Components and regulation of nuclear transport processes

Membrane Nups

Membrane Nups (POMs) anchor the symmetric part of the NPC to the pore membrane where the inner and outer nuclear membranes fuse to form the nuclear pore binding the assembly complex to the NE. Membrane Nups contain transmembrane α‐helices that allow the protein to anchor onto the membrane while large regions extend toward the luminal and pore sides of the membrane 27, 28.

Scaffold Nups

The scaffold Nups (coat Nups and adaptor Nups) form the skeleton of the NPC connecting the membrane Nups to the barrier Nups. The scaffold Nups contain mainly α‐solenoid and β‐propeller folds and are classified into outer ring Nups, inner ring Nups and linker Nups according to their location and function. The high flexibility of the outer ring Nup components allows for conformational changes of the scaffold that enable large cargo to pass through the central channel 29.

Barrier Nups

Barrier Nups (channel Nups) are phenylalanineglycine Nups (FG‐Nups) and form the innermost cylindrical layer that acts as a selective gatekeeper for nuclear transport regulation. FG‐Nups contain multiple stretches of FG sequences that form intrinsically disordered regions. These motifs are present in about one‐third of Nups forming an unstructured meshwork lining the central channel. The tentacle‐like structures provide several low affinity, high specificity interactions with transport receptors that escort cargo proteins through the nuclear pore. Collectively, FG‐rich regions build the diffusion barrier of the NPC.

Asymmetric Nups

Asymmetric Nups (formed by nuclear basket Nups and cytoplasmic filament Nups) are key components in establishing the directionality of the nucleo‐cytoplasmic transport process. These structures mediate specific interactions with transport complexes and several asymmetric Nups that contain FG repeats serving as binding sites with important roles in cargo–NPC interactions.

Classical NLSs

The first nuclear transport signals were described in the SV40 large T antigen and nucleoplasmin in the early 1980s comprising a short lysine‐rich sequence classified as classical NLSs 31, 38. These bind the armadillo (ARM) domain in the C terminus of Imp‐α. The adaptor protein Imp‐α also binds the transport receptor Imp‐β through its N‐terminal αIBB domain forming a ternary complex 39. The small size and relatively simple sequence patterns of these basic NLSs have facilitated identification of similar signals in many proteins. The classical NLSs contain one or two clusters of positively charged amino acids, typically lysine or arginine, and are divided into two classes, monopartite and bipartite classical NLSs 40. The monopartite classical NLS is a short and highly basic signal. The two stretches of basic amino acids within a bipartite classical NLS are separated by a linker region that is usually 10–12 residues long, although longer linker sequences have been reported 41. In addition, several atypical NLSs that bind to classical NLS binding sites in Imp‐α have been characterized, including the hydrophobic NLS from phospholipid scramblase 1 40.

Non‐classical NLSs

Some cargo proteins bypass the requirement for an adaptor protein and bind directly to transport receptors through non‐classical NLSs. Proteins that are directly recognized by Imp‐β include ribosomal proteins, CREB, the human immunodeficiency virus (HIV) Rev and Tat, SREBP‐2, the human T‐cell leukemia virus type 1 (HTLV‐1) protein Rex, PTHrP, cyclin B1, Smad3, TRF and SRY 40. In contrast to the interaction between classical NLS and Imp‐α, proteins that bind to Imp‐β directly do not obey strict rules and the NLSs that confer Imp‐β recognition vary significantly in both size and charge 40.

Many proteins that are imported into the nucleus can bind directly to the transport receptor transportin‐1. Common characteristics between the apparently disparate signals recognized by transportin‐1 were described unifying them into a new class of NLS termed PY‐NLS 34. The consensus motif of PY‐NLS consists of a loose N‐terminal hydrophobic motif, a central arginine residue and a C‐terminal PY sequence 42. The physical rules that describe the PY‐NLSs as structurally disordered in free cargoes, positively charged and with weak consensus motifs predicted approximately 100 candidate transportin‐1 cargoes 34. Several of these have been confirmed experimentally 34, 43, 44. Interestingly, arginine–glycine‐rich NLSs known as RG‐NLSs in the yeast proteins Hrp1 and Nab2 and the 38 amino acid long NLS of the hnRNP A1 protein, designated M9, were also shown to have the same characteristics as the PY‐NLS 42, 45. Recently other non‐classical NLSs such as the extensive coiled‐coil domain of STAT5a 46 have been characterized. Furthermore, a number of proteins have been identified that contain both classical and non‐classical NLS motifs that can interact directly with both Imp‐α and Imp‐β family members.

Leucine‐rich NESs containing cargoes

The first signals that direct the nuclear export of a protein were identified in HIV Rev and protein kinase inhibitor A 32, 35. The consensus sequence for NES is ϕ1‐X_(2–3)‐ϕ2‐X_(2–3)‐ϕ3‐X‐ϕ4 (where ϕ represents one of the hydrophobic residues L, V, I, F or M, and X can be any amino acid but preferentially is charged, polar or a small amino acid) 47, 48, 49.

More complex export signatures incorporate the three‐dimensional features of the whole protein that are recognized and targeted for export 50, 51. Proteins containing these NESs are recognized and exported by CRM1 52, 53, 54, 55 (Fig. 2). Güttler et al. showed that CRM1 contains five pockets for binding the conserved hydrophobic residues of NESs. Accordingly, a structure‐based NES consensus with an additional hydrophobic position (five instead of four) has been proposed 56. Kosugi et al. generated a large number of NES peptides in a random peptide library screen and delineated multiple distinct consensus sequences that described more than 80% of known functional NESs 57. A predictor, NESsential, that uses sequence derived meta‐features, such as predicted disorder and solvent accessibility, in addition to a primary sequence that can identify promising NES‐containing candidate proteins 58, 59, http://validness.ym.edu.tw/index.php. Other recently developed NES predictor tools include wregex, which uses position‐specific scoring matrices for motif prediction 60, and nesmapper (http://sourceforge.net/projects/nesmapper), which has been developed based on the activity profile of all classes of NESs 61.

CRM1 (chromosome region maintenance 1)

The most extensively characterized export receptor is chromosome region maintenance 1 (CRM1), a member of the karyopherin‐β family of receptor proteins. CRM1 exports proteins that contain leucine‐rich NESs from the nucleus into the cytoplasm 53, 55. CRM1 has 20 HEAT domains that allow RanGTP to bind. Cargo binding takes place outside of this HEAT domain ring in a hydrophobic cleft producing a generic NES docking site 56. CRM1 recognizes NESs that are present in a large range of proteins that are structurally unrelated 72. CRM1 can also be recruited by adaptor molecules in situations where it does not bind directly to a protein that is to be exported from the nucleus (e.g. Exp5 cooperates with CRM1 to export large ribosomal subunits 73). CRM1 also participates in the export of the 40s and 60s pre‐ribosomal subunits as well as essential RNPs 74, 75, 76, 77. With two adaptors [the Cap binding complex (CBC) complex and PHAX] CRM1 is also essential for the maturation of the spliceosomal U snRNPs 78, 79. Furthermore, CRM1 actively maintains the exclusive cytoplasmic localization of RanBP1, RanGAP and other translation factors that contribute to the identity of the nuclear compartment 80, 81, 82, 83. Another key function of CRM1 is its role in SPN1 recycling (an adaptor for U snRNPs) back to the cytoplasm 51. Intriguingly, overexpression of CRM1 has been reported in various tumor types and has been correlated with poor prognosis and resistance to therapy 4, 5. In addition CRM1 is hijacked during infection by many viruses 32, 84, 85, 86.

Other exportins

In addition to CRM1, a number of other alternative export receptors have been characterized. Exportin 2 recycles Imp‐α from the nucleus into the cytoplasm allowing Imp‐α to mediate another round of nuclear import if required 87, 88, 89. Together with the adaptor STRADα exportin 7 regulates the distribution of LKB1 kinase 90 and regulates the leakage of RhoGAP1 and 14‐3‐3‐σ into the nucleus 91. Exportins are also dedicated to RNA transit. For example, exportin‐t is dedicated to export fully mature tRNA with the 5′‐ and 3′‐ends correctly processed 92, 93, 94, 95. A second tRNA (alone or with eEF1A) exporter is exportin 5 which displays a different binding specificity compared to exportin‐t 80, 96. Exportin 5 also exports double strand RNA, pre‐miRNAs 97, 98, 99 and cooperates with CRM1 as described previously. Exportin 4 exports eIF5A and Smad3 but can also act as an importin for Sox‐type transcription factors 100, 101. Importin 13, despite its namesake, also demonstrates a bi‐directional transit capability directing eIF1A nuclear export and the import of the heterodimer component of the exon junction complex Mago‐Y14 102, 103.

Nuclear import

The recognition of NLSs by Imp‐α and heterodimerization with Imp‐β initiates the process of nuclear import. The import complex, consisting of the cargo protein and the Imp‐α/β complex, localizes to the nuclear envelope, binds RanGDP and docks at the nuclear pore. The Imp‐α/β complex mediates the binding to and translocation through the NPC. Once translocation through the pore is executed, dissociation of the import complex is stimulated by RanGTP in the nucleus. Importin‐α is recycled back to the cytoplasm through the nuclear exporter CAS, whereas Imp‐β is separately transported back to the cytoplasm together with RanGTP. RanGAP1‐facilitated GTP hydrolysis of Ran on the cytoplasmic side causes the release of Imp‐β for the next cycle.

Nuclear export

The process of nuclear export is carried out according to principles which are analogous to those of nuclear import, using specific nuclear export receptors like CRM1 that recognize NES sequences on cargo proteins 131, 132. The affinity of CRM1 for most NESs is low and formation of the export complex is promoted by RanBP3 which links CRM1 to the chromatin binding protein RCC1 133, 134 and increases the active concentration of RanGTP. This promotes the affinity of the NES cargo for the export receptor 135, 136. This complex moves through the NPC via the interaction with FG repeat proteins 137. Within the cytoplasm, the CRM1 complex binds to the cytoplasmic filament complex (Nup88, Nup214 and Nup358 84, 138, 139, 140, 141) and interacts with RanGAP causing the hydrolysis of GTP which promotes the dissociation of the protein complex. Following this dissociation, the cargo is released in the cytoplasm 36, 142.

Export of RNAs

RNAs transcribed in the nucleus have to be exported, either to fulfill their function in protein synthesis or to mature into functional particles 143. The pre‐mRNA is processed and packaged into messenger ribonucleoprotein (mRNP) complexes to be exported through bulk or specific export. The majority of poly‐A transcripts are exported via the non‐karyopherin heterodimer Nxf1/Nxt1 independently of the RanGTP gradient. Conversely, a specific subset of endogenous transcripts is exported from the nucleus via CRM1. For efficient export, RNAs must undergo processing that includes splicing, 3′‐end formation of the poly‐A tail and the addition of a methyl‐7‐guanosine (m7G) cap structure to their 5′‐ends 144, 145, 146. For the majority of transcripts, the m7G cap recruits the CBC, which then activates export factors that allow the export mRNPs to bind to the NPC and traverse the hydrophobic central channel. Nxf1 (also known as TAP) 147) is the major driver of interaction between the export mRNP and the NPC. The Nxf1/Nxt1 heterodimer is recruited to the mRNP via the transcription‐export (TREX) complex 79, 144, 145, 148, 149, 150. The TREX complex consists of UAP56, REF/a Aly, CIP29 and THO multi‐subunit complex composed of THOC1/Hpr1, hTho2, THOC5, THOC6, THOC7 and Tex1 144, 151, 152, 153. REF/Aly and THO complexes promote the interaction of cargo mRNAs with the Nxf1 receptor 144, 151 that associates mRNPs with the nuclear basket via the ribonucleic acid export protein Rae1 and Nup98 to permit passage through the central channel (Fig. 2) 154, 155. At the cytoplasmic face, the cargo mRNPs are released and the export factors are recycled. RanBP2 associates with Nup88 and Nup214 141 and plays a very important role in cargo release and recycling after bulk mRNA export.

An alternative to the Nxf1‐driven export pathway involves the serine‐ and arginine‐rich SR proteins SRp20 and 9G8 that mediate the export of H2a mRNA and of some spliced transcripts 156, 157. Although the majority of mRNAs use the Nxf1 receptor to cross the NPC, subsets of transcripts are exported via the general CRM1 pathway. Additionally, through protein cofactors, CRM1 is involved in the export of specific types of mRNAs, small nuclear RNAs (U snRNAs) and ribosomal RNAs. CRM1 does not bind RNA directly but via NES‐containing adaptor proteins that bind to RNA or other RNA binding proteins 148. Once this step is completed, CRM1–cargo complexes dissociate, permitting the RNA to enter the cytoplasm and to recycle export factors 148. As in bulk mRNA export, Nup88, Nup214 and RanBP2 play critical roles in the recycling and release steps for CRM1‐dependent export 138, 158.

Regulation of the nucleo‐cytoplasmic trafficking

The nucleo‐cytoplasmic transport processes are regulated by cellular signaling systems via cargo protein modification(s) and the transport machinery, including the transport receptors, the NPC and the Ran system. Indeed targeting nucleo‐cytoplasmic transport has directed the development of new exciting therapeutic avenues to treat cancers that display aberrant protein subcellular localization 159, 160.

Modification of the cargo proteins

Importin‐NLS/exportin‐NES interactions can be modulated by conformational changes in both NLS/NES regions and in the substrate binding site of karyopherins 161. In some cases the three‐dimensional structure of a protein masks its own transport signal as in the case of p105 161, the precursor p50 subunit of NF‐κB. Upon stimulation, p105 is phosphorylated, the C‐terminal part of the protein is degraded and the NLS becomes accessible allowing the p50 protein to be recognized by the Imp‐α/Imp‐β complex 162. Furthermore, phosphorylation within or close to the NLS/NES can promote intramolecular masking. In the case of Hog1p (high osmolarity glycerol pathway‐signaling protein) phosphorylation at Thr174 and Tyr176 renders Hog1p‐NES inaccessible for binding to CRM1 and prevents its export from the nucleus 163. Similarly NF‐AT2 (nuclear factor of activated T cell 2) contains two NLSs that can be masked by phosphorylation at a low calcium concentration. Following a calcium concentration increase, calcineurin is dephosphorylated and NF‐AT2 is imported into the nucleus 164, 165, 166. In the canonical NF‐κB pathway, I‐κB masks the NLS sequence of NF‐κB p65 167. In resting conditions, I‐κB is phosphorylated, ubiquitinated and degraded by proteasome resulting in NLS unmasking and NF‐κB p65 nuclear import 168, 169. One of the multiple regulatory mechanisms of p53 is its homo‐tetramerization that masks one of its NES sequences. Dissociation of this tetramer is required for its nuclear export 170. Binding of proteins to RNA and DNA can also modulate the intermolecular masking of localization signal. For example, the NLS of the Rev protein, implicated in HIV mRNA translocation, is masked when Rev is linked to mRNA and recycling of Rev is possible only after release of the transported mRNA 171. Conversely, nucleo‐cytoplasmic transport can be enhanced by phosphorylation of SV40 virus large T antigen 172 or Pho4 factor 173 increasing the affinity of their NLS/NES signals to the corresponding karyopherin receptors 172.

Viral regulation of nucleo‐cytoplasmic trafficking

Many viruses have evolved elegant strategies to exploit the host nucleo‐cytoplasmic transport pathways to evade the cellular anti‐viral response or to facilitate viral replication. Some viruses such as the vesicular stomatitis virus interact with Nups to inhibit the export of host mRNAs that encode anti‐viral factors and make the translation machinery available for expression of viral mRNAs 174. Similarly, the ICP27 protein of herpes simplex virus interacts with Nup62 and blocks nuclear import of proteins via Imp‐α/β1 and Imp‐β2 pathways 175, 176. Conversely, poliovirus and human rhinovirus block the nuclear import of proteins via the Imp‐α/β1 and Imp‐β2 pathways by viral‐mediated proteolytic cleavage of specific Nups 21, 177, 178, 179, 180, 181, 182, 183. Other viruses inhibit the nuclear import of the STAT proteins that are known to be key regulators of the cell anti‐virus response. Furthermore, the severe acute respiratory syndrome (SARS) virus disrupts the nuclear import of STAT1 by tethering the tyrosine‐phosphorylated STAT1–Imp‐α/Imp‐β complex to endoplasmic reticulum/Golgi membranes 184, 185, 186, 187. Yet another way exploited by viruses to prevent STAT1 nuclear localization is to bind viral proteins to import receptors. The Ebola virus VP24 protein binds Imp‐α to block its interaction with phosphorylated STAT1 and hnRNP C1/C2 188, 189, 190. The L1 protein of the human papilloma virus type 11 binds Imp‐β2/β3 and disrupts cargo import. Encephalomyocarditis virus exerts its inhibitory effect on the nuclear protein import of infected cells via its L protein that hyper‐phosphorylates Nups and binds Ran 191, 192, 193. In addition herpes viruses and HIV promote viral mRNA export by reprogramming the cellular transport pathways. The HIV‐1 Rev protein facilitates nuclear export of unspliced or partially spliced viral mRNAs through the Rev‐responsive element, an RNA signature within these viral mRNAs 85, 194, 195. As a result, Rev‐bound viral RNA binds to CRM1 and RanGTP and is transported through the NPC.