The nucleus is separated from the cytoplasm by two mem branes, which form the nuclear envelope. The nuclear envelope is continuous with the ER and forms a part of it. Transport of proteins from the cytoplasm into the nucleus and movement of macromolecules, including mRNAs, tRNAs, and ribosomal subunits, out of the nucleus occur through nuclear pores, which span both membranes of the nuclear envelope. Import of proteins into the nucleus shares some fundamental features with protein import into other organelles. For example, imported nuclear proteins carry specific targeting sequences known as nuclear-localization signals (NLSs). However, proteins are imported into the nucleus in a folded state, and thus nuclear import differs fundamentally from protein translocation across the membranes of the ER, mitochondrion, and chloroplast, during which proteins are unfolded. In this section, we discuss the main mechanism by which proteins enter and exit the nucleus. We also discuss the process by which mRNAs and other ribonuclear protein complexes are exported from the nucleus, which differs mechanistically from nuclear protein import.
Large and Small Molecules Enter and Leave the Nucleus via Nuclear Pore Complexes
Numerous nuclear pores perforate the nuclear envelope in all eukaryotic cells. Each nuclear pore is formed from an elaborate structure termed the nuclear pore complex (NPC), which is one of the largest protein assemblages in the cell. The total mass of the pore structure is 60,000–80,000 kDa in vertebrates, which is about 16 times larger than a ribosome. An NPC is made up of multiple copies of some 30 different proteins called nucleoporins. Electron micrographs of nuclear pore complexes reveal a membrane-embedded ring structure that surrounds a largely aqueous pore (Figure1). Eight approximately 100-nm-long filaments extend into the nucleoplasm with the distal ends of these filaments joined by a terminal ring, forming a structure called the nuclear basket. Cytoplasmic filaments extend from the cytoplasmic side of the NPC into the cytosol.
Fig1. Nuclear pore complex at different levels of resolution. (a) Nuclear envelopes from the large nuclei of Xenopus oocytes, visualized by scanning electron microscopy. Top: View of the cytoplasmic face reveals the octagonal shape of the membrane embedded portion of nuclear pore complexes. Bottom: View of the nucleoplasmic face shows the nuclear basket that extends from the membrane-embedded portion. (b) Cutaway model of the nuclear pore complex, showing the major structural features formed by membrane nucleoporins, structural nucleoporins, and FG-nucleoporins. (c) Sixteen copies of the Y-complex form a major part of the structural scaffold of the nuclear pore complex. The three-dimensional structure of the Y-complex is modeled into the pore structure. Note the twofold symmetry across the double membrane of the nucleus (left) and the eightfold rotational symmetry around the axis of the pore (right). (d) The FG-nucleoporins have extended disordered structures that are composed of repeats of the sequence Phe–Gly interspersed with hydrophilic regions (left). The FG-nucleoporins are most abundant in the central part of the pore, and the FG-repeat sequences are thought to fill the central channel with a gel-like matrix (right). See K. Ribbeck and D. Görlich, 2001, EMBO J. 20:1320–1330 and M. P. Rout and J. D. Atchison, 2001, J. Biol. Chem. 276:16593. [Part (a) republished with permission of Elsevier, from Doye, V. and Hurt, E., “Nucleoporins to Nuclear Pore Complexes,” Curr. Opin. Cell Biol. 9(3):401-411, 1997; permission conveyed through the Copyright Clearance Center, Inc.]
Ions, small metabolites, and globular proteins up to about 40 kDa can diffuse passively through the central aqueous region of the nuclear pore complex. However, large proteins and ribonucleoprotein complexes cannot diffuse in and out of the nucleus. Rather, these macromolecules are actively trans ported through the NPC with the assistance of soluble trans port proteins that bind macromolecules and also interact with nucleoporins. The capacity and efficiency of the NPC for such active transport is remarkable. In one minute, each NPC is estimated to import 60,000 protein molecules into the nucleus, while exporting 50–250 mRNA molecules, 10–20 ribosomal subunits, and 1000 tRNAs out of the nucleus.
In general terms, the nucleoporins are of three types: structural nucleoporins, membrane nucleoporins, and FG- nucleoporins. The structural nucleoporins form the scaffold of the nuclear pore, which is a ring of eightfold rotational symmetry that traverses both membranes of the nuclear envelope, creating an annulus. The inner and outer membranes of the nuclear envelope are connected at the NPC by a highly curved region of membrane that contains the embedded mem brane nucleoporins (Figure 1b). A set of seven structural nucleoporins forms a Y-shaped structure about the size of the ribosome, known as the Y-complex. Sixteen copies of the Y-complex form the basic structural scaffold of the pore, which has bilateral symmetry across the nuclear envelope and eightfold rotational symmetry in the plane of the envelope (Figure 1c). A structural motif repeated several times within the Y-complex is closely related to a structure found in the COPII proteins that drive the formation of coated vesicles within cells. This primordial relationship between structural nucleoproteins and vesicle coat proteins suggests that the two types of membrane coat complexes share a common origin. The basic function of this element may be to form a protein lattice that, in a complex with membrane nucleoporins, deforms the membrane into a highly curved structure.
The FG-nucleoporins, which line the channel of the nuclear pore complex and are also found associated with the nuclear basket and the cytoplasmic filaments, contain multiple repeats of short hydrophobic sequences that are rich in phenylalanine (F) and glycine (G) residues (FG-repeats).
The hydrophobic FG-repeats are thought to occur in regions of extended, otherwise hydrophilic polypeptide chains that fill the central transporter channel. The FG-nucleoporins are essential for the function of the NPC; however, the NPC remains functional even if up to half of the FG-repeats have been deleted. The FG-nucleoporins are thought to form a flexible gel-like matrix with bulk properties that allow the diffusion of small molecules while excluding unchaperoned hydrophilic proteins larger than 40 kDa (Figure 1d).
Nuclear Transport Receptors Escort Proteins Containing Nuclear-Localization Signals into the Nucleus
All proteins found in the nucleus—such as histones, transcription factors, and DNA and RNA polymerases—are synthesized in the cytoplasm and imported into the nucleus through nuclear pore complexes. Such proteins contain a nuclear-localization signal that directs their selective trans port into the nucleus. NLSs were first discovered through the analysis of mutations of the gene for large T-antigen encoded by simian virus 40 (SV40). The wild-type form of large T-antigen is localized to the nucleus in virus-infected cells, whereas some mutated forms of large T-antigen accumulate in the cytoplasm (Figure 2). The mutations responsible for this altered cellular localization all occur within a specific seven-residue sequence rich in basic amino acids near the C-terminus of the protein: Pro-Lys-Lys-Lys-Arg-Lys-Val. Experiments with chimeric proteins in which this sequence was fused to a cytosolic protein demonstrated that it directs transport into the nucleus and consequently functions as an NLS. NLS sequences were subsequently identified in numerous other proteins imported into the nucleus. Many of these sequences are similar to the basic NLS in SV40 large T-antigen, whereas others are chemically quite different. For instance, an NLS in the RNA-binding protein hnRNP A1 is hydrophobic. Accordingly, there are multiple mechanisms for the recognition of these very different sequences.
Fig2. Nuclear-localization signals (NLSs) direct proteins to the cell nucleus. Cytoplasmic proteins can be transported to the nucleus if they are fused to a nuclear-localization signal. (a) Normal pyruvate kinase, here visualized by immunofluorescence after cultured cells were treated with a specific antibody (yellow), is localized to the cytoplasm. This very large cytosolic protein functions in carbohydrate metabolism. (b) When a chimeric pyruvate kinase containing the SV40 NLS at its N-terminus was expressed in cells, it was localized to the nucleus. The chimeric protein was expressed from a transfected engineered gene produced by fusing a viral gene fragment encoding the SV40 NLS to the pyruvate kinase gene. [Republished with permission of Elsevier, from D. Kalderon et al., “A Short Amino Acid Sequence Able to Specify Nuclear Location,” Cell 39(3 Pt 2): 499–509, 1984; permission conveyed through the Copyright Clearance Center Inc.]
Early work on the mechanism of nuclear import showed that proteins containing a basic NLS, similar to the one in SV40 large T-antigen, were efficiently transported into isolated nuclei only if they were provided with a cytosolic extract (Figure 3). Using this assay system, researchers purified two required cytosolic components: Ran and a nuclear trans port receptor. Ran is a small monomeric G protein that exists in either a GTP-bound or a GDP-bound conformation. It is the cycling of Ran between GTP-bound and GDP-bound conformations, leading to the net hydro lysis of GTP to GDP, that ultimately provides the energy to drive unidirectional transport of macromolecules through the nuclear pore. A nuclear transport receptor binds to the NLS on a cargo protein to be transported into the nucleus and to FG-repeats on nucleoporins. By a physical process that is not well understood, by binding transiently to FG-repeats, nuclear transport receptors have the ability to rapidly traverse the FG repeat-containing matrix in the central channel of the nuclear pore, whereas proteins of similar size that lack this property are excluded from the central channel. Nuclear transport receptors can be monomeric, consisting of a single polypeptide that can bind both to an NLS and to FG-repeats, or they can be dimeric, with one subunit binding to the NLS and the other binding to FG-repeats.
Fig3. Cytosolic proteins are required for nuclear transport. The failure of nuclear transport to occur in permeabilized cultured cells in the absence of cell lysate demonstrates the involvement of soluble cytosolic components in the process. (a) Phase-contrast micrographs of untreated and digitonin permeabilized HeLa cells. Treatment of a monolayer of cultured cells with the mild non-ionic detergent digitonin permeabilizes the plasma membrane so that cytosolic constituents leak out, but leaves the nuclear envelope and NPCs intact. (b) Fluorescence micrographs of digitonin-permeabilized HeLa cells incubated with a fluorescent protein chemically coupled to a synthetic SV40 T-antigen NLS peptide in the presence and absence of cell lysate (cytosol). Accumulation of the transport substrate in the nucleus occurred only when cytosol was present (right). [©1990 S. Adam et al., The Journal of Cell Biology 111:807–816. doi:10.1083/jcb.111.3.807.]
The mechanism for the import of cytoplasmic cargo proteins mediated by a nuclear transport receptor known as importin is shown in Figure 4. Free importin in the cytoplasm binds to its cognate NLS in a cargo protein, forming an importin-cargo complex. The cargo complex then translocates through the NPC channel as the importin interacts with FG repeats. The cargo complex rapidly reaches the nucleoplasm, and there the importin interacts with Ran⋅GTP, which causes a conformational change in the importin that displaces the NLS, releasing the cargo protein into the nucleoplasm. The importin Ran⋅GTP complex then diffuses back through the NPC. Once the importin-Ran⋅GTP complex reaches the cytoplasmic side of the NPC, Ran interacts with a specific GTPase-activating protein (Ran-GAP) that is a component of the NPC cytoplasmic filaments. This interaction stimulates Ran to hydrolyze its bound GTP to GDP, which causes it to convert to a conformation that has low affinity for the importin, so that the importin is released into the cytoplasm, where it can participate in another cycle of import. Ran⋅GDP travels back through the pore to the nucleoplasm, where it encounters a specific guanine nucleotide exchange factor (Ran-GEF) that causes Ran to release its bound GDP in favor of GTP. The net result of this series of reactions is the coupling of the hydrolysis of GTP to the transfer of an NLS bearing protein from the cytoplasm to the nuclear interior, thus providing the energy to drive nuclear import.
Fig4. Mechanism for nuclear import of proteins. In the cytoplasm (top), free importin binds to the NLS of a cargo protein, forming an importin-cargo complex. The importin-cargo complex dif fuses through the NPC by transiently interacting with FG-nucleoporins. In the nucleoplasm, Ran⋅GTP binds to the importin, causing a conformational change that decreases the importin’s affinity for the NLS and releasing the cargo. To support another cycle of import, the importin Ran⋅GTP complex is transported back to the cytoplasm. A GTPase activating protein (GAP) associated with the cytoplasmic filaments of the NPC stimulates Ran to hydrolyze the bound GTP. This generates a conformational change that causes Ran to dissociate from the nuclear transport receptor, which can then initiate another round of import. Ran⋅GDP is returned to the nucleoplasm, where a guanine nucleotide exchange factor (GEF) causes release of GDP and rebinding of GTP.
Although the importin-cargo complex travels through the pore by random diffusion, the overall process of transport of cargo into the nucleus is unidirectional. Because of the rapid dissociation of the complex when it reaches the nucleoplasm, there is a concentration gradient of importin-cargo complex across the NPC: high in the cytoplasm, where the complex assembles, and low in the nucleoplasm, where it dissociates. This concentration gradient is responsible for the unidirectional nature of nuclear import. A similar concentration gradient is responsible for driving importin from the nucleus back into the cytoplasm. The concentration of the importin-Ran⋅GTP complex is higher in the nucleoplasm, where it assembles, than on the cytoplasmic side of the NPC, where it dissociates. Ultimately, the direction of the transport processes depends on the localization of the Ran-GEF predominantly in the nucleoplasm and the Ran-GAP predominantly in the cytoplasm. Ran-GEF in the nucleoplasm maintains Ran in the Ran⋅GTP state, where it promotes dissociation of the cargo complex. Ran-GAP on the cytoplasmic side of the NPC converts Ran⋅GTP to Ran⋅GDP, dissociating the importin-Ran⋅GTP complex and releasing free importin into the cytosol.
A Second Type of Nuclear Transport Receptor Escorts Proteins Containing Nuclear-Export Signals Out of the Nucleus
A mechanism very similar to the one we have just described is used to export proteins, tRNAs, and ribosomal subunits from the nucleus to the cytoplasm. This mechanism was initially elucidated by studies of certain ribonuclear protein complexes that “shuttle” between the nucleus and the cytoplasm. Such “shuttling” proteins contain a nuclear-export signal (NES) that stimulates their export from the nucleus to the cytoplasm through nuclear pores, in addition to an NLS that results in their uptake into the nucleus. Experiments with engineered hybrid genes encoding a nucleus-restricted protein fused to various segments of a shuttling protein have identified at least three different types of NESs: a leucine-rich sequence found in PKI (an inhibitor of protein kinase A) and in the Rev protein of human immunodeficiency virus (HIV), and two other sequences identified in two different heterogeneous ribonucleoprotein particles (hnRNPs). The precise structural features that determine the recognition of each type of sequence for nuclear export remain poorly understood.
The mechanism whereby shuttling proteins are exported from the nucleus is best understood for those containing a leucine-rich NES. According to the current model, shown in Figure 5a, a specific nuclear transport receptor, called exportin 1, first forms a complex with Ran⋅GTP in the nucleus and then binds the NES in a cargo protein. Binding of exportin 1 to Ran⋅GTP causes a conformational change in exportin 1 that increases its affinity for the NES, so that a trimolecular cargo complex is formed. Like other nuclear transport receptors, exportin 1 interacts transiently with FG repeats in FG-nucleoporins and diffuses through the NPC. The cargo complex dissociates when it encounters the Ran GAP associated with the NPC cytoplasmic filaments, which stimulates Ran to hydrolyze the bound GTP, shifting it into a conformation that has low affinity for exportin 1. After Ran⋅GDP dissociates from the trimolecular cargo complex, exportin 1 changes its conformation to one that has low affinity for the NES, releasing the cargo into the cytosol. The direction of the export process is driven by this dissociation of the cargo from exportin 1 in the cytoplasm, which causes a concentration gradient of the cargo complex across the NPC that is high in the nucleoplasm and low in the cytoplasm. Exportin 1 and Ran⋅GDP are then transported back into the nucleus through the NPC.
Fig5. Ran-dependent and Ran-independent nuclear export. (a) 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. (b) Ran-independent nuclear export of mRNAs. The heterodimeric NXF1/NXT1 complex binds to mRNA-protein complexes (mRNPs) in the nucleus. 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. 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 in Figure 4.
By comparing this model for nuclear export with that in Figure 13-36 for nuclear import, we can see one obvious difference: Ran⋅GTP is part of the cargo complex during export, but not during import. Apart from this difference, the two transport processes are remarkably similar. In both processes, association of a nuclear transport receptor with Ran⋅GTP in the nucleoplasm causes a conformational change that affects its affinity for the transport signal. During import, the interaction causes release of the cargo, whereas during export, the interaction promotes association with the cargo. In both export and import, stimulation of Ran⋅GTP hydrolysis in the cytoplasm by Ran-GAP produces a conformational change in Ran that releases the nuclear transport receptor. During nuclear export, the cargo is also released. Localization of Ran-GAP and Ran-GEF to the cytoplasm and nucleus, respectively, is the basis for the unidirectional import and export of cargo proteins across the NPC.
In keeping with their similarity in function, the two types of nuclear transport receptors—importins and exportins— are highly homologous in sequence and structure. The family of nuclear transport receptors has 14 members in yeast and more than 20 in mammalian cells. The NESs or NLSs to which they bind have been determined for only a fraction of them. Some individual nuclear transport receptors function in both import and export.
A similar shuttling mechanism has been shown to export other cargoes from the nucleus. For example, exportin-t functions to export tRNAs. Exportin-t binds fully processed tRNAs in a complex with Ran⋅GTP that diffuses through NPCs and dissociates when it interacts with Ran-GAP in the NPC cytoplasmic filaments, releasing the tRNA into the cytosol. A Ran-dependent process is also required for the nuclear export of ribosomal subunits through NPCs once the protein and RNA components have been properly assembled in the nucleolus. Likewise, certain specific mRNAs that associate with particular hnRNP proteins can be exported by a Ran-dependent mechanism.
Most mRNAs Are Exported from the Nucleus by a Ran-Independent Mechanism
Once the processing of an mRNA is completed in the nucleus, it remains associated with specific proteins in a messenger ribonuclear protein complex, or mRNP. The principal trans porter of mRNPs out of the nucleus is the mRNP exporter, a heterodimeric protein composed of a large subunit called nuclear export factor 1 (NXF1) and a small subunit called nuclear export transporter 1 (NXT1). Multiple NXF1/NXT1 dimers bind to nuclear mRNPs through cooperative interactions with the RNA and other mRNP adapter proteins that associate with nascent pre-mRNAs during transcriptional elongation and pre-mRNA processing. In many respects, the subunits of NXF1/NXT1 act like a nuclear transport receptor that binds to an NLS or NES, since both subunits interact with the FG-repeats of FG-nucleoporins, and this interaction al lows them to diffuse through the central channel of the NPC.
The process of mRNP export does not require Ran, and thus the unidirectional transport of mRNA out of the nucleus requires a source of energy other than GTP hydrolysis by Ran. Once the mRNP-NXF1/NXT1 complex reaches the cytoplasmic side of the NPC, NXF1 and NXT1 dissociate from the mRNP with the help of the RNA helicase Dbp5, which is associated with cytoplasmic NPC filaments. Recall that RNA helicases use the energy derived from hydrolysis of ATP to move along RNA molecules, separating double stranded RNA chains and dissociating RNA-protein complexes. This leads to the simple idea that Dpb5, which is associated with the cytoplasmic side of the nuclear pore complex, acts as an ATP-driven motor to re move NXF1/NXT1 from the mRNP complexes as they emerge on the cytoplasmic side of the NPC. The assembly of NXF1/NXT1 onto mRNPs on the nucleoplasmic side of the NPC and the subsequent ATP-dependent removal of NXF1/ NXT1 from mRNPs on the cytoplasmic side of the NPC creates a concentration gradient of mRNP-NXF1/NXT1, which drives unidirectional export. After being removed from the mRNP, the free NXF1 and NXT1 subunits that have been stripped from the mRNA by Dbp5 helicase are imported back into the nucleus by a process that depends on Ran and a nuclear transport receptor (Figure 5b).
In Ran-dependent nuclear export (discussed in the previous subsection), hydrolysis of GTP by Ran on the cytoplasmic side of the NPC causes dissociation of the nuclear transport receptor from its cargo. In basic outline, the Ran independent nuclear export discussed here operates by a similar mechanism, except that Dbp5p on the cytosolic side of the NPC uses hydrolysis of ATP to dissociate the mRNP exporter from mRNA.
