears to contain a plug-like structure. It is yet unknown whether this corresponds to an actual plug or is merely cargo caught in transit.
Size and complexity
The entire nuclear pore complex (NPC) has a diameter of about 120 nanometers, the diameter of the opening (functional diameter) is about 9 nanometers wide and its "depth" is about 200 nanometers. It had been suggested that the pore can be dilated to around 26 nanometers to allow molecule passage. The molecular massMolecular mass
The molecular mass of a substance is the mass of one molecule of that substance, in unified atomic mass unit u...
of the mammalian NPC is about 120 megadaltons
Atomic mass unit
The unified atomic mass unit or dalton is a unit that is used for indicating mass on an atomic or molecular scale. It is defined as one twelfth of the rest mass of an unbound neutral atom of carbon-12 in its nuclear and electronic ground state, and has a value of...
and it contains approximately 30 different protein components, each in multiple copies. In contrast, the yeast model organism
Model organism
A model organism is a non-human species that is extensively studied to understand particular biological phenomena, with the expectation that discoveries made in the organism model will provide insight into the workings of other organisms. Model organisms are in vivo models and are widely used to...
Saccharomyces cerevisiae
Saccharomyces cerevisiae
Saccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes...
is smaller, weighing only 66MDa.
Transport through the nuclear pore complex
Importin
Importin is a type of protein that moves other protein molecules into the nucleus by binding to a specific recognition sequence, called the nuclear localization signal . Importin is classified as a karyopherin....
s or exportins are part of the Importin-β super-family which all share a similar three-dimensional structure.
Three models have been suggested to explain the translocation mechanism:
- Affinity gradients along the central plug
- Brownian affinity gating
- Selective phase
Import of proteins
Any cargo with a nuclear localization signalNuclear localization signal
A nuclear localization signal or sequence is an amino acid sequence which 'tags' a protein for import into the cell nucleus by nuclear transport. Typically, this signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface. Different...
(NLS) exposed will be destined for quick and efficient transport through the pore. Several NLS sequences are known, generally containing a conserved polypeptide sequence with basic residues such as PKKKRKV. Any material with an NLS will be taken up by importins to the nucleus.
The classical scheme of NLS-protein importation begins with Importin-α first binding to the NLS sequence, and acts as a bridge for Importin-β to attach. The importinβ—importinα—cargo complex is then directed towards the nuclear pore and diffuses through it. Once the complex is in the nucleus, RanGTP binds to Importin-β and displaces it from the complex. Then the cellular apoptosis susceptibility protein
Cellular apoptosis susceptibility protein
The cellular apoptosis susceptibility protein is an exportin which in the nucleus is bound to RanGTP....
(CAS), an exportin which in the nucleus is bound to RanGTP, displaces Importin-α from the cargo. The NLS-protein is thus free in the nucleoplasm. The Importinβ-RanGTP and Importinα-CAS-RanGTP complex diffuses back to the cytoplasm where GTP
Guanosine triphosphate
Guanosine-5'-triphosphate is a purine nucleoside triphosphate. It can act as a substrate for the synthesis of RNA during the transcription process...
s are hydrolyzed to GDP leading to the release of Importinβ and Importinα which become available for a new NLS-protein import round.
Although cargo passes through the pore with the assistance of chaperone proteins, the translocation through the pore itself is not energy dependent. However, the whole import cycle needs the hydrolysis of 2 GTPs and is thus energy dependent and has to be considered as active transport
Active transport
Active transport is the movement of a substance against its concentration gradient . In all cells, this is usually concerned with accumulating high concentrations of molecules that the cell needs, such as ions, glucose, and amino acids. If the process uses chemical energy, such as from adenosine...
. The import cycle is powered by the nucleo-cytoplasmic RanGTP gradient. This gradient arises from the exclusive nuclear localization of RanGEFs, proteins that exchange GDP to GTP on Ran molecules. Thus there is an elevated RanGTP concentration in the nucleus compared to the cytoplasm.
Export of proteins
Some nuclear proteins need to be exported from the nucleus to the cytoplasm, as do ribosomeRibosome
A ribosome is a component of cells that assembles the twenty specific amino acid molecules to form the particular protein molecule determined by the nucleotide sequence of an RNA molecule....
subunits and messenger RNA
Messenger RNA
Messenger RNA is a molecule of RNA encoding a chemical "blueprint" for a protein product. mRNA is transcribed from a DNA template, and carries coding information to the sites of protein synthesis: the ribosomes. Here, the nucleic acid polymer is translated into a polymer of amino acids: a protein...
s. Thus there is an export mechanism similar to the import mechanism.
In the classical export scheme, proteins with an nuclear export sequence (NES) can bind in the nucleus to form a heterotrimeric complex with an exportin and RanGTP (for example the exportin CRM1). The complex can then diffuse to the cytoplasm where GTP is hydrolysed and the NES-protein is released. CRM1-RanGDP diffuses back to the nucleus where GDP is exchanged to GTP by RanGEFs. This process is also energy dependent as it consumes one GTP. Export with the exportin CRM1 can be inhibited by Leptomycin
Leptomycin
Leptomycin B is a secondary metabolite produced by Streptomyces spp.Leptomycin B was originally discovered as a potent anti-fungal antibiotic. Leptomycin B was found to cause cell elongation of the fission yeast Schizosaccharomyces pombe. Since then this elongation effect has been used for the...
B.
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Export of RNA
There are different export pathways through the NPC for each RNARNA
Ribonucleic acid , or RNA, is one of the three major macromolecules that are essential for all known forms of life....
class that exists. RNA export is also signal mediated (NES); the NES is in RNA-binding proteins (except for tRNA which has no adapter). It is notable that all viral RNAs and cellular RNAs (tRNA, rRNA, U snRNA, microRNA) except mRNA are dependent on RanGTP. Conseved mRNA export factors are necessary for mRNA nuclear export. Export factors are Mex67/Tap (large subunit) and Mtr2/p15 (small subunit).
Assembly of the NPC
As the NPC controls access to the genome, it is essential that it exists in large amounts in areas of the cell cycle where plenty of transcription is necessary. For example, cycling mammalian and yeast cells double the amount of NPC in the nucleus between the G1 and G2 phase of cell MitosisMitosis
Mitosis is the process by which a eukaryotic cell separates the chromosomes in its cell nucleus into two identical sets, in two separate nuclei. It is generally followed immediately by cytokinesis, which divides the nuclei, cytoplasm, organelles and cell membrane into two cells containing roughly...
. And oocytes accumulate large numbers of NPCs to prepare for the rapid mitosis that exists in the early stages of development. Interphase
Interphase
Interphase is the phase of the cell cycle in which the cell spends the majority of its time and performs the majority of its purposes including preparation for cell division. In preparation for cell division, it increases its size and makes a copy of its DNA...
cells must also keep up a level of NPC generation to keep the levels of NPC in the cell constant as some may get damaged. Some cells can even increase the NPC numbers due to increased transcriptional demand.
Theories of assembly
So how are these vast proteins complexes assembled? As the immunodepletion of certain protein complexes, such as the Nup 107–160 complex, leads to the formation of poreless nuclei, it seems likely that the Nup complexes are involved in fusing the outer membrane of the nuclear envelope with the inner and not that the fusing of the membrane begins the formation of the pore. There are several ways that this could lead to the formation of the full NPC.- One possibility is that as a protein complex it binds to the chromatinChromatinChromatin is the combination of DNA and proteins that make up the contents of the nucleus of a cell. The primary functions of chromatin are; to package DNA into a smaller volume to fit in the cell, to strengthen the DNA to allow mitosis and meiosis and prevent DNA damage, and to control gene...
. It is then inserted into the double membrane close to the chromatin. This, in turn, leads to the fusing of that membrane. Around this protein complex others eventually bind forming the NPC. This method is possible during every phases of mitosis as the double membrane is present around the chromatin before the membrane fusion proteins complex can insert. Post mitotic cells could form a membrane first with pores being inserted into after formation.
- Another model for the formation of the NPC is the production of a prepore as a start as opposed to a single protein complex. This prepore would form when several Nup complexes come together and bind to the chromatin. This would have the double membrane form around it in during mitotic reassembly. Possible prepore structures have been observed on chromatinChromatinChromatin is the combination of DNA and proteins that make up the contents of the nucleus of a cell. The primary functions of chromatin are; to package DNA into a smaller volume to fit in the cell, to strengthen the DNA to allow mitosis and meiosis and prevent DNA damage, and to control gene...
before nuclear envelopeNuclear envelopeA nuclear envelope is a double lipid bilayer that encloses the genetic material in eukaryotic cells. The nuclear envelope also serves as the physical barrier, separating the contents of the nucleus from the cytosol...
(NE) formation using electron microscopy. During the interphase of the cell cycle the formation of the prepore would happen within the nucleus, each component being transported in through existing NPCs. These Nups would bind to an importin, once formed, preventing the assembly of a prepore in the cytoplasm. Once transported into the nucleus Ran GTP would bind to the importin and cause it to release the cargo. This Nup would be free to from a prepore. The binding of importins has at least been shown to bring Nup 107 and the Nup 153 nucleoporins into the nucleus. NPC assembly is a very rapid process yet defined intermediate states occur which leads to the idea that this assembly occurs in a stepwise fashion.
- A third possible method of NPC assembly is splitting. This method seems to be tailor made for NPC formation during the interphase. It happens when more protomers are added on to an existing NPC. The eightfold symmetry of the NPC has been shown to have a degree of plasticity and will allow this. Eventually enough protomers will add and allow a new NPC to split off the original. This method of NPC assembly can only happen during the interphase of the cell cycle.