Limits...
A deep proteomics perspective on CRM1-mediated nuclear export and nucleocytoplasmic partitioning.

Kırlı K, Karaca S, Dehne HJ, Samwer M, Pan KT, Lenz C, Urlaub H, Görlich D - Elife (2015)

Bottom Line: In addition, we quantified the partitioning of ≈5000 unique proteins between nucleus and cytoplasm of Xenopus oocytes.There are also numerous new instances where CRM1 appears to act in regulatory circuits.Altogether, our dataset allows unprecedented insights into the nucleocytoplasmic organisation of eukaryotic cells, into the contributions of an exceedingly promiscuous exportin and it provides a new basis for NES prediction.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

ABSTRACT
CRM1 is a highly conserved, RanGTPase-driven exportin that carries proteins and RNPs from the nucleus to the cytoplasm. We now explored the cargo-spectrum of CRM1 in depth and identified surprisingly large numbers, namely >700 export substrates from the yeast S. cerevisiae, ≈1000 from Xenopus oocytes and >1050 from human cells. In addition, we quantified the partitioning of ≈5000 unique proteins between nucleus and cytoplasm of Xenopus oocytes. The data suggest new CRM1 functions in spatial control of vesicle coat-assembly, centrosomes, autophagy, peroxisome biogenesis, cytoskeleton, ribosome maturation, translation, mRNA degradation, and more generally in precluding a potentially detrimental action of cytoplasmic pathways within the nuclear interior. There are also numerous new instances where CRM1 appears to act in regulatory circuits. Altogether, our dataset allows unprecedented insights into the nucleocytoplasmic organisation of eukaryotic cells, into the contributions of an exceedingly promiscuous exportin and it provides a new basis for NES prediction.

No MeSH data available.


Related in: MedlinePlus

Identification of cargo candidates as direct CRM1-binders.The H14-ZZ-Sumo tagged candidate proteins ARP2/3 1b (Q6GNU1), eRF3a (Q91855), Haus1 (Q3B8L5), pRKAr2a (F7CZT8), Septin-2 (B7ZR20) were expressed in E. coli, purified, immobilised on anti-zz beads, and incubated with CRM1 in the absence or presence of RanGTP. Immobilised candidate proteins were released, after washing, by Sumo-protease cleavage and co-eluting materials were analysed by SDS-PAGE (Note that Septin-2 elution was less efficient than the others). An unfused H14-zz-Sumo module served as a negative and a fusion with a PKI-NES as a positive control for CRM1-binding.DOI:http://dx.doi.org/10.7554/eLife.11466.008
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4764573&req=5

fig5: Identification of cargo candidates as direct CRM1-binders.The H14-ZZ-Sumo tagged candidate proteins ARP2/3 1b (Q6GNU1), eRF3a (Q91855), Haus1 (Q3B8L5), pRKAr2a (F7CZT8), Septin-2 (B7ZR20) were expressed in E. coli, purified, immobilised on anti-zz beads, and incubated with CRM1 in the absence or presence of RanGTP. Immobilised candidate proteins were released, after washing, by Sumo-protease cleavage and co-eluting materials were analysed by SDS-PAGE (Note that Septin-2 elution was less efficient than the others). An unfused H14-zz-Sumo module served as a negative and a fusion with a PKI-NES as a positive control for CRM1-binding.DOI:http://dx.doi.org/10.7554/eLife.11466.008

Mentions: eRF3a binds also purified CRM1 in a RanGTP-dependent manner (Figure 5), suggesting that the interaction is direct and not bridged by another factor. Due to its efficient binding from Xenopus oocyte extract to CRM1, eRF3a was classified as a ‘category A’ cargo. Next, we also confirmed a far lower scoring ‘category B’ candidate, namely the 1b subunit of the Arp2/3 complex, as a directly-binding, bona fide CRM1 export cargo with an NES at its extreme C-terminus. In the light of the rather weak CRM1-binding of Arp2/3-1b (Supplementary file 2), its NES turned out to be surprisingly strong. When fused to GFP, it behaved like a supraphysiological NES (Engelsma et al., 2004) and produced pronounced transport intermediates at NPCs (best visible in weakly expressing cells). This difference is, however, plausible in the context of the Arp2/3 complex structure (pdb 1K8K; Robinson et al., 2001), which shows this NES packing against the rest of the chain. The rather loose packing and high local B-factor suggest, however, that this NES region is sufficiently mobile to get transiently exposed for subsequent CRM1-binding.10.7554/eLife.11466.008Figure 5.Identification of cargo candidates as direct CRM1-binders.


A deep proteomics perspective on CRM1-mediated nuclear export and nucleocytoplasmic partitioning.

Kırlı K, Karaca S, Dehne HJ, Samwer M, Pan KT, Lenz C, Urlaub H, Görlich D - Elife (2015)

Identification of cargo candidates as direct CRM1-binders.The H14-ZZ-Sumo tagged candidate proteins ARP2/3 1b (Q6GNU1), eRF3a (Q91855), Haus1 (Q3B8L5), pRKAr2a (F7CZT8), Septin-2 (B7ZR20) were expressed in E. coli, purified, immobilised on anti-zz beads, and incubated with CRM1 in the absence or presence of RanGTP. Immobilised candidate proteins were released, after washing, by Sumo-protease cleavage and co-eluting materials were analysed by SDS-PAGE (Note that Septin-2 elution was less efficient than the others). An unfused H14-zz-Sumo module served as a negative and a fusion with a PKI-NES as a positive control for CRM1-binding.DOI:http://dx.doi.org/10.7554/eLife.11466.008
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4764573&req=5

fig5: Identification of cargo candidates as direct CRM1-binders.The H14-ZZ-Sumo tagged candidate proteins ARP2/3 1b (Q6GNU1), eRF3a (Q91855), Haus1 (Q3B8L5), pRKAr2a (F7CZT8), Septin-2 (B7ZR20) were expressed in E. coli, purified, immobilised on anti-zz beads, and incubated with CRM1 in the absence or presence of RanGTP. Immobilised candidate proteins were released, after washing, by Sumo-protease cleavage and co-eluting materials were analysed by SDS-PAGE (Note that Septin-2 elution was less efficient than the others). An unfused H14-zz-Sumo module served as a negative and a fusion with a PKI-NES as a positive control for CRM1-binding.DOI:http://dx.doi.org/10.7554/eLife.11466.008
Mentions: eRF3a binds also purified CRM1 in a RanGTP-dependent manner (Figure 5), suggesting that the interaction is direct and not bridged by another factor. Due to its efficient binding from Xenopus oocyte extract to CRM1, eRF3a was classified as a ‘category A’ cargo. Next, we also confirmed a far lower scoring ‘category B’ candidate, namely the 1b subunit of the Arp2/3 complex, as a directly-binding, bona fide CRM1 export cargo with an NES at its extreme C-terminus. In the light of the rather weak CRM1-binding of Arp2/3-1b (Supplementary file 2), its NES turned out to be surprisingly strong. When fused to GFP, it behaved like a supraphysiological NES (Engelsma et al., 2004) and produced pronounced transport intermediates at NPCs (best visible in weakly expressing cells). This difference is, however, plausible in the context of the Arp2/3 complex structure (pdb 1K8K; Robinson et al., 2001), which shows this NES packing against the rest of the chain. The rather loose packing and high local B-factor suggest, however, that this NES region is sufficiently mobile to get transiently exposed for subsequent CRM1-binding.10.7554/eLife.11466.008Figure 5.Identification of cargo candidates as direct CRM1-binders.

Bottom Line: In addition, we quantified the partitioning of ≈5000 unique proteins between nucleus and cytoplasm of Xenopus oocytes.There are also numerous new instances where CRM1 appears to act in regulatory circuits.Altogether, our dataset allows unprecedented insights into the nucleocytoplasmic organisation of eukaryotic cells, into the contributions of an exceedingly promiscuous exportin and it provides a new basis for NES prediction.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Logistics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.

ABSTRACT
CRM1 is a highly conserved, RanGTPase-driven exportin that carries proteins and RNPs from the nucleus to the cytoplasm. We now explored the cargo-spectrum of CRM1 in depth and identified surprisingly large numbers, namely >700 export substrates from the yeast S. cerevisiae, ≈1000 from Xenopus oocytes and >1050 from human cells. In addition, we quantified the partitioning of ≈5000 unique proteins between nucleus and cytoplasm of Xenopus oocytes. The data suggest new CRM1 functions in spatial control of vesicle coat-assembly, centrosomes, autophagy, peroxisome biogenesis, cytoskeleton, ribosome maturation, translation, mRNA degradation, and more generally in precluding a potentially detrimental action of cytoplasmic pathways within the nuclear interior. There are also numerous new instances where CRM1 appears to act in regulatory circuits. Altogether, our dataset allows unprecedented insights into the nucleocytoplasmic organisation of eukaryotic cells, into the contributions of an exceedingly promiscuous exportin and it provides a new basis for NES prediction.

No MeSH data available.


Related in: MedlinePlus