Kevin -Nuclear transport-exam-answer PDF

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Summary

Describe the mechanism of nuclear protein import including two key experimental approaches that have given rise to the model. Be sure to include discussion of Ran GTPase and the nuclear pore complex. Nuclear Import The transport of macromolecules in and out of the nucleus is essential for the viabil...

Description

Describe the mechanism of nuclear protein import including two key experimental approaches that have given rise to the model. Be sure to include discussion of Ran GTPase and the nuclear pore complex.

Nuclear Import The transport of macromolecules in and out of the nucleus is essential for the viability of eukaryotic cells in order to permit gene transcription in the nucleus and protein translation within the cytoplasm. The transport of cargo proteins is mediated by several distinct transport signals that are recognised by transport receptors or adaptor proteins. These transport receptors can then interact with compounds of the nuclear pore complex (NPC) and with the Ras family GTPase Ran. Many of the steps within this process have potential to be therapeutically targeted for innovative anti-cancer and anti-viral therapies. The nuclear envelope surrounds the nucleus and is composed of two phospholipid membranes that are 30 nm apart and acts as a selective barrier between the nucleus and the cytoplasm. The outer membrane is continuous with the ER while the inner membrane associates with the nuclear lamina. The NE functions as a selectively permeable barrier allowing macromolecules to move between the cytoplasm and the nucleus via the gatekeeper NPC. The NE is studded with NPC, and there are 2000-5000 per somatic cell. The NPC is 100150 nm in diameter and is highly conserved in eukaryotes. The NPC is a huge protein complex that fuses internal and external nuclear membranes to form an aqueous channel. The NPC has a doughnut shaped central core with an eight fold rotational symmetry. The NPC has been extensively studied by electron microscopy. There are eight protein filaments attached to the cytoplasmic side as well as the nuclear side that converge into a ring-like structure call the nuclear basket. The NPC can be divided into symmetric and asymmetric parts. The NPC contains 30 different types of proteins known as nucleoporins (Nups) that are present in multiple copies. Nups from the symmetric NPC include: membrane Nups, scaffold Nups, adaptor Nups, and channel (barrier) Nups. A macromolecule (protein) has an NLS and is hence destined to be transported into the nucleus. It binds to importin, where the NLS of the cargo proteins binds to imp-a which acts as an adaptor protein and imp-B interacts with components of the NPC. The cargo protein, complexed with Imp-a/B localises to the nuclear envelope, where it binds to Nups at binding sites rich in FG-repeats, in the NPC. The cargo-bearing importin is then shuttled across into the nucleus. Once through the pore, the complex interacts with Ran GTP. Ran GTP binds the importin, which has sort of stacked alpha helix motif forming a spring-like structure. The spring like structure has a conformational change and the cargo protein is released to its destination. After this exchange, the imp-B bound to Ran-GTP is transported back to the cytoplasm. Ran-GTP is hydrolysed to Ran-GDP and inorganic phosphate and dissociates from imp-b. Imp-a is transported back to the cytoplasm through the nuclear exporter CAS. The importin is thus recycled and can be used to transfer the next cargo.

The transport of macromolecules into the nucleus depends on a nuclear localisation signal (NLS) that is recognised by importin receptors from the karyopherin family which have 22 putative members in humans. The import receptors are soluble cytosolic proteins that bind to both NLSs on the cargo protein as well as NPC barrier Nups that contain large number of phenylalanine and glycine (FG) repeats that serve as the binding sites for importin receptors, known as FG-Nups. FG-Nups act as a selective gatekeeper for nuclear transport regulation – ‘virtual gating’. The FG repeat proteins create a barrier to proteins that have no specific affinity. The FG repeats are present in 1/3 of Nups and importin-cargo complexes move along the transport path created by FG repeats by constantly binding and dissociating to adjacent repeats to move through the nuclear pore. The known NLSs can be classified into either classical or non-classical NLSs. Classical NLSs can be further subdivided into monopartite or bipartite NLSs. Classical NLSs were first identified in the SV40 T antigen as short lysine-rich sequences (PKKKRKV) that bind to the armadillo (ARM) domain in the C-terminal of the adaptor protein, Importin-α (Imp-a). Imp-a also binds to the receptor imp-B forming a ternary complex. In contrast to classical NLSs and imp-a, proteins that do not require an adaptor protein bind directly to Imp-B through nonclassical NLSs and do not obey strict rules. The karyopherin-B proteins (imp-B) are multi-domain transport factors that contain a cargo binding domain, an NPC domain, and a binding domain for the small Ras-like GTPase Ran. The loading and unloading of importin receptors to cargo molecules is controlled by the small Ras-like GTPase, Ran and requires GTP hydrolysis. Ran is a 25kDa protein that exists in the RanGDP bound state or the RanGTP bound state. Ran itself is inefficient at GTP hydrolysis and requires the regulatory factors Ran-GAP1 and RanBP1 which increase GTP hydrolysis by Ran, and thus the release of the nuclear import receptor. Conversely, RanGTP exchange factor (RanGEF) accelerates the exchange of nucleotides restoring the pool of RanGTP. Because RanGAP1 is present in the cytosol and RanGEF is located in the nucleus, this localises Ran-GDP and Ran-GTP respectively which greats a directionality

gradient that drives nuclear transport. Import-cargo complexes are dissociated by binding to RanGTP directly and use the metabolic energy supplied by the Ran GTPase system for directional transport. Importins bind their cargo at low RanGTP level in the cytoplasm and traverse the NPC as dimeric complexes transporting cargo.

Nuclear Export For nuclear export to occur proteins also require specific transport signals called a nuclear export signal (NES). Nuclear export is organized in a very similar fashion to nuclear import, except that transport receptors bind cargo in the presence of Ran-GTP and discharge in the presence of Ran-GDP. The most extensively characterized export receptor is chromosome region maintenance 1 (CRM1), which is also a member of the karyopherin-B family. CRMI1 .;]shares homology with importin B but cargo is bound in the presence of Ran-GTP. CRM1 exports proteins that contain leucine rich NESs from the nucleus to the cytoplasm. CRM1 has HEAT domains that allow Ran GTP to bind, and cargo binding takes place outside of these HEAT domains in a hydrophobic cleft cleft (NES docking site). The affinity of CRM1 for most NESs is low and formation of the export complex is promoted by RanBP3. RanBP3 linksCRM1 to the chromatin binding protein RCC1 and increases the active concentration of RanGTP. This promotes the affinity of the NES cargo for CRM1. This complex moves through the NPC via the interaction with FG repeat proteins. Within the cytoplasm, the CRM1 complex binds to the cytoplasmic filament complex 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. RNA export RNA transcribed in the nucleus also has to be exported. RNA export occurs through two principle mechanisms either through Ran independent mechanism or Ran-dependent mechanisms. mRNP (messenger ribonucleoprotein) is transported from the nucleus via the non-karyopherin heterodimer Nxf1/Nxt1 independently of RanGTP. Nxf1 is the major driver of interaction between the export of mRNP and the NPC. The Nxf1/Nxt1 heterodimer is recruited to the mRNP via the transcription-export (TREX). The TREX complexes promote the interaction of cargo mRNPs with the nuclear basket to permit passage through the central channel. Cargo mRNPs are released at the cytoplasmic face. Although the majority of mRNAs use the Nxf1 receptor to cross the NPC, some transcripts are exported by the general Crm1 pathway, in a Ran-dependant mechanism. CRM1 is involved in the export of specific types of mRNA such as U snRNAs.

Experimental approaches that gave rise to the model

Electron microscopists first described the presence of and first coined the term ‘nuclear pore complex’. They revealed that the NPC was an octagonally symmetric cylinder, with eight spokes conjoined by coaxial rings, and a central channel through which all cargoes were shown to pass. This overall architecture was also shown to be well conserved in all the eukaryotes analyzed. From this ‘core’ structure, eight filaments project from both the

nucleoplasmic and the cytoplasmic faces; the nuclear filaments conjoin at a distal ring to form the ‘nuclear basket’. Atomic force microscopy is helping to define the physical and mechanical properties of the NPC. Using immunoelectron microscopy, most Nups were at least roughly localized to portions of the NPC, and importantly such localizations also showed that a particular class of Nups, termed ‘FG Nups’ is found in and around the NPCs central channel. Recently, crystallographic and NMR spectroscopy have revealed data on numerous Nups and transport factors, opening up the prospect of an atomic-level understanding of nuclear transport. Several fragments from peripheral Nups have been crystallized, either alone or in complexes with transport factors. But the main targets for crystallographic studies have been the Nups forming the architectural core of the NPC From lecture: Using mass spectrometry ~30 NPC proteins were identified. Each was tagged and localisation examined by EM A statistically based map of protein location was superimposed on NPC outline Crystallography and other structural analysis identifies • 8 different protein fold types account for >95% of components • 3 groups: • transmembrane group • central scaffold group • FG repeat group...