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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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Effects of microinjected anti-gp210 and WGA on reporter accumulation at the NE. HeLa cells were cotransfected with reporter and trap plasmids and grown for 20 h. Control cultures, or cells that had been injected in the cytoplasm with anti-gp210 antibodies or WGA, were incubated for another 3 h, treated with or without rapamycin for 30 min, and fixed. The NE accumulation of reporter is represented by a ratio of the NE to cytoplasmic fluorescence intensity (see Materials and methods). Shown are the average values and SD measured for 12 or more separate cells for each experiment. (A) Uninjected cells, examined without (−) or with (+) rapamycin treatment. (B) Rapamycin-treated cells: either uninjected (−) or injected in the cytoplasm (+) with ∼1 mg/ml of affinity-purified anti-gp210 or with anti-gp210 mixed with the antigen peptide. (C) Rapamycin-treated cells: either uninjected (−) or injected in the cytoplasm (+) with either 0.3 mg/ml or 1 mg/ml WGA. (D) Micrographs showing representative cells from the experiment in B, depicting the reporter localization in cells injected with anti-gp210 or with anti-gp210 + peptide (arrows). Microinjected cells were detected by immunofluorescent localization of the injected rabbit IgG. Uninjected cells in the same fields depict control levels of rapamycin-induced accumulation at the NE.
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fig8: Effects of microinjected anti-gp210 and WGA on reporter accumulation at the NE. HeLa cells were cotransfected with reporter and trap plasmids and grown for 20 h. Control cultures, or cells that had been injected in the cytoplasm with anti-gp210 antibodies or WGA, were incubated for another 3 h, treated with or without rapamycin for 30 min, and fixed. The NE accumulation of reporter is represented by a ratio of the NE to cytoplasmic fluorescence intensity (see Materials and methods). Shown are the average values and SD measured for 12 or more separate cells for each experiment. (A) Uninjected cells, examined without (−) or with (+) rapamycin treatment. (B) Rapamycin-treated cells: either uninjected (−) or injected in the cytoplasm (+) with ∼1 mg/ml of affinity-purified anti-gp210 or with anti-gp210 mixed with the antigen peptide. (C) Rapamycin-treated cells: either uninjected (−) or injected in the cytoplasm (+) with either 0.3 mg/ml or 1 mg/ml WGA. (D) Micrographs showing representative cells from the experiment in B, depicting the reporter localization in cells injected with anti-gp210 or with anti-gp210 + peptide (arrows). Microinjected cells were detected by immunofluorescent localization of the injected rabbit IgG. Uninjected cells in the same fields depict control levels of rapamycin-induced accumulation at the NE.

Mentions: For this analysis, we microinjected cells with antibodies or a lectin that bind to the NPC and could potentially impose a steric block to reporter movement. We monitored NE accumulation of the standard reporter. Because it was not technically feasible to carry out live cell imaging in these experiments, we examined cells that were fixed 30 min after rapamycin addition (Fig. 8 A). Our quantification method in these experiments involved comparing the fluorescence intensity at the NE to the intensity over cytoplasmic regions located in an area between 4–6 μm from the NE (see Materials and methods). By this method, in uninjected cells the NE to cytoplasmic fluorescence intensity ratio had an average value of approximately three before rapamycin treatment and increased to a value of approximately eight after rapamycin treatment (Fig. 8 A).


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)

Effects of microinjected anti-gp210 and WGA on reporter accumulation at the NE. HeLa cells were cotransfected with reporter and trap plasmids and grown for 20 h. Control cultures, or cells that had been injected in the cytoplasm with anti-gp210 antibodies or WGA, were incubated for another 3 h, treated with or without rapamycin for 30 min, and fixed. The NE accumulation of reporter is represented by a ratio of the NE to cytoplasmic fluorescence intensity (see Materials and methods). Shown are the average values and SD measured for 12 or more separate cells for each experiment. (A) Uninjected cells, examined without (−) or with (+) rapamycin treatment. (B) Rapamycin-treated cells: either uninjected (−) or injected in the cytoplasm (+) with ∼1 mg/ml of affinity-purified anti-gp210 or with anti-gp210 mixed with the antigen peptide. (C) Rapamycin-treated cells: either uninjected (−) or injected in the cytoplasm (+) with either 0.3 mg/ml or 1 mg/ml WGA. (D) Micrographs showing representative cells from the experiment in B, depicting the reporter localization in cells injected with anti-gp210 or with anti-gp210 + peptide (arrows). Microinjected cells were detected by immunofluorescent localization of the injected rabbit IgG. Uninjected cells in the same fields depict control levels of rapamycin-induced accumulation at the NE.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172626&req=5

fig8: Effects of microinjected anti-gp210 and WGA on reporter accumulation at the NE. HeLa cells were cotransfected with reporter and trap plasmids and grown for 20 h. Control cultures, or cells that had been injected in the cytoplasm with anti-gp210 antibodies or WGA, were incubated for another 3 h, treated with or without rapamycin for 30 min, and fixed. The NE accumulation of reporter is represented by a ratio of the NE to cytoplasmic fluorescence intensity (see Materials and methods). Shown are the average values and SD measured for 12 or more separate cells for each experiment. (A) Uninjected cells, examined without (−) or with (+) rapamycin treatment. (B) Rapamycin-treated cells: either uninjected (−) or injected in the cytoplasm (+) with ∼1 mg/ml of affinity-purified anti-gp210 or with anti-gp210 mixed with the antigen peptide. (C) Rapamycin-treated cells: either uninjected (−) or injected in the cytoplasm (+) with either 0.3 mg/ml or 1 mg/ml WGA. (D) Micrographs showing representative cells from the experiment in B, depicting the reporter localization in cells injected with anti-gp210 or with anti-gp210 + peptide (arrows). Microinjected cells were detected by immunofluorescent localization of the injected rabbit IgG. Uninjected cells in the same fields depict control levels of rapamycin-induced accumulation at the NE.
Mentions: For this analysis, we microinjected cells with antibodies or a lectin that bind to the NPC and could potentially impose a steric block to reporter movement. We monitored NE accumulation of the standard reporter. Because it was not technically feasible to carry out live cell imaging in these experiments, we examined cells that were fixed 30 min after rapamycin addition (Fig. 8 A). Our quantification method in these experiments involved comparing the fluorescence intensity at the NE to the intensity over cytoplasmic regions located in an area between 4–6 μm from the NE (see Materials and methods). By this method, in uninjected cells the NE to cytoplasmic fluorescence intensity ratio had an average value of approximately three before rapamycin treatment and increased to a value of approximately eight after rapamycin treatment (Fig. 8 A).

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