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The adapter importin-alpha provides flexible control of nuclear import at the expense of efficiency.

Riddick G, Macara IG - Mol. Syst. Biol. (2007)

Bottom Line: However, computer simulations predicted the opposite outcome, and showed that direct transport is faster than adapter-mediated transport.These predictions were validated experimentally.The data, together with previous analyses of nuclear protein import, suggest that the use of adapters such as importin-alpha provides the cell with increased dynamic range for control of nuclear import rates, but at the expense of efficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Virginia, Charlottesville, VA 22908, USA.

ABSTRACT
Although there exists a large family of nuclear transport receptors (Karyopherins), the majority of known import cargoes use an adapter protein, Importin-alpha (Impalpha), which links the cargo to a karyopherin, Importin-beta (Impbeta). The reason for the existence of transport adapters is unknown. One hypothesis is that, as Impalpha re-export is coupled to GTP hydrolysis, it can drive a higher concentration of nuclear cargo than could be achieved by direct cargo binding to Importin-beta. However, computer simulations predicted the opposite outcome, and showed that direct transport is faster than adapter-mediated transport. These predictions were validated experimentally. The data, together with previous analyses of nuclear protein import, suggest that the use of adapters such as importin-alpha provides the cell with increased dynamic range for control of nuclear import rates, but at the expense of efficiency.

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Effect of transport protein levels on the steady-state accumulation of the shuttling GST-NES-GFP-NLS cargo. (A) HeLa cells were injected with a mixture of 20 μM GST-NES-GFP-NLS and 4 μM recombinant transport protein. Cells were kept at 37°C in physiological saline for the duration of the experiment. N/C ratios were measured 30 min post-injection. Simulation results using identical conditions (white) are displayed next to experimental results (black). (B) Upper panel: HeLa cells were transfected with siRNA for Impβ. After incubation in DMEM (5% CS, 5% FCS, 1% PS) for 72 h, cells were injected with the GST-NES-GFP-NLS cargo. After further incubation at 37°C for 30 min, cells were fixed with formalin and immunostained with anti-Impβ and an Alexa-546 conjugated secondary antibody. N/C ratio of the cargo was defined by the ratio of mean pixel intensity in the nucleus divided by that of the cytoplasm. IF intensity was measured as the mean pixel value of the entire cell. IF intensity, which reflects the relative abundance of Impβ, was plotted against N/C ratio of GST-NES-GFP-NLS. The slope was fit by linear regression. Gray areas represent 99% CI of the regression line. (B) Lower panel: HeLa cells were co-injected with 20 μM GST-NES-GFP-NLS and various concentrations of recombinant Impβ (1.25, 2.5, 5 and 10 μM). Intracellular concentration of Impβ was estimated using the approximate average cargo concentration (3 μM) from fluorescence as described previously and the known molar ratio of cargo to recombinant protein. (C) Left panel: HeLa cells were transfected with siRNA for Impα1, microinjected with GST-NES-GFP-NLS, and immunostained with anti-Impα1 as previously described. (C) Right panel: HeLa cells were co-injected with 20 μM GST-NES-GFP-NLS and various concentrations of recombinant Impα1 (1.25, 2.5, 5, 10, and 20 μM). Intracellular concentration of Impα1 was estimated using the approximate average cargo concentration (3 μM) from fluorescence as described previously and the known molar ratio of cargo to recombinant protein.
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f3: Effect of transport protein levels on the steady-state accumulation of the shuttling GST-NES-GFP-NLS cargo. (A) HeLa cells were injected with a mixture of 20 μM GST-NES-GFP-NLS and 4 μM recombinant transport protein. Cells were kept at 37°C in physiological saline for the duration of the experiment. N/C ratios were measured 30 min post-injection. Simulation results using identical conditions (white) are displayed next to experimental results (black). (B) Upper panel: HeLa cells were transfected with siRNA for Impβ. After incubation in DMEM (5% CS, 5% FCS, 1% PS) for 72 h, cells were injected with the GST-NES-GFP-NLS cargo. After further incubation at 37°C for 30 min, cells were fixed with formalin and immunostained with anti-Impβ and an Alexa-546 conjugated secondary antibody. N/C ratio of the cargo was defined by the ratio of mean pixel intensity in the nucleus divided by that of the cytoplasm. IF intensity was measured as the mean pixel value of the entire cell. IF intensity, which reflects the relative abundance of Impβ, was plotted against N/C ratio of GST-NES-GFP-NLS. The slope was fit by linear regression. Gray areas represent 99% CI of the regression line. (B) Lower panel: HeLa cells were co-injected with 20 μM GST-NES-GFP-NLS and various concentrations of recombinant Impβ (1.25, 2.5, 5 and 10 μM). Intracellular concentration of Impβ was estimated using the approximate average cargo concentration (3 μM) from fluorescence as described previously and the known molar ratio of cargo to recombinant protein. (C) Left panel: HeLa cells were transfected with siRNA for Impα1, microinjected with GST-NES-GFP-NLS, and immunostained with anti-Impα1 as previously described. (C) Right panel: HeLa cells were co-injected with 20 μM GST-NES-GFP-NLS and various concentrations of recombinant Impα1 (1.25, 2.5, 5, 10, and 20 μM). Intracellular concentration of Impα1 was estimated using the approximate average cargo concentration (3 μM) from fluorescence as described previously and the known molar ratio of cargo to recombinant protein.

Mentions: Knockdown of Impβ reduced nuclear accumulation in agreement with the sensitivity analysis (Figure 3B). Knockdown of Impα1 shows that changing Impα1 levels exert larger corresponding changes in nuclear cargo accumulation (Figure 3C), indicating a larger dynamic range of control.


The adapter importin-alpha provides flexible control of nuclear import at the expense of efficiency.

Riddick G, Macara IG - Mol. Syst. Biol. (2007)

Effect of transport protein levels on the steady-state accumulation of the shuttling GST-NES-GFP-NLS cargo. (A) HeLa cells were injected with a mixture of 20 μM GST-NES-GFP-NLS and 4 μM recombinant transport protein. Cells were kept at 37°C in physiological saline for the duration of the experiment. N/C ratios were measured 30 min post-injection. Simulation results using identical conditions (white) are displayed next to experimental results (black). (B) Upper panel: HeLa cells were transfected with siRNA for Impβ. After incubation in DMEM (5% CS, 5% FCS, 1% PS) for 72 h, cells were injected with the GST-NES-GFP-NLS cargo. After further incubation at 37°C for 30 min, cells were fixed with formalin and immunostained with anti-Impβ and an Alexa-546 conjugated secondary antibody. N/C ratio of the cargo was defined by the ratio of mean pixel intensity in the nucleus divided by that of the cytoplasm. IF intensity was measured as the mean pixel value of the entire cell. IF intensity, which reflects the relative abundance of Impβ, was plotted against N/C ratio of GST-NES-GFP-NLS. The slope was fit by linear regression. Gray areas represent 99% CI of the regression line. (B) Lower panel: HeLa cells were co-injected with 20 μM GST-NES-GFP-NLS and various concentrations of recombinant Impβ (1.25, 2.5, 5 and 10 μM). Intracellular concentration of Impβ was estimated using the approximate average cargo concentration (3 μM) from fluorescence as described previously and the known molar ratio of cargo to recombinant protein. (C) Left panel: HeLa cells were transfected with siRNA for Impα1, microinjected with GST-NES-GFP-NLS, and immunostained with anti-Impα1 as previously described. (C) Right panel: HeLa cells were co-injected with 20 μM GST-NES-GFP-NLS and various concentrations of recombinant Impα1 (1.25, 2.5, 5, 10, and 20 μM). Intracellular concentration of Impα1 was estimated using the approximate average cargo concentration (3 μM) from fluorescence as described previously and the known molar ratio of cargo to recombinant protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1911202&req=5

f3: Effect of transport protein levels on the steady-state accumulation of the shuttling GST-NES-GFP-NLS cargo. (A) HeLa cells were injected with a mixture of 20 μM GST-NES-GFP-NLS and 4 μM recombinant transport protein. Cells were kept at 37°C in physiological saline for the duration of the experiment. N/C ratios were measured 30 min post-injection. Simulation results using identical conditions (white) are displayed next to experimental results (black). (B) Upper panel: HeLa cells were transfected with siRNA for Impβ. After incubation in DMEM (5% CS, 5% FCS, 1% PS) for 72 h, cells were injected with the GST-NES-GFP-NLS cargo. After further incubation at 37°C for 30 min, cells were fixed with formalin and immunostained with anti-Impβ and an Alexa-546 conjugated secondary antibody. N/C ratio of the cargo was defined by the ratio of mean pixel intensity in the nucleus divided by that of the cytoplasm. IF intensity was measured as the mean pixel value of the entire cell. IF intensity, which reflects the relative abundance of Impβ, was plotted against N/C ratio of GST-NES-GFP-NLS. The slope was fit by linear regression. Gray areas represent 99% CI of the regression line. (B) Lower panel: HeLa cells were co-injected with 20 μM GST-NES-GFP-NLS and various concentrations of recombinant Impβ (1.25, 2.5, 5 and 10 μM). Intracellular concentration of Impβ was estimated using the approximate average cargo concentration (3 μM) from fluorescence as described previously and the known molar ratio of cargo to recombinant protein. (C) Left panel: HeLa cells were transfected with siRNA for Impα1, microinjected with GST-NES-GFP-NLS, and immunostained with anti-Impα1 as previously described. (C) Right panel: HeLa cells were co-injected with 20 μM GST-NES-GFP-NLS and various concentrations of recombinant Impα1 (1.25, 2.5, 5, 10, and 20 μM). Intracellular concentration of Impα1 was estimated using the approximate average cargo concentration (3 μM) from fluorescence as described previously and the known molar ratio of cargo to recombinant protein.
Mentions: Knockdown of Impβ reduced nuclear accumulation in agreement with the sensitivity analysis (Figure 3B). Knockdown of Impα1 shows that changing Impα1 levels exert larger corresponding changes in nuclear cargo accumulation (Figure 3C), indicating a larger dynamic range of control.

Bottom Line: However, computer simulations predicted the opposite outcome, and showed that direct transport is faster than adapter-mediated transport.These predictions were validated experimentally.The data, together with previous analyses of nuclear protein import, suggest that the use of adapters such as importin-alpha provides the cell with increased dynamic range for control of nuclear import rates, but at the expense of efficiency.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Virginia, Charlottesville, VA 22908, USA.

ABSTRACT
Although there exists a large family of nuclear transport receptors (Karyopherins), the majority of known import cargoes use an adapter protein, Importin-alpha (Impalpha), which links the cargo to a karyopherin, Importin-beta (Impbeta). The reason for the existence of transport adapters is unknown. One hypothesis is that, as Impalpha re-export is coupled to GTP hydrolysis, it can drive a higher concentration of nuclear cargo than could be achieved by direct cargo binding to Importin-beta. However, computer simulations predicted the opposite outcome, and showed that direct transport is faster than adapter-mediated transport. These predictions were validated experimentally. The data, together with previous analyses of nuclear protein import, suggest that the use of adapters such as importin-alpha provides the cell with increased dynamic range for control of nuclear import rates, but at the expense of efficiency.

Show MeSH
Related in: MedlinePlus