Binding dynamics of structural nucleoporins govern nuclear pore complex permeability and may mediate channel gating - PubMed (original) (raw)

Binding dynamics of structural nucleoporins govern nuclear pore complex permeability and may mediate channel gating

Nataliya Shulga et al. Mol Cell Biol. 2003 Jan.

Abstract

The nuclear pore complex (NPC) is a permeable sieve that can dilate to facilitate the bidirectional translocation of a wide size range of receptor-cargo complexes. The binding of receptors to FG nucleoporin docking sites triggers channel gating by an unknown mechanism. Previously, we used deoxyglucose and chilling treatments to implicate Nup170p and Nup188p in the control of NPC sieving in Saccharomyces cerevisiae. Here, we report that aliphatic alcohols increase the permeability of wild-type and nup170Delta NPCs. In conjunction with increases in permeability, aliphatic alcohols, deoxyglucose, and chilling trigger the reversible dissociation of several nucleoporins from nup170Delta NPCs. These results are consistent with the hypothesis that NPC gating occurs when molecular latches composed of FG repeats and structural nucleoporins dissociate.

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Figures

FIG. 1.

FIG. 1.

Aliphatic alcohols promote equilibration of cNLS-GFP. (A) Dose-dependent capacity of various alcohols to induce the equilibration of cNLS-GFP across wt nuclear envelopes. ○, methanol; ◊, ethanol; ▪, isopropanol; ▴, HD; •, _n_-butanol. Cells were exposed to the alcohols mixed with SC-Glu, and the percentage of cells exhibiting predominately nuclear localization of cNLS-GFP (% Nuclear cells) after 10 min was plotted as a function of alcohol concentration (vol/vol). The inset shows the relationship between the relative hydrophobicities (log P) (29) of five alcohols and the molar concentrations at which each induced 50% delocalization of cNLS-GFP in 10 min. Data for cyclohexanol (▵) are included instead of data for HD, for which a partition coefficient is not available. (B) Confocal images of wt cells treated with 0, 1, and 2% HD for 10 min and of cells after a 30-min recovery after 10 min in 5% alcohol. (C) Time course of cNLS-GFP localization in wt cells incubated in either 5% ethanol (▴) or 2% HD (⋄). Also shown are data for cells expressing GAL1::SSA1 in 5% ethanol (▵) or 2% HD (⋄). (D) Adaptive response of wt cells continuously incubated for 6 h in either 2% HD or 5% ethanol. Untreated control cells (white bars), HD-pretreated cells (grey bars), and ethanol-pretreated cells (black bars) were subsequently challenged with either 2% HD or 5% ethanol (EtOH) for 10 min before cNLS-GFP localization was scored.

FIG. 2.

FIG. 2.

Alcohol-induced increases in the sieving diameters of wt and _nup170_Δ NPCs. (A) Cells expressing NES-GFP reporters of various sizes were incubated for 10 min in the presence of 5% concentrations of ethanol, 2-propanol, or HD or 2% butanol. Minus signs indicate that the reporter was not excluded from the large majority of nuclei, plus signs indicate that the reporter was excluded from nuclei, and +− indicates partial equilibration. (B) Confocal images of wt and _nup170_Δ cells expressing the 66-kDa NES-GFP reporter and treated for 10 min with each of the indicated alcohols at the concentrations stated above. Arrowheads indicate location of nuclei. Scoring of cells is indicated in the lower right corner of each panel. The large black vacuolar compartments serve as a control for complete exclusion of the GFP reporter.

FIG. 3.

FIG. 3.

Reversible dissociation of nups in _nup170_Δ cells is induced by HD. Shown are confocal images revealing the localization of 14 different Nup-EYFP and Nup-GFP reporters in wt and _nup170_Δ cells after 10 min in the presence of 5% HD or after recovery from the alcohol (see Material and Methods). Nups are indicated by the numbers associated with their gene designations (e.g., 53 indicates Nup53p-EYFP), and the numbers for Nups containing FG repeats are circled.

FIG. 4.

FIG. 4.

Dissociation of nups in _nup170_Δ cells induced by deoxyglucose and chilling. Shown are confocal images revealing the localization of four Nup-EYFP reporters in _nup170_Δ cells in SC-Glu medium before (untreated) and after treatment with 20 mM deoxyglucose for 30 min at 30°C (Deoxyglucose) or chilled on ice for 30 min (0°C). The nups are designated as described in the legend to Fig. 3.

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