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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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Mobility of reporter constructs in the peripheral ER and at the NE. HeLa cells were cotransfected with reporter and trap plasmids and examined after 20 h. (A) FRAP was used to measure the mobility of reporter constructs in the peripheral ER and NE at 37°C. We analyzed mobility in the peripheral ER in the absence of rapamycin (−rap) of the standard reporter (left column), reporter with PK fused to the lumenal domain (second column), and reporter with PK fused to the cytosolic domain (third column). We also analyzed mobility of the fluorescent reporter in the NE at 30 min after addition of rapamycin (+rap, right column). Fluorescence photobleaching was performed in the designated boxed areas, and the fluorescence recovery was measured at various times thereafter. Shown are cells immediately before and after photobleaching, and at 1 and 5 min thereafter. (B) Time course of recovery of fluorescence intensities in the photobleached cellular areas shown in A. Fluorescence intensities are normalized to the prebleach intensity.
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fig4: Mobility of reporter constructs in the peripheral ER and at the NE. HeLa cells were cotransfected with reporter and trap plasmids and examined after 20 h. (A) FRAP was used to measure the mobility of reporter constructs in the peripheral ER and NE at 37°C. We analyzed mobility in the peripheral ER in the absence of rapamycin (−rap) of the standard reporter (left column), reporter with PK fused to the lumenal domain (second column), and reporter with PK fused to the cytosolic domain (third column). We also analyzed mobility of the fluorescent reporter in the NE at 30 min after addition of rapamycin (+rap, right column). Fluorescence photobleaching was performed in the designated boxed areas, and the fluorescence recovery was measured at various times thereafter. Shown are cells immediately before and after photobleaching, and at 1 and 5 min thereafter. (B) Time course of recovery of fluorescence intensities in the photobleached cellular areas shown in A. Fluorescence intensities are normalized to the prebleach intensity.

Mentions: We used this assay to monitor the kinetics of accumulation of the reporter at the NE after rapamycin addition by real-time imaging (Fig. 3). The initial fluorescence level at the NE was normalized to a value of 1. Based on the data presented in Figs. 3 and 4, this initial NE pool apparently represents protein that is in equilibrium between the INM, ONM, and peripheral ER. With rapamycin addition the NE staining was observed to increase rapidly over the first ∼10 min, concomitant with a decrease in the level of peripheral ER staining (Fig. 3 A). Quantification revealed that reporter accumulation at the NE reached a plateau at ∼10–20 min, at an average intensity value ∼1.4-fold higher than the initial NE labeling (Fig. 3 C).


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)

Mobility of reporter constructs in the peripheral ER and at the NE. HeLa cells were cotransfected with reporter and trap plasmids and examined after 20 h. (A) FRAP was used to measure the mobility of reporter constructs in the peripheral ER and NE at 37°C. We analyzed mobility in the peripheral ER in the absence of rapamycin (−rap) of the standard reporter (left column), reporter with PK fused to the lumenal domain (second column), and reporter with PK fused to the cytosolic domain (third column). We also analyzed mobility of the fluorescent reporter in the NE at 30 min after addition of rapamycin (+rap, right column). Fluorescence photobleaching was performed in the designated boxed areas, and the fluorescence recovery was measured at various times thereafter. Shown are cells immediately before and after photobleaching, and at 1 and 5 min thereafter. (B) Time course of recovery of fluorescence intensities in the photobleached cellular areas shown in A. Fluorescence intensities are normalized to the prebleach intensity.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172626&req=5

fig4: Mobility of reporter constructs in the peripheral ER and at the NE. HeLa cells were cotransfected with reporter and trap plasmids and examined after 20 h. (A) FRAP was used to measure the mobility of reporter constructs in the peripheral ER and NE at 37°C. We analyzed mobility in the peripheral ER in the absence of rapamycin (−rap) of the standard reporter (left column), reporter with PK fused to the lumenal domain (second column), and reporter with PK fused to the cytosolic domain (third column). We also analyzed mobility of the fluorescent reporter in the NE at 30 min after addition of rapamycin (+rap, right column). Fluorescence photobleaching was performed in the designated boxed areas, and the fluorescence recovery was measured at various times thereafter. Shown are cells immediately before and after photobleaching, and at 1 and 5 min thereafter. (B) Time course of recovery of fluorescence intensities in the photobleached cellular areas shown in A. Fluorescence intensities are normalized to the prebleach intensity.
Mentions: We used this assay to monitor the kinetics of accumulation of the reporter at the NE after rapamycin addition by real-time imaging (Fig. 3). The initial fluorescence level at the NE was normalized to a value of 1. Based on the data presented in Figs. 3 and 4, this initial NE pool apparently represents protein that is in equilibrium between the INM, ONM, and peripheral ER. With rapamycin addition the NE staining was observed to increase rapidly over the first ∼10 min, concomitant with a decrease in the level of peripheral ER staining (Fig. 3 A). Quantification revealed that reporter accumulation at the NE reached a plateau at ∼10–20 min, at an average intensity value ∼1.4-fold higher than the initial NE labeling (Fig. 3 C).

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