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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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Temperature and ATP dependence of reporter movement to the INM. (A) HeLa cells cotransfected with reporter and trap plasmids were preincubated at 20°C for 10 min (left) or in glucose-free medium containing sodium azide at 37°C (−ATP) for 15 min (right). Subsequently, rapamycin was added and the localization of reporter proteins was followed by monitoring the GFP fluorescence at the times indicated. (B) Fluorescence intensities of the reporter at the NE were quantified at various times after rapamycin addition in cells maintained at 37°C in normal medium (closed circles), at 20°C in normal medium (closed triangles), or at 37°C in glucose-free medium containing sodium azide (−ATP, open diamonds). Shown are the average intensities and SD measured for 10 or more cells for each condition.
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fig5: Temperature and ATP dependence of reporter movement to the INM. (A) HeLa cells cotransfected with reporter and trap plasmids were preincubated at 20°C for 10 min (left) or in glucose-free medium containing sodium azide at 37°C (−ATP) for 15 min (right). Subsequently, rapamycin was added and the localization of reporter proteins was followed by monitoring the GFP fluorescence at the times indicated. (B) Fluorescence intensities of the reporter at the NE were quantified at various times after rapamycin addition in cells maintained at 37°C in normal medium (closed circles), at 20°C in normal medium (closed triangles), or at 37°C in glucose-free medium containing sodium azide (−ATP, open diamonds). Shown are the average intensities and SD measured for 10 or more cells for each condition.

Mentions: To investigate the molecular mechanism for movement of integral proteins from the peripheral ER to the INM, we examined the effects of decreased temperature or ATP depletion on this process (Fig. 5). Either depleting ATP or incubating cells at 16–22°C strongly inhibits vesicular trafficking in cultured cells, although passive diffusion of cargo proteins in membranes of the secretory pathway is not significantly affected (Lippincott-Schwartz et al., 2000). We found that movement of the standard reporter to the INM was completely inhibited by ATP depletion, and also was strongly inhibited by cooling cells to 20°C (Fig. 5, A and B). However, neither depletion of ATP nor decrease in temperature to 20°C detectably affected the mobility of the reporter protein in the peripheral ER, as determined by FRAP (Fig. 6). The diffusion constants of the reporter protein under these conditions (Table II) were not significantly different from those measured in untreated cells at 37°C (Table I). These results indicate that the reporter proteins retained their long-range mobility in the peripheral ER under the ATP-depletion and reduced temperature conditions where transit to the INM was strongly inhibited. These findings clearly indicate that the ATP-depletion and reduced temperature do not limit access of the reporter to the ONM, and therefore, that lack of accumulation at the INM reflects a more fundamental aspect of the transport mechanism.


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)

Temperature and ATP dependence of reporter movement to the INM. (A) HeLa cells cotransfected with reporter and trap plasmids were preincubated at 20°C for 10 min (left) or in glucose-free medium containing sodium azide at 37°C (−ATP) for 15 min (right). Subsequently, rapamycin was added and the localization of reporter proteins was followed by monitoring the GFP fluorescence at the times indicated. (B) Fluorescence intensities of the reporter at the NE were quantified at various times after rapamycin addition in cells maintained at 37°C in normal medium (closed circles), at 20°C in normal medium (closed triangles), or at 37°C in glucose-free medium containing sodium azide (−ATP, open diamonds). Shown are the average intensities and SD measured for 10 or more cells for each condition.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172626&req=5

fig5: Temperature and ATP dependence of reporter movement to the INM. (A) HeLa cells cotransfected with reporter and trap plasmids were preincubated at 20°C for 10 min (left) or in glucose-free medium containing sodium azide at 37°C (−ATP) for 15 min (right). Subsequently, rapamycin was added and the localization of reporter proteins was followed by monitoring the GFP fluorescence at the times indicated. (B) Fluorescence intensities of the reporter at the NE were quantified at various times after rapamycin addition in cells maintained at 37°C in normal medium (closed circles), at 20°C in normal medium (closed triangles), or at 37°C in glucose-free medium containing sodium azide (−ATP, open diamonds). Shown are the average intensities and SD measured for 10 or more cells for each condition.
Mentions: To investigate the molecular mechanism for movement of integral proteins from the peripheral ER to the INM, we examined the effects of decreased temperature or ATP depletion on this process (Fig. 5). Either depleting ATP or incubating cells at 16–22°C strongly inhibits vesicular trafficking in cultured cells, although passive diffusion of cargo proteins in membranes of the secretory pathway is not significantly affected (Lippincott-Schwartz et al., 2000). We found that movement of the standard reporter to the INM was completely inhibited by ATP depletion, and also was strongly inhibited by cooling cells to 20°C (Fig. 5, A and B). However, neither depletion of ATP nor decrease in temperature to 20°C detectably affected the mobility of the reporter protein in the peripheral ER, as determined by FRAP (Fig. 6). The diffusion constants of the reporter protein under these conditions (Table II) were not significantly different from those measured in untreated cells at 37°C (Table I). These results indicate that the reporter proteins retained their long-range mobility in the peripheral ER under the ATP-depletion and reduced temperature conditions where transit to the INM was strongly inhibited. These findings clearly indicate that the ATP-depletion and reduced temperature do not limit access of the reporter to the ONM, and therefore, that lack of accumulation at the INM reflects a more fundamental aspect of the transport mechanism.

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