Structural basis for Nup2p function in cargo release and karyopherin recycling in nuclear import

Matsuura, Yasushi; Lange, Allison; Harreman, Michelle T.; Corbett, Anita H.; Stewart, Murray

doi:10.1093/emboj/cdg538

Cited by 91 publications

(121 citation statements)

References 38 publications

(78 reference statements)

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“…Another sequence that resembles a monopartite classical NLS, albeit suboptimal, is 320 KTNKSHK 326 , found at the C terminus of HuR. Recently, the repertoire of sequences that bind in an NLS-like manner to importin ␣ has been expanded to include the MNRRKIAMPKRRMAFK sequence of nucleoporin Nup2p, which binds in a different but overlapping site from the classical NLS (57). Therefore, it is conceivable that importin ␣ binds cargo ligands containing NLS other than the well known mono-and bipartite classical NLSs.…”

Section: Discussionmentioning

confidence: 99%

AMP-activated Protein Kinase-regulated Phosphorylation and Acetylation of Importin α1

Wang

Yang

Kawai

et al. 2004

Journal of Biological Chemistry

123

101

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Nuclear import of HuR, a shuttling RNA-binding protein, is associated with reduced stability of its target mRNAs. Increased function of the AMP-activated protein kinase (AMPK), an enzyme involved in responding to metabolic stress, was recently shown to reduce the cytoplasmic levels of HuR. Here, we provide evidence that importin ␣1, an adaptor protein involved in nuclear import, contributes to the nuclear import of HuR through two AMPK-modulated mechanisms. First, AMPK triggered the acetylation of importin ␣1 on Lys 22 , a process dependent on the acetylase activity of p300. Second, AMPK phosphorylated importin ␣1 on Ser 105 . Accordingly, expression of importin ␣1 proteins bearing K22R or S105A mutations failed to mediate the nuclear import of HuR in intact cells. Our results point to importin ␣1 as a critical downstream target of AMPK and key mediator of AMPK-triggered HuR nuclear import.

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Section: Discussionmentioning

confidence: 99%

AMP-activated Protein Kinase-regulated Phosphorylation and Acetylation of Importin α1

Wang

Yang

Kawai

et al. 2004

Journal of Biological Chemistry

123

101

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“…This result was not predicted from biochemical studies. Although numerous investigators found that NUP50 destabilizes the Imp ␣/cargo interaction (16,22,23,25,26), this destabilization seems to be insufficient to cause Imp ␣/cargo complex dissociation in the context of a functioning NPC on the time scale of transport.…”

Section: Cas Localization Bias In the Npc Can Explain The Imp ␣1 And mentioning

confidence: 98%

“…Numerous groups have postulated that NUP50 (Nup2p) catalyzes Imp ␣1/cargo complex dissociation when it reaches the nuclear basket region of the NPC (16,(22)(23)(24)(25)(26)(27). In control experiments, Ϸ50% of EGFP2-mNup50 (fusion of 2ϫEGFP with mouse Nup50) was retained by the NPCs for at least 20 min after permeabilization (Fig.…”

Section: Cas Localization Bias In the Npc Can Explain The Imp ␣1 And mentioning

confidence: 99%

“…In principle, this mechanism could function within the NPC or within the nucleoplasm. According to a third mechanism, NUP50 (Nup2p in yeast), a nuclear pore protein, promotes dissociation of the Imp ␣/cargo complex (22)(23)(24)(25)(26)(27). Because NUP50 is located on the nuclear basket, this model predicts that the Imp ␣/cargo dissociation event occurs near the end of cargo translocation through the NPC.…”

mentioning

confidence: 99%

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Single-molecule measurements of importin α/cargo complex dissociation at the nuclear pore

Sun

Yang

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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Macromolecules are transported between the cytoplasm and the nucleoplasm of eukaryotic cells through nuclear pore complexes (NPCs). Large (more than Ϸ40 kDa) transport cargoes imported into the nucleus typically form a complex with at least one soluble transport cofactor of the importin (Imp) ␤ superfamily. Many cargoes require an accessory cofactor, Imp ␣, which binds to Imp ␤ and to the nuclear localization sequence on the cargo. We previously reported the use of narrow-field epifluorescence microscopy to directly monitor cargoes in transit through NPCs in permeabilized cells. We now report an expanded approach in which singlemolecule fluorescence resonance energy transfer (FRET) is used to detect the disassembly of Imp ␣/cargo complexes as they transit through NPCs. We found that CAS, the recycling cofactor for Imp ␣, and RanGTP are essential for this dissociation process. After Imp ␣/cargo complex dissociation, most Imp ␣ and cargo molecules entered the nucleoplasm. In contrast, the majority of Imp ␣/cargo complexes that did not dissociate at the NPC in the presence of CAS and RanGTP returned to the cytoplasm. These data are consistent with a model in which Imp ␣/cargo complexes are dissociated on the nucleoplasmic side of the NPC, and this dissociation requires both CAS and RanGTP.FRET ͉ nuclear transport ͉ single-molecule fluorescence N uclear pore complexes (NPCs) mediate the bidirectional transport of proteins and RNAs across the doublemembrane nuclear envelope (NE) of eukaryotic cells. They are comprised of Ϸ30 different nuclear pore proteins (Nups), each present in an integer multiple of eight copies (1-3). The pore itself is Ϸ90 nm long and is Ϸ50 nm wide at its narrowest point. Flexible filaments extend out from the pore Ϸ50 nm into the cytoplasm, and a basket structure extends Ϸ75 nm into the nucleoplasm (1, 4). The pore is filled by natively unfolded (disordered) and highly flexible protein structures containing thousands of phenylalanine-glycine (FG) repeat motifs (3,(5)(6)(7)(8). This FG-network provides a selectivity filter, allowing small molecules (less than Ϸ20-40 kDa) to diffuse through and rejecting most larger molecules. Larger molecules (up to Ϸ25 MDa) penetrate the FG-network with the assistance of transport receptors of the importin (Imp) ␤ superfamily, which directly interact with the FG repeats (9-11). Import complexes consisting of Imp ␤, Imp ␣, and cargo are dissociated after transport by RanGTP, the GTP-bound form of the G protein Ran (12). Imp ␤ is recycled to the cytoplasm in a complex with RanGTP, and Imp ␣ is recycled in a complex with CAS and RanGTP. Activation of the Ran GTPase by the cytoplasmically located RanGAP frees Imp ␤ and Imp ␣ for another round of cargo transport (13-15; for recent reviews, see refs. 9-11).Numerous mechanisms of Imp ␣/cargo complex dissociation have been proposed. One possibility is that Imp ␣/cargo complexes dissociate spontaneously, and an autoinhibitory sequence in the N-terminal Imp ␤ binding domain of Imp ␣ inhibits cargo rebinding (16-18). Spo...

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“…Such an approach shows that F(l) is the free energy of the total system constrained to a particular value of l. D is the diffusion constant of the cargo-carrier complex. The rate of transport from cytoplasm to nucleoplasm k C→N is then the inverse of the mean exit time [17] from the pore at steady state obtained from (1) for absorbing boundary conditions at l N on the nucleoplasmic side, representing the rapid disassembly of the cargo-complex catalyzed by RanGTP and nuclear porins associated with the nuclear basket [18,19]. It can be derived from Eq.…”

Section: Modelmentioning

confidence: 99%

Managing Free-energy Barriers in Nuclear Pore Transport

2006

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The Nuclear Pore Complexes (NPC) facilitate highly selective gateways for transport of macromolecules across the Nuclear Envelope (NE). Based on the current accumulated knowledge of the architecture of NPC we have established a minimal physical model of the pore and the transport mechanism. The barrier properties of the NPC model are analyzed by the recently established WangLandau Monte Carlo computer simulation technique and the transport properties are extracted by employing Kramers' theory of reaction rates. We show that our physical model can account for a range of characteristics observed for nuclear pore transport.

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Structural basis for Nup2p function in cargo release and karyopherin recycling in nuclear import

Cited by 91 publications

References 38 publications

AMP-activated Protein Kinase-regulated Phosphorylation and Acetylation of Importin α1

AMP-activated Protein Kinase-regulated Phosphorylation and Acetylation of Importin α1

Single-molecule measurements of importin α/cargo complex dissociation at the nuclear pore

Managing Free-energy Barriers in Nuclear Pore Transport

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