Yeast nucleoporins involved in passive nuclear envelope permeability - PubMed (original) (raw)

Yeast nucleoporins involved in passive nuclear envelope permeability

N Shulga et al. J Cell Biol. 2000.

Abstract

The vertebrate nuclear pore complex (NPC) harbors an approximately 10-nm diameter diffusion channel that is large enough to admit 50-kD polypeptides. We have analyzed the permeability properties of the Saccharomyces cerevisiae nuclear envelope (NE) using import (NLS) and export (NES) signal-containing green fluorescent protein (GFP) reporters. Compared with wild-type, passive export rates of a classical karyopherin/importin (Kap) Kap60p/Kap95p-targeted NLS-GFP reporter (cNLS-GFP) were significantly faster in nup188-Delta and nup170-Delta cells. Similar results were obtained using two other NLS-GFP reporters, containing either the Kap104p-targeted Nab2p NLS (rgNLS) or the Kap121p-targeted Pho4p NLS (pNLS). Elevated levels of Hsp70 stimulated cNLS-GFP import, but had no effect on the import of rgNLS-GFP. Thus, the role of Hsp70 in NLS-directed import may be NLS- or targeting pathway-specific. Equilibrium sieving limits for the diffusion channel were assessed in vivo using NES-GFP reporters of 36-126 kD and were found to be greater than wild-type in nup188-Delta and nup170-Delta cells. We propose that Nup170p and Nup188p are involved in establishing the functional resting diameter of the NPC's central transport channel.

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Figures

Figure 1

Figure 1

Export kinetics of cNLS-GFP as a function of temperature in azide/deoxyglucose. Cells expressing cNLS-GFP were grown at 24°C, pelleted, washed, and suspended in glucose-free SC medium containing 10 mM azide and 10 mM 2-deoxyglucose that had been warmed or chilled at the assay temperature. Export was quantified as described in Materials and Methods.

Figure 2

Figure 2

Steady-state localization of cNLS-GFP in wt, _nup170-_Δ, and _nup188-_Δ cells was determined before induction of GAL1-SSA1 (23°C), after induction (23°C +SSA1), and incubated on ice after induction (0°C +SSA1). GFP fluorescence and Hoechst stain images were obtained by confocal microscopy (see Materials and Methods).

Figure 3

Figure 3

Temperature dependence of cNLS-GFP nuclear accumulation in wt and mutant cells. A, Dynamics of cNLS-GFP localization in wt and _nup188-_Δ cells ± GAL1-SSA1 induction were quantified as described in Materials and Methods. At time = 0, cells were shifted from 23°C to an ice bath (0°C). Wt cells were maintained at 0°C for the duration of the time course. Arrows on the time line indicate when _nup188-_Δ cells were shifted from 0 to 23°C (15 min) and then back to 0°C (30 min). B, Relaxation kinetics of cNLS-GFP redistribution in _nup188-_Δ + SSA1 cells after shifting initial incubation temperature from 30 to 20, 10, or 0°C. C, Export kinetics of cNLS-GFP at 0°C in additional null strains.

Figure 3

Figure 3

Temperature dependence of cNLS-GFP nuclear accumulation in wt and mutant cells. A, Dynamics of cNLS-GFP localization in wt and _nup188-_Δ cells ± GAL1-SSA1 induction were quantified as described in Materials and Methods. At time = 0, cells were shifted from 23°C to an ice bath (0°C). Wt cells were maintained at 0°C for the duration of the time course. Arrows on the time line indicate when _nup188-_Δ cells were shifted from 0 to 23°C (15 min) and then back to 0°C (30 min). B, Relaxation kinetics of cNLS-GFP redistribution in _nup188-_Δ + SSA1 cells after shifting initial incubation temperature from 30 to 20, 10, or 0°C. C, Export kinetics of cNLS-GFP at 0°C in additional null strains.

Figure 4

Figure 4

Import kinetics of cNLS-GFP at 0°C. Wt, _nup170-_Δ, and _nup188-_Δ cells were treated for 40 min with 10 mM 2-deoxyglucose at 30°C to equilibrate cNLS-GFP. Cells were then washed and resuspended in ice-cold complete medium and incubated at 0°C. Cells were harvested at various times and assayed for cNLS-GFP nuclear accumulation as described in Materials and Methods. Also shown are fluorescence (GFP) and light (DIC) images of wt cells before and after 2-deoxyglucose-induced equilibration, and after 20 h on ice.

Figure 4

Figure 4

Import kinetics of cNLS-GFP at 0°C. Wt, _nup170-_Δ, and _nup188-_Δ cells were treated for 40 min with 10 mM 2-deoxyglucose at 30°C to equilibrate cNLS-GFP. Cells were then washed and resuspended in ice-cold complete medium and incubated at 0°C. Cells were harvested at various times and assayed for cNLS-GFP nuclear accumulation as described in Materials and Methods. Also shown are fluorescence (GFP) and light (DIC) images of wt cells before and after 2-deoxyglucose-induced equilibration, and after 20 h on ice.

Figure 5

Figure 5

Effects of chilling, _GAL1_-SSA1 expression, and azide/deoxyglucose on the nuclear localization of rgNLS-GFP in wt, _nup188-_Δ, and _nup170-_Δ cells. A, Localization of rgNLS-GFP in wt, _nup170-_Δ, and _nup188-_Δ cells was determined before induction of GAL1-SSA1 (23°C), after induction (23°C +SSA1), after incubation on ice after induction (0°C +SSA1), and after incubation in azide/deoxyglucose. GFP and DIC images were captured by confocal microscopy (see Materials and Methods). B, Kinetic import assay of rgNLS-GFP in wt and mutant cells after equilibration in azide/deoxyglucose. Import was assayed in _nup170-_Δ and _nup188-_Δ cells ± GAL1-SSA1 induction. Kinetic assays were performed at 37°C as described in Materials and Methods.

Figure 5

Figure 5

Effects of chilling, _GAL1_-SSA1 expression, and azide/deoxyglucose on the nuclear localization of rgNLS-GFP in wt, _nup188-_Δ, and _nup170-_Δ cells. A, Localization of rgNLS-GFP in wt, _nup170-_Δ, and _nup188-_Δ cells was determined before induction of GAL1-SSA1 (23°C), after induction (23°C +SSA1), after incubation on ice after induction (0°C +SSA1), and after incubation in azide/deoxyglucose. GFP and DIC images were captured by confocal microscopy (see Materials and Methods). B, Kinetic import assay of rgNLS-GFP in wt and mutant cells after equilibration in azide/deoxyglucose. Import was assayed in _nup170-_Δ and _nup188-_Δ cells ± GAL1-SSA1 induction. Kinetic assays were performed at 37°C as described in Materials and Methods.

Figure 6

Figure 6

Effects of chilling on the nuclear localization of pNLS-GFP in wt, _nup188-_Δ, and _nup170-_Δ cells. Cells were grown at 23°C and incubated at 0°C as described in Materials and Methods. GFP fluorescence and Hoechst stain images were obtained by confocal microscopy.

Figure 7

Figure 7

NE sieving limits in wt, _nup188-_Δ, and _nup170-_Δ cells with NES-GFP molecular size probes. A, Localization of NES-GFP66 at 23 and 0°C in wt, _nup188-_Δ, and _nup170-_Δ cells. NES-GFP66 fluorescence (GFP) and Hoechst stain images were obtained by confocal microscopy. B, Time course of reexport of equilibrated NES-GFP66 in _nup188-_Δ cells. _nup188-_Δ cells incubated at 0°C for 1 h were transferred to a slide and the localization of NES-GFP66 observed as the cells warmed to room temperature (∼23°C). C, Quantification of nucleocytoplasmic distributions of NES-GFP size probes in wt, _nup188-_Δ, and _nup170-_Δ cells at 23 and 0°C. NES-GFP66, 81-, and 126-kD probes were expressed in wt and mutant cells at 23°C and then shifted to 0°C for 1 h. The top value in each ratio is the [C]/[N] ratio at 23°C and the bottom value is the [C]/[N] ratio at 0°C (the values of the ratios of these ratios are indicated to the right). In each case, nuclear and cytoplasmic levels of GFP fluorescence ([C]/[N]) at 23 and 0°C were quantified using the averaged pixel density of three different areas within the nuclei and cytoplasms of 10–15 different cells using the region tool in MetaMorph.

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