A non-canonical mechanism for Crm1-export cargo complex assembly
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Abstract:
The transport receptor Crm1 mediates the export of diverse cargos containing leucine-rich nuclear export signals (NESs) through complex formation with RanGTP. To ensure efficient cargo release in the cytoplasm, NESs have evolved to display low affinity for Crm1. However, mechanisms that overcome low affinity to assemble Crm1-export complexes in the nucleus remain poorly understood. In this study, we reveal a new type of RanGTP-binding protein, Slx9, which facilitates Crm1 recruitment to the 40S pre-ribosome-associated NES-containing adaptor Rio2. In vitro, Slx9 binds Rio2 and RanGTP, forming a complex. This complex directly loads Crm1, unveiling a non-canonical stepwise mechanism to assemble a Crm1-export complex. A mutation in Slx9 that impairs Crm1-export complex assembly inhibits 40S pre-ribosome export. Thus, Slx9 functions as a scaffold to optimally present RanGTP and the NES to Crm1, therefore, triggering 40S pre-ribosome export. This mechanism could represent one solution to the paradox of weak binding events underlying rapid Crm1-mediated export.Keywords:
Ran
Nuclear export signal
Nuclear pore
Abstract During mitotic exit, thousands of nuclear pore complexes (NPCs) assemble concomitant with the nuclear envelope to build a transport-competent nucleus. We show here that Nup50 plays a crucial role in NPC assembly that is independent of its well-established function in nuclear transport. RNAi-mediated downregulation in cells or immunodepletion of the protein in Xenopus egg extracts interferes with NPC assembly. We define a conserved central region of 46 residues in Nup50 that is crucial for Nup153 and MEL28/ELYS binding, and NPC interaction. Surprisingly, neither NPC interaction nor binding of Nup50 to importin α, β, the GTPase Ran or chromatin is crucial for its function in the assembly process. Instead, we discovered that an N-terminal fragment of Nup50 can stimulate the Ran guanine exchange factor RCC1 and NPC assembly, indicating that Nup50 acts via the Ran system in mitotic NPC reformation. In support of this conclusion, Nup50 mutants defective in RCC1 binding and stimulation cannot replace the wild type protein in in vitro NPC assembly assays.
Ran
Nuclear pore
Nucleoporin
Importin
Karyopherin
Small GTPase
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Abstract The nuclear pore complex (NPC) serves as a central gate for mRNAs to transit from the nucleus to the cytoplasm. The ability for mRNAs to get exported is linked to various upstream nuclear processes including co‐transcriptional RNP assembly and processing, and only export competent mRNPs are thought to get access to the NPC. While the nuclear pore is generally viewed as a monolithic structure that serves as a mediator of transport driven by transport receptors, more recent evidence suggests that the NPC might be more heterogenous than previously believed, both in its composition or in the selective treatment of cargo that seek access to the pore, providing functional plasticity to mRNA export. In this review, we consider the interconnected processes of nuclear mRNA metabolism that contribute and mediate export competence. Furthermore, we examine different aspects of NPC heterogeneity, including the role of the nuclear basket and its associated complexes in regulating selective and/or efficient binding to and transport through the pore. This article is categorized under: RNA Export and Localization > Nuclear Export/Import RNA Turnover and Surveillance > Turnover/Surveillance Mechanisms RNA Interactions with Proteins and Other Molecules > Protein‐RNA Interactions: Functional Implications
Nuclear pore
Nuclear export signal
Nucleoporin
Mediator
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Ran
Nuclear export signal
Karyopherin
Nucleoporin
Importin
Nuclear pore
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Ran
Importin
Karyopherin
Nuclear export signal
Nuclear pore
Nucleoporin
Small GTPase
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Ran
Nucleoporin
Nuclear pore
Karyopherin
Nuclear export signal
Importin
Small GTPase
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Citations (60)
The small GTPase Ran is essential for virtually all nucleocytoplasmic transport events. It is hypothesized that Ran drives vectorial transport of macromolecules into and out of the nucleus via the establishment of a Ran gradient between the cytoplasm and nucleoplasm. Although Ran shuttles between the nucleus and cytoplasm, it is concentrated in the nucleus at steady state. We show that nuclear transport factor 2 (NTF2) is required to concentrate Ran in the nucleus in the budding yeast, Saccharomyces cerevisiae. To analyze the mechanism of Ran import into the nucleus by NTF2, we use mutants in a variety of nuclear transport factors along with biochemical analyses of NTF2 complexes. We find that Ran remains concentrated in the nucleus when importin-mediated protein import is disrupted and demonstrate that NTF2 does not form a stable complex with the transport receptor, importin-β. Consistent with a critical role for NTF2 in establishing and maintaining the Ran gradient, we show that NTF2 is required for early embryogenesis inCaenorhabditis elegans. Our data distinguish between two possible mechanisms for Ran import by NTF2 and demonstrate that Ran import is independent from importin-β-mediated protein import.
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Importin
Nucleoplasm
Small GTPase
Nucleoporin
Nuclear pore
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Soluble karyopherins of the importin-β (impβ) family use RanGTP to transport cargos directionally through the nuclear pore complex (NPC). Whether impβ or RanGTP regulate the permeability of the NPC itself has been unknown. In this study, we identify a stable pool of impβ at the NPC. A subpopulation of this pool is rapidly turned-over by RanGTP, likely at Nup153. Impβ, but not transportin-1 (TRN1), alters the pore's permeability in a Ran-dependent manner, suggesting that impβ is a functional component of the NPC. Upon reduction of Nup153 levels, inert cargos more readily equilibrate across the NPC yet active transport is impaired. When purified impβ or TRN1 are mixed with Nup153 in vitro, higher-order, multivalent complexes form. RanGTP dissolves the impβ•Nup153 complexes but not those of TRN1•Nup153. We propose that impβ and Nup153 interact at the NPC's nuclear face to form a Ran-regulated mesh that modulates NPC permeability.
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Importin
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Nuclear pore
Nucleoporin
Importin
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Docking (animal)
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Active transport of macromolecules between the nucleus and cytoplasm requires signals for import and export and their recognition by shuttling receptors. Each class of macromolecule is thought to have a distinct receptor that mediates the transport reaction. Assembly and disassembly reactions of receptor-substrate complexes are coordinated by Ran, a GTP-binding protein whose nucleotide state is regulated catalytically by effector proteins. Ran function is modulated in a noncatalytic fashion by NTF2, a protein that mediates nuclear import of Ran-GDP. Here we characterize a novel component of the Ran system that is 26% identical to NTF2, which based on its function we refer to as NTF2-related export protein 1 (NXT1). In contrast to NTF2, NXT1 preferentially binds Ran-GTP, and it colocalizes with the nuclear pore complex (NPC) in mammalian cells. These properties, together with the fact that NXT1 shuttles between the nucleus and the cytoplasm, suggest an active role in nuclear transport. Indeed, NXT1 stimulates nuclear protein export of the NES-containing protein PKI in vitro. The export function of NXT1 is blocked by the addition of leptomycin B, a compound that selectively inhibits the NES receptor Crm1. Thus, NXT1 regulates the Crm1-dependent export pathway through its direct interaction with Ran-GTP.
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Nuclear export signal
Importin
Nucleoporin
Nuclear pore
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Importin
Nuclear pore
Nuclear export signal
Family member
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