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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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Comparison of import rates and steady-state nuclear accumulation for NLS, CBP80, and IBB protein cargoes. (A) Left panel: simulation results predict that cargo imported directly by Impβ has greater initial rate and steady-state level of nuclear accumulation than cargo imported via the Impα adapter. Right panel: the bipartite NLS, CPB80, is predicted to accumulate more efficiently than the monopartite NLS. (B) A total of 20 μM GST-NES-GFP-NLS, GST-GFP-NES-CBP80, or GST-NES-GFP-IBB was injected into HeLa cell cytoplasm. Confocal images were collected 30 min post-injection. N/C ratios were calculated from mean pixel intensities for the nuclear and cytoplasmic compartments. Values are means of 15 cells±1 s.d. (C) Experimental validation of the computer predictions. A portion of 20 μM cargo was injected into the cytoplasm of HeLa cells. Images were collected every 20 s. Pixel intensity was converted to concentration by use of an external series of standards. Initial rate was defined as the rate of change of nuclear concentration during the first 30 s. Initial concentration for each cell was plotted against initial rate and the slope was fit by linear regression. Each data point represents an individual cell. Gray areas indicate the 99% confidence interval of the regression line.
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f2: Comparison of import rates and steady-state nuclear accumulation for NLS, CBP80, and IBB protein cargoes. (A) Left panel: simulation results predict that cargo imported directly by Impβ has greater initial rate and steady-state level of nuclear accumulation than cargo imported via the Impα adapter. Right panel: the bipartite NLS, CPB80, is predicted to accumulate more efficiently than the monopartite NLS. (B) A total of 20 μM GST-NES-GFP-NLS, GST-GFP-NES-CBP80, or GST-NES-GFP-IBB was injected into HeLa cell cytoplasm. Confocal images were collected 30 min post-injection. N/C ratios were calculated from mean pixel intensities for the nuclear and cytoplasmic compartments. Values are means of 15 cells±1 s.d. (C) Experimental validation of the computer predictions. A portion of 20 μM cargo was injected into the cytoplasm of HeLa cells. Images were collected every 20 s. Pixel intensity was converted to concentration by use of an external series of standards. Initial rate was defined as the rate of change of nuclear concentration during the first 30 s. Initial concentration for each cell was plotted against initial rate and the slope was fit by linear regression. Each data point represents an individual cell. Gray areas indicate the 99% confidence interval of the regression line.

Mentions: To investigate cargo gradients in both types of import, we developed a 3-compartment in silico transport model (Figure 1). Details of the model are in Materials and methods, and the complete schematic for the cargo import, Ran transport, and Karyopherin transport modules can be found in the Supplementary Data by Riddick and Macara (2005). Addition of either type of cargo to the cytoplasm was simulated by instantaneously stepping its concentration from 0 to 4 μM and measuring nuclear accumulation over 1800 s. Unexpectedly, cargo imported directly by Impβ had a greater initial rate and a higher steady-state nuclear accumulation than cargo imported via the adapter, Impα (Figure 2A). This difference results from the greater reaction rate for a bimolecular interaction, faster cycling time of Impβ between the nucleus and the cytoplasm, and the slightly higher permeability for the Impβ–cargo complex through the NPC, as compared to the Impα/β–cargo complex.


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

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

Comparison of import rates and steady-state nuclear accumulation for NLS, CBP80, and IBB protein cargoes. (A) Left panel: simulation results predict that cargo imported directly by Impβ has greater initial rate and steady-state level of nuclear accumulation than cargo imported via the Impα adapter. Right panel: the bipartite NLS, CPB80, is predicted to accumulate more efficiently than the monopartite NLS. (B) A total of 20 μM GST-NES-GFP-NLS, GST-GFP-NES-CBP80, or GST-NES-GFP-IBB was injected into HeLa cell cytoplasm. Confocal images were collected 30 min post-injection. N/C ratios were calculated from mean pixel intensities for the nuclear and cytoplasmic compartments. Values are means of 15 cells±1 s.d. (C) Experimental validation of the computer predictions. A portion of 20 μM cargo was injected into the cytoplasm of HeLa cells. Images were collected every 20 s. Pixel intensity was converted to concentration by use of an external series of standards. Initial rate was defined as the rate of change of nuclear concentration during the first 30 s. Initial concentration for each cell was plotted against initial rate and the slope was fit by linear regression. Each data point represents an individual cell. Gray areas indicate the 99% confidence interval of the regression line.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Comparison of import rates and steady-state nuclear accumulation for NLS, CBP80, and IBB protein cargoes. (A) Left panel: simulation results predict that cargo imported directly by Impβ has greater initial rate and steady-state level of nuclear accumulation than cargo imported via the Impα adapter. Right panel: the bipartite NLS, CPB80, is predicted to accumulate more efficiently than the monopartite NLS. (B) A total of 20 μM GST-NES-GFP-NLS, GST-GFP-NES-CBP80, or GST-NES-GFP-IBB was injected into HeLa cell cytoplasm. Confocal images were collected 30 min post-injection. N/C ratios were calculated from mean pixel intensities for the nuclear and cytoplasmic compartments. Values are means of 15 cells±1 s.d. (C) Experimental validation of the computer predictions. A portion of 20 μM cargo was injected into the cytoplasm of HeLa cells. Images were collected every 20 s. Pixel intensity was converted to concentration by use of an external series of standards. Initial rate was defined as the rate of change of nuclear concentration during the first 30 s. Initial concentration for each cell was plotted against initial rate and the slope was fit by linear regression. Each data point represents an individual cell. Gray areas indicate the 99% confidence interval of the regression line.
Mentions: To investigate cargo gradients in both types of import, we developed a 3-compartment in silico transport model (Figure 1). Details of the model are in Materials and methods, and the complete schematic for the cargo import, Ran transport, and Karyopherin transport modules can be found in the Supplementary Data by Riddick and Macara (2005). Addition of either type of cargo to the cytoplasm was simulated by instantaneously stepping its concentration from 0 to 4 μM and measuring nuclear accumulation over 1800 s. Unexpectedly, cargo imported directly by Impβ had a greater initial rate and a higher steady-state nuclear accumulation than cargo imported via the adapter, Impα (Figure 2A). This difference results from the greater reaction rate for a bimolecular interaction, faster cycling time of Impβ between the nucleus and the cytoplasm, and the slightly higher permeability for the Impβ–cargo complex through the NPC, as compared to the Impα/β–cargo complex.

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