9.A.14 The Nuclear Pore Complex (NPC) Family

The nuclear pore complex (NPC), present in the nuclear membranes of interphase eukaryotic cells, is a 120 megadalton supramolecular assembly (Tran and Wente, 2006). It is believed to mediate bidirectional molecular trafficking of fully folded proteins, RNA, large ribonucleoprotein particle complexes and small molecules between the 'nucleoplasm' or 'perinuclear space' and the cytoplasm. Most nuclearly synthesized RNA molecules are exported to the cytoplasm while proteins required for nuclear function are imported from the cytoplasm. Ions and small molecules may passively diffuse across the NPC while large proteins, RNAs (up to about 60 kDa) and ribonucleoproteins may be actively transported via a signal mediated, gated, channel mechanism. The inner diameter of the inner NPC channel has been estimated by various workers to be 9-26 nm, and the length to be about 45 nm.

Numerous NPC proteins, called nucleoporins, have been identified and characterized from vertebrates and yeast. Thirty such proteins are recognized constituents of the yeast NPC, and at least 50 nucleopore proteins have been characterized from vertebrates. Many of these proteins have been tabulated for (1) S. cerevisiae and (2) vertebrates by Stoffler et al. (1999) and Tran and Wente (2006). How they function in transport is poorly defined. It is known, however, that nuclear proteins contain short sequences called 'nuclear localization sequences' (NLS) that target them for nuclear import. A nuclear localization sequence receptor and several cytosolic factors appear to play roles in nuclear import of NLS-bearing proteins. Nuclear export signals (NESs) have also been identified. Several different forms of each type of targeting signal have been identified that lack homology to each other and may be recognized by different receptors. NLS and NES receptors (termed importins and exportins, respectively) may all be homologous and are members of the karyopherin-β/importin-βsuperfamily (Sorokin et al., 2008). The many protein constituents of NPCs have been discussed from structural, topological and functional standpoints by Panté and Aebi (1996), Nigg (1997) and Stoffler et al. (1999). Evidence for an involvement of the specific receptors or shuttling vectors, such as the importin-β-family member, Msn5 (spP52918), has been presented (Kaffman et al., 1998). Structural features and functional correlates have been discussed by Talcott and Moore (1999) as well as Gorlich and Kutay (Gorlich and Kutay 1999).

Members of the importin-β family of transport receptors mediate NPC passage of cargo by interacting with nucleoporins and a small GTPase, Ran. Ran acts as a molecular switch by interconverting between a GTP and GDP binding state, regulated by a nuclear GTP/GDP exchange factor, RCC1, and a cytoplasmic GTPase-activating factor, RanGAP. The asymmetric distribution of these proteins insures that nuclear Ran is primarily in the GTP-bound form, but cytoplasmic Ran is in the GDP-bound form. This gradient of Ran-GTP ensures release of cargo from the transport importin-β receptors which bind NLS-substrate/importin-β complex in the cytoplasm, and this ternary complex dissociates by binding RanGTP to importin-β in the nucleus. While ATP (or GTP) is required for nuclear export of importin-β, it is not required for nuclear import.

Mediators of import into the nucleus (importins) and export mediators (exportins) interact with RanGTP but respond to the nucleocytoplasmic RanGTP gradient in diametrically opposed ways (Mingot et al., 2004). Importins bind cargo at low RanGTP levels in the cytoplasm and release cargo upon RanGTP binding in the nucleus. In contrast, exportins recruit cargo at high RanGTP concentrations, as ternary cargo/exportin/RanGTP complexes, in the nuclear compartment and release cargo when the Ran-bound GTP molecule is hydrolyzed in the cytoplasm. This active control of cargo binding and release by the RanGTPase system constitutes the sole input of metabolic enegy into these transport cycles and is sufficient to allow importins and exportins to accumulate cargoes actively against gradients of chemical activity.

Transport through the NPC occurs by facilitated diffusion of the soluble carrier proteins or carrier-cargo complexes (Macara, 2001). Vectorality is provided by compartment-specific assembly and disassembly of the carrier-cargo complexes, often mediated by the Ran GTPase as noted above. The carriers recognize localization signals on the cargo and bind to pore proteins (Macara, 2001). While the yeast NPC is complex, those in plants and animals are much more so with hundreds of proteins functioning in various capacities. Many of the yeast NPC constituents can be found in other eukaryotes (e.g., vertebrate centrins function as does Cdc31p of yeast and plays a role in mRNA and protein export) (Resendes et al., 2008). The RNA U small nuclear (sn)RNA export adaptor protein, or the phosphorylated adaptor for RNA export, regulates U snRNA nuclear export to the cytoplasm in metazoa. It is phosphorylated in the nucleus and exported as part of the U snRNA export complex where it is dephosphorylated, causing complex disassembly (Kitao et al., 2008).

Targeting of newly synthesized integral membrane proteins to the appropriate cellular compartment is specified by discrete sequence elements, many of which have been well characterized. An understanding of the signals required to direct integral membrane proteins to the inner nuclear membrane (INM) represent a notable exception. King et al. (2006) have shown that integral INM proteins possess basic sequence motifs that resemble 'classical' nuclear localization signals. These sequences can mediate direct binding to karyopherin-β and are essential for the passage of integral membrane proteins to the INM. Furthermore, karyopherin-β, karyopherin-β1 and the Ran GTPase cycle are required for INM targeting, underscoring parallels between mechanisms governing the targeting of integral INM proteins and soluble nuclear transport. King et al. (2006) provided evidence that specific nuclear pore complex proteins contribute to this process, suggesting a role for signal-mediated alterations in the nuclear pore complex to allow for passage of INM proteins along the pore membrane.

The transport receptor Mex67-Mtr2 functions in mRNA export, and also, using a loop-confined surface on the heterodimer, it binds to and exports pre-60S particles. Mex67-Mtr2, through the same surface that recruits pre-60S particles, interacts with the Nup84 complex, a structural module of the nuclear pore complex devoid of Phe-Gly domains (Yao et al., 2007). In vitro, pre-60S particles and the Nup84 complex compete for an overlapping binding site on the loop-extended Mex67-Mtr2 surface. Nup85 is the subunit in the Nup84 complex that binds to the Mex67 loop, an interaction that is crucial for mRNA export.

NPCs are proteinaceous assemblies of approximately 50 MDa of 456 known constituents that selectively transport cargoes across the nuclear envelope. Half of the NPC is made up of a core scaffold, which is structurally analogous to vesicle-coating complexes. This scaffold forms an interlaced network that coats the entire curved surface of the nuclear envelope membrane within which the NPC is embedded. The selective barrier for transport is formed by large numbers of proteins with disordered regions that line the inner face of the scaffold (Alber et al., 2007). The NPC consists of only a few structural modules that resemble each other in terms of the configuration of their homologous constituents. The most striking of these is a 16-fold repetition of 'columns'.

Trafficking of nucleic acids and large proteins through nuclear pore complexes (NPCs) requires interactions with NPC proteins that harbor FG (phenylalanine-glycine) repeat domains. Specialized transport receptors that recognize cargo and bind FG domains facilitate these interactions. Terry and Wente (2007) generated in S. cerevisiae a set of more minimal pore (mmp) mutants lacking specific FG domains. A comparison of messenger RNA (mRNA) export versus protein import reveals unique subsets of mmp mutants with functional defects in specific transport receptors. Thus, multiple functionally independent NPC translocation routes exist for different transport receptors. mRNA export also requires two NPC substructures-one on the nuclear NPC face and one in the NPC central core.

A novel family of NPC proteins, the FG-nucleoporins (FG-Nups), coordinates and potentially regulates NPC translocation. The extensive repeats of phenylalanine-glycine (FG) in each FG-Nup directly bind to shuttling transport receptors moving through the NPC. In addition, FG-Nups are essential components of the nuclear permeability barrier. Terry & Wente (2009) reviewed the structural features, cellular functions, and evolutionary conservation of the FG-Nups.