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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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Rapamycin-induced accumulation of reporter protein at the NE. HeLa cells were cotransfected with the reporter and trap plasmids, and after 20 h were incubated with or without 150 ng/ml rapamycin (rap) at 37°C for 2 h. Cells were washed in buffer lacking or containing 1% Triton X-100 and fixed. The reporter protein was visualized by GFP fluorescence (green), whereas the trap protein was visualized by immunofluorescent staining with anti-FLAG mAb (red). Similar results were obtained upon treatment of cells with rapamycin for 30 min instead of 2 h (not depicted).
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fig2: Rapamycin-induced accumulation of reporter protein at the NE. HeLa cells were cotransfected with the reporter and trap plasmids, and after 20 h were incubated with or without 150 ng/ml rapamycin (rap) at 37°C for 2 h. Cells were washed in buffer lacking or containing 1% Triton X-100 and fixed. The reporter protein was visualized by GFP fluorescence (green), whereas the trap protein was visualized by immunofluorescent staining with anti-FLAG mAb (red). Similar results were obtained upon treatment of cells with rapamycin for 30 min instead of 2 h (not depicted).

Mentions: The reporter and trap proteins were coexpressed in HeLa cells by transient transfection, and cells were examined by fluorescence microscopy (Fig. 2). Consistent with previous work showing that the short segment of LAP2β present in the reporter lacks NE targeting/retention sequences (Furukawa et al., 1998), the reporter was distributed throughout both the peripheral ER and the NE in the absence of rapamycin. This yielded a vesicular labeling pattern in fixed cells (Fig. 2, −Triton X-100, −rap). In contrast, the trap protein was highly concentrated in the nucleus, with elevated localization at the NE, and was essentially absent from the cytoplasm. When nonrapamycin-treated cells were washed with Triton X-100 before fixation, the reporter protein was almost completely extracted from cells. The extracted protein included the reporter initially present at the nuclear rim (Fig. 2, +Triton X-100, −rap). This behavior contrasts with that of most lamina-associated proteins, which remain bound to the lamina after Triton X-100 extraction (Furukawa et al., 1998).


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)

Rapamycin-induced accumulation of reporter protein at the NE. HeLa cells were cotransfected with the reporter and trap plasmids, and after 20 h were incubated with or without 150 ng/ml rapamycin (rap) at 37°C for 2 h. Cells were washed in buffer lacking or containing 1% Triton X-100 and fixed. The reporter protein was visualized by GFP fluorescence (green), whereas the trap protein was visualized by immunofluorescent staining with anti-FLAG mAb (red). Similar results were obtained upon treatment of cells with rapamycin for 30 min instead of 2 h (not depicted).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172626&req=5

fig2: Rapamycin-induced accumulation of reporter protein at the NE. HeLa cells were cotransfected with the reporter and trap plasmids, and after 20 h were incubated with or without 150 ng/ml rapamycin (rap) at 37°C for 2 h. Cells were washed in buffer lacking or containing 1% Triton X-100 and fixed. The reporter protein was visualized by GFP fluorescence (green), whereas the trap protein was visualized by immunofluorescent staining with anti-FLAG mAb (red). Similar results were obtained upon treatment of cells with rapamycin for 30 min instead of 2 h (not depicted).
Mentions: The reporter and trap proteins were coexpressed in HeLa cells by transient transfection, and cells were examined by fluorescence microscopy (Fig. 2). Consistent with previous work showing that the short segment of LAP2β present in the reporter lacks NE targeting/retention sequences (Furukawa et al., 1998), the reporter was distributed throughout both the peripheral ER and the NE in the absence of rapamycin. This yielded a vesicular labeling pattern in fixed cells (Fig. 2, −Triton X-100, −rap). In contrast, the trap protein was highly concentrated in the nucleus, with elevated localization at the NE, and was essentially absent from the cytoplasm. When nonrapamycin-treated cells were washed with Triton X-100 before fixation, the reporter protein was almost completely extracted from cells. The extracted protein included the reporter initially present at the nuclear rim (Fig. 2, +Triton X-100, −rap). This behavior contrasts with that of most lamina-associated proteins, which remain bound to the lamina after Triton X-100 extraction (Furukawa et al., 1998).

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