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Energy- and temperature-dependent transport of integral proteins to the inner nuclear membrane via the nuclear pore.

Ohba T, Schirmer EC, Nishimoto T, Gerace L - J. Cell Biol. (2004)

Bottom Line: However, increasing the size of either domain by 47 kD strongly inhibited movement.Reduced temperature and ATP depletion also inhibited movement, which is characteristic of membrane fusion mechanisms, but pharmacological inhibition of vesicular trafficking had no effect.Because reporter accumulation at the INM was inhibited by antibodies to the nuclear pore membrane protein gp210, our results support a model wherein transport of integral proteins to the INM involves lateral diffusion in the lipid bilayer around the nuclear pore membrane, coupled with active restructuring of the nuclear pore complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
Resident integral proteins of the inner nuclear membrane (INM) are synthesized as membrane-integrated proteins on the peripheral endoplasmic reticulum (ER) and are transported to the INM throughout interphase using an unknown trafficking mechanism. To study this transport, we developed a live cell assay that measures the movement of transmembrane reporters from the ER to the INM by rapamycin-mediated trapping at the nuclear lamina. Reporter constructs with small (<30 kD) cytosolic and lumenal domains rapidly accumulated at the INM. However, increasing the size of either domain by 47 kD strongly inhibited movement. Reduced temperature and ATP depletion also inhibited movement, which is characteristic of membrane fusion mechanisms, but pharmacological inhibition of vesicular trafficking had no effect. Because reporter accumulation at the INM was inhibited by antibodies to the nuclear pore membrane protein gp210, our results support a model wherein transport of integral proteins to the INM involves lateral diffusion in the lipid bilayer around the nuclear pore membrane, coupled with active restructuring of the nuclear pore complex.

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Time course of accumulation of reporters at the NE after rapamycin treatment. HeLa cells were cotransfected with reporter and trap plasmids and examined after 20 h. (A) Localization of the reporter protein was followed for up to 20 min after rapamycin treatment (as indicated) at 37°C by monitoring the GFP fluorescence using real-time imaging. (B) Localization of reporter with PK fused to the lumenal domain (reporter-PK(L)) or to the cytosolic domain (reporter-PK(C)) was also followed as in A. (C) NE fluorescence intensities of the reporter (closed circles), reporter-PK(L) (closed squares), or reporter-PK(C) (open squares) were plotted as a function of time after rapamycin addition (+rap). For each time course, the fluorescence intensities of the NE were quantified using NIH image, with the 0 min time point set at 1, and with the following time points normalized as the relative increase. The fluorescence mean and SD are given, based on the measurement of seven or more separate cells for each condition.
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fig3: Time course of accumulation of reporters at the NE after rapamycin treatment. HeLa cells were cotransfected with reporter and trap plasmids and examined after 20 h. (A) Localization of the reporter protein was followed for up to 20 min after rapamycin treatment (as indicated) at 37°C by monitoring the GFP fluorescence using real-time imaging. (B) Localization of reporter with PK fused to the lumenal domain (reporter-PK(L)) or to the cytosolic domain (reporter-PK(C)) was also followed as in A. (C) NE fluorescence intensities of the reporter (closed circles), reporter-PK(L) (closed squares), or reporter-PK(C) (open squares) were plotted as a function of time after rapamycin addition (+rap). For each time course, the fluorescence intensities of the NE were quantified using NIH image, with the 0 min time point set at 1, and with the following time points normalized as the relative increase. The fluorescence mean and SD are given, based on the measurement of seven or more separate cells for each condition.

Mentions: When cells were treated with rapamycin, there was substantial accumulation of the reporter at the NE. The percentage of the total fluorescent reporter at the NE with rapamycin varied from cell to cell (see Fig. 3 A). This variety was apparently due to differences in the levels of reporter and/or trap protein expression, as the percentage of NE-associated reporter was always high in cells that expressed low levels of the reporter (Fig. 2, −Triton X-100, +rap; see also Fig. 3 A). When rapamycin-treated cells were extracted with Triton X-100 before fixation, a significant amount of reporter remained at the NE (Fig. 2, +Triton X-100, +rap), in contrast to nonrapamycin treated cells. This finding suggests that rapamycin-induced dimerization of the reporter with the trap protein led to immobilization of the reporter at the INM.


Energy- and temperature-dependent transport of integral proteins to the inner nuclear membrane via the nuclear pore.

Ohba T, Schirmer EC, Nishimoto T, Gerace L - J. Cell Biol. (2004)

Time course of accumulation of reporters at the NE after rapamycin treatment. HeLa cells were cotransfected with reporter and trap plasmids and examined after 20 h. (A) Localization of the reporter protein was followed for up to 20 min after rapamycin treatment (as indicated) at 37°C by monitoring the GFP fluorescence using real-time imaging. (B) Localization of reporter with PK fused to the lumenal domain (reporter-PK(L)) or to the cytosolic domain (reporter-PK(C)) was also followed as in A. (C) NE fluorescence intensities of the reporter (closed circles), reporter-PK(L) (closed squares), or reporter-PK(C) (open squares) were plotted as a function of time after rapamycin addition (+rap). For each time course, the fluorescence intensities of the NE were quantified using NIH image, with the 0 min time point set at 1, and with the following time points normalized as the relative increase. The fluorescence mean and SD are given, based on the measurement of seven or more separate cells for each condition.
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Related In: Results  -  Collection

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fig3: Time course of accumulation of reporters at the NE after rapamycin treatment. HeLa cells were cotransfected with reporter and trap plasmids and examined after 20 h. (A) Localization of the reporter protein was followed for up to 20 min after rapamycin treatment (as indicated) at 37°C by monitoring the GFP fluorescence using real-time imaging. (B) Localization of reporter with PK fused to the lumenal domain (reporter-PK(L)) or to the cytosolic domain (reporter-PK(C)) was also followed as in A. (C) NE fluorescence intensities of the reporter (closed circles), reporter-PK(L) (closed squares), or reporter-PK(C) (open squares) were plotted as a function of time after rapamycin addition (+rap). For each time course, the fluorescence intensities of the NE were quantified using NIH image, with the 0 min time point set at 1, and with the following time points normalized as the relative increase. The fluorescence mean and SD are given, based on the measurement of seven or more separate cells for each condition.
Mentions: When cells were treated with rapamycin, there was substantial accumulation of the reporter at the NE. The percentage of the total fluorescent reporter at the NE with rapamycin varied from cell to cell (see Fig. 3 A). This variety was apparently due to differences in the levels of reporter and/or trap protein expression, as the percentage of NE-associated reporter was always high in cells that expressed low levels of the reporter (Fig. 2, −Triton X-100, +rap; see also Fig. 3 A). When rapamycin-treated cells were extracted with Triton X-100 before fixation, a significant amount of reporter remained at the NE (Fig. 2, +Triton X-100, +rap), in contrast to nonrapamycin treated cells. This finding suggests that rapamycin-induced dimerization of the reporter with the trap protein led to immobilization of the reporter at the INM.

Bottom Line: However, increasing the size of either domain by 47 kD strongly inhibited movement.Reduced temperature and ATP depletion also inhibited movement, which is characteristic of membrane fusion mechanisms, but pharmacological inhibition of vesicular trafficking had no effect.Because reporter accumulation at the INM was inhibited by antibodies to the nuclear pore membrane protein gp210, our results support a model wherein transport of integral proteins to the INM involves lateral diffusion in the lipid bilayer around the nuclear pore membrane, coupled with active restructuring of the nuclear pore complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
Resident integral proteins of the inner nuclear membrane (INM) are synthesized as membrane-integrated proteins on the peripheral endoplasmic reticulum (ER) and are transported to the INM throughout interphase using an unknown trafficking mechanism. To study this transport, we developed a live cell assay that measures the movement of transmembrane reporters from the ER to the INM by rapamycin-mediated trapping at the nuclear lamina. Reporter constructs with small (<30 kD) cytosolic and lumenal domains rapidly accumulated at the INM. However, increasing the size of either domain by 47 kD strongly inhibited movement. Reduced temperature and ATP depletion also inhibited movement, which is characteristic of membrane fusion mechanisms, but pharmacological inhibition of vesicular trafficking had no effect. Because reporter accumulation at the INM was inhibited by antibodies to the nuclear pore membrane protein gp210, our results support a model wherein transport of integral proteins to the INM involves lateral diffusion in the lipid bilayer around the nuclear pore membrane, coupled with active restructuring of the nuclear pore complex.

Show MeSH
Related in: MedlinePlus