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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 membrane trafficking inhibitors on accumulation of reporter protein at the NE. HeLa cells cotransfected with reporter and trap plasmids were preincubated with 50 μM BAPTA-AM for 30 min (closed triangles), with 10 mM NEM for 20 min (open triangles), or were maintained in normal growth medium (closed circles). Cells were then treated with rapamycin, and accumulation of fluorescent reporter at the NE at 37°C was followed as a function of time. Shown are the average intensities and SD measured for seven or more cells for each condition.
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fig7: Effects of membrane trafficking inhibitors on accumulation of reporter protein at the NE. HeLa cells cotransfected with reporter and trap plasmids were preincubated with 50 μM BAPTA-AM for 30 min (closed triangles), with 10 mM NEM for 20 min (open triangles), or were maintained in normal growth medium (closed circles). Cells were then treated with rapamycin, and accumulation of fluorescent reporter at the NE at 37°C was followed as a function of time. Shown are the average intensities and SD measured for seven or more cells for each condition.

Mentions: These results were consistent with a membrane fusion–based mechanism for movement of transmembrane proteins to the INM (Introduction). To further explore this possibility, reporter protein movement to the INM was measured after pretreatment of cells with membrane-permeable inhibitors of vesicular trafficking that block fusion between cytosolic membrane surfaces, N-ethylmaleimide (NEM; Beckers et al., 1989; Macaulay and Forbes, 1996), and 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl) ester (BAPTA-AM; Chen et al., 2002; Fig. 7). NEM inactivates vesicular trafficking machinery by modifying sulfhydryls of proteins such as p97/NSF (Dalal et al., 2004), whereas BAPTA-AM chelates calcium. Pretreatment of cells with 10 mM NEM or with 50 μM BAPTA-AM caused no significant inhibition of reporter movement to the INM (Fig. 7). However, these conditions efficiently inhibited the movement of the vesicular stomatitis virus (VSV) G protein from the ER to the Golgi in transfected HeLa cells, in which we performed a temperature shift with the temperature-sensitive ts045 G protein mutant (de Silva et al., 1990) to synchronize ER egress (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200409149/DC1). Thus, membrane fusion involving cytosolic membrane surfaces is unlikely to be involved in integral protein trafficking to the INM, despite the fact that this process requires ATP and is inhibited at 20°C. These results prompted us to investigate whether or not protein trafficking to the INM might involve movement around the nuclear pore membrane in the plane of the lipid bilayer.


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 membrane trafficking inhibitors on accumulation of reporter protein at the NE. HeLa cells cotransfected with reporter and trap plasmids were preincubated with 50 μM BAPTA-AM for 30 min (closed triangles), with 10 mM NEM for 20 min (open triangles), or were maintained in normal growth medium (closed circles). Cells were then treated with rapamycin, and accumulation of fluorescent reporter at the NE at 37°C was followed as a function of time. Shown are the average intensities and SD measured for seven or more cells for each condition.
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Related In: Results  -  Collection

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fig7: Effects of membrane trafficking inhibitors on accumulation of reporter protein at the NE. HeLa cells cotransfected with reporter and trap plasmids were preincubated with 50 μM BAPTA-AM for 30 min (closed triangles), with 10 mM NEM for 20 min (open triangles), or were maintained in normal growth medium (closed circles). Cells were then treated with rapamycin, and accumulation of fluorescent reporter at the NE at 37°C was followed as a function of time. Shown are the average intensities and SD measured for seven or more cells for each condition.
Mentions: These results were consistent with a membrane fusion–based mechanism for movement of transmembrane proteins to the INM (Introduction). To further explore this possibility, reporter protein movement to the INM was measured after pretreatment of cells with membrane-permeable inhibitors of vesicular trafficking that block fusion between cytosolic membrane surfaces, N-ethylmaleimide (NEM; Beckers et al., 1989; Macaulay and Forbes, 1996), and 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis (acetoxymethyl) ester (BAPTA-AM; Chen et al., 2002; Fig. 7). NEM inactivates vesicular trafficking machinery by modifying sulfhydryls of proteins such as p97/NSF (Dalal et al., 2004), whereas BAPTA-AM chelates calcium. Pretreatment of cells with 10 mM NEM or with 50 μM BAPTA-AM caused no significant inhibition of reporter movement to the INM (Fig. 7). However, these conditions efficiently inhibited the movement of the vesicular stomatitis virus (VSV) G protein from the ER to the Golgi in transfected HeLa cells, in which we performed a temperature shift with the temperature-sensitive ts045 G protein mutant (de Silva et al., 1990) to synchronize ER egress (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200409149/DC1). Thus, membrane fusion involving cytosolic membrane surfaces is unlikely to be involved in integral protein trafficking to the INM, despite the fact that this process requires ATP and is inhibited at 20°C. These results prompted us to investigate whether or not protein trafficking to the INM might involve movement around the nuclear pore membrane in the plane of the lipid bilayer.

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