Nuclear mRNA export requires specific FG nucleoporins for translocation through the nuclear pore complex - PubMed (original) (raw)

Nuclear mRNA export requires specific FG nucleoporins for translocation through the nuclear pore complex

Laura J Terry et al. J Cell Biol. 2007.

Abstract

Trafficking of nucleic acids and large proteins through nuclear pore complexes (NPCs) requires interactions with NPC proteins that harbor FG (phenylalanine-glycine) repeat domains. Specialized transport receptors that recognize cargo and bind FG domains facilitate these interactions. Whether different transport receptors utilize preferential FG domains in intact NPCs is not fully resolved. In this study, we use a large-scale deletion strategy in Saccharomyces cerevisiae to generate a new set of more minimal pore (mmp) mutants that lack specific FG domains. A comparison of messenger RNA (mRNA) export versus protein import reveals unique subsets of mmp mutants with functional defects in specific transport receptors. Thus, multiple functionally independent NPC translocation routes exist for different transport receptors. Our global analysis of the FG domain requirements in mRNA export also finds a requirement for two NPC substructures-one on the nuclear NPC face and one in the NPC central core. These results pinpoint distinct steps in the mRNA export mechanism that regulate NPC translocation efficiency.

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Figures

Figure 1.

Figure 1.

The more minimal NPC (mmp) FGΔ mutants have temperature-sensitive growth defects. (A) Wild-type, ΔNΔC, and new mmp FGΔ yeast strains were spotted onto YPD in fivefold serial dilutions and grown at the temperatures shown. (B) Schematic representation of the distribution of FG Nups within the NPC.

Figure 2.

Figure 2.

The mmp FGΔ NPC mutants have distinct defects in Kap104 and Kap121 steady-state import. (A) Indirect immunofluorescence with an anti-Nab2 antibody in yeast mmp FGΔ strains was conducted after a 1-h shift to 37°C. Nab2 localization, indicating Kap104 import, and DAPI-staining panels are shown. (B) Localization of a Spo12-NLS-GFP reporter, which is imported by Kap121, was evaluated at 23°C and after a 1-h shift to 37°C in mmp FGΔ strains.

Figure 3.

Figure 3.

mRNA export is inhibited in the symmetric FGΔ mutants and the mmp mutant ΔNΔ C nup57Δ GLFG. In situ hybridization with an oligo d(T) probe was conducted in the FGΔ NPC mutants after a 1-h shift to 37°C. Signal for the oligo d(T) probe indicates the subcellular distribution of poly(A)+ RNA in comparison with the nuclear signal (by coincident DAPI staining).

Figure 4.

Figure 4.

mRNA export requires the FG domains of Nup57 and nuclear face Nups. In situ hybridization with an oligo d(T) probe was conducted with the FGΔ strains indicated after a 1-h shift to 37°C. The percentage of cells showing the accumulation of poly(A)+ RNA was calculated based on fields of >100 cells in three independent trials. Deletion of the nuclear face FG domains (nup1ΔFXFG, nup2ΔFXFG, and nup60ΔFXF) is abbreviated as ΔN. Deletion of the cytoplasmic face FG domains (nup42ΔFG and nup159ΔFG) is abbreviated as ΔC. Error bars represent SEM.

Figure 5.

Figure 5.

Mex67 binds the GLFG domain of Nup57. Bacterially expressed GST, GST-GLFG-NUP57, and GST-GLFG-NUP116 were each immobilized on glutathione agarose beads. Recombinant purified MBP-Mex67 was added, and the bound fraction was eluted. 10% of the input (MBP-Mex67) and the eluted fractions was resolved by SDS-PAGE and stained with Coomassie blue. Molecular mass (kilodaltons) markers are shown at the left (M r).

Figure 6.

Figure 6.

Mex67-GFP recruitment to the NE/NPC is severely inhibited in both the ΔNΔ C nup57Δ GLFG mutant and ΔN nup57Δ GLFG mutant. (A–D) Mex67-GFP localization in representative wild-type (A), ΔNΔC (B), ΔNΔC nup57ΔGLFG (C), and ΔN nup57ΔGLFG (D) cells before the assay (untreated; left), after energy depletion (middle), or after 5-6 min of recovery from energy depletion (right). For each, the coincident localization of the ER marker dsRed-HDEL is shown. (E) As controls, the localization of GFP-Nic96 and Nup170-GFP or Nup49-GFP under the same conditions was evaluated. (F) A schematic diagram of the energy depletion assay for Mex67-GFP localization is shown. (G) The kinetics of Mex67-GFP recovery to the nuclear rim over time after energy depletion was determined. For three independent experiments, >150 cells were scored for the subcellular distribution of GFP signal at each time point. Error bars represent SEM. DIC, differential interference contrast.

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