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Sec16 influences transitional ER sites by regulating rather than organizing COPII.

Bharucha N, Liu Y, Papanikou E, McMahon C, Esaki M, Jeffrey PD, Hughson FM, Glick BS - Mol. Biol. Cell (2013)

Bottom Line: An upstream conserved region (UCR) localizes Sec16 to tER sites.We propose that Sec16 does not in fact organize COPII.Instead, regulation of COPII turnover can account for the influence of Sec16 on tER sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637 Department of Molecular Biology, Princeton University, Princeton, NJ 08544.

ABSTRACT
During the budding of coat protein complex II (COPII) vesicles from transitional endoplasmic reticulum (tER) sites, Sec16 has been proposed to play two distinct roles: negatively regulating COPII turnover and organizing COPII assembly at tER sites. We tested these ideas using the yeast Pichia pastoris. Redistribution of Sec16 to the cytosol accelerates tER dynamics, supporting a negative regulatory role for Sec16. To evaluate a possible COPII organization role, we dissected the functional regions of Sec16. The central conserved domain, which had been implicated in coordinating COPII assembly, is actually dispensable for normal tER structure. An upstream conserved region (UCR) localizes Sec16 to tER sites. The UCR binds COPII components, and removal of COPII from tER sites also removes Sec16, indicating that COPII recruits Sec16 rather than the other way around. We propose that Sec16 does not in fact organize COPII. Instead, regulation of COPII turnover can account for the influence of Sec16 on tER sites.

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Removal of Sec16 from tER sites by anchoring on cytosolic ribosomes. (A) Drug-induced displacement of Sec16 from tER sites to ribosomes. Endogenous Sec16 was modified with an FRB-GFP dual tag to visualize Sec16 at tER sites in the absence of rapamycin (–Rap). Rapamycin was then added to 1 μg/ml, and cells were imaged after 10 min of incubation with the drug (+Rap). Fluorescence and differential interference contrast images (DIC) were combined, with the same exposure times for both panels. (B) tER dispersal induced by anchoring away Sec16. Gene replacement was used to tag Sec16 with two tandem copies of FRB and to tag Sec31 with GFP. tER sites were then visualized in the absence of rapamycin or after incubation for 10 min in the presence of 1 μg/ml rapamycin. Fluorescence and DIC images were combined, with the same exposure times for both panels. The tER sites per cell were counted in ∼50 cells from each sample. Plotted are mean and SEM. Scale bars, 5 μm.
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Figure 7: Removal of Sec16 from tER sites by anchoring on cytosolic ribosomes. (A) Drug-induced displacement of Sec16 from tER sites to ribosomes. Endogenous Sec16 was modified with an FRB-GFP dual tag to visualize Sec16 at tER sites in the absence of rapamycin (–Rap). Rapamycin was then added to 1 μg/ml, and cells were imaged after 10 min of incubation with the drug (+Rap). Fluorescence and differential interference contrast images (DIC) were combined, with the same exposure times for both panels. (B) tER dispersal induced by anchoring away Sec16. Gene replacement was used to tag Sec16 with two tandem copies of FRB and to tag Sec31 with GFP. tER sites were then visualized in the absence of rapamycin or after incubation for 10 min in the presence of 1 μg/ml rapamycin. Fluorescence and DIC images were combined, with the same exposure times for both panels. The tER sites per cell were counted in ∼50 cells from each sample. Plotted are mean and SEM. Scale bars, 5 μm.

Mentions: As a control to visualize ribosomal anchoring, we tagged Sec16 by gene replacement with an FRB-GFP dual tag. Within 5–10 min after rapamycin addition, most of the Sec16-FRB-GFP molecules redistributed from tER sites to a diffuse cytosolic localization (Figure 7A). Similar results were obtained when Sec16 was tagged with two copies of FRB followed by GFP (FRBx2-GFP; unpublished data). Addition of rapamycin inhibited growth of the Sec16-FRB-GFP strain but not of the parental strain (Supplemental Figure S6A). These results confirm that FRB-tagged Sec16 can be functionally inactivated by anchoring on ribosomes.


Sec16 influences transitional ER sites by regulating rather than organizing COPII.

Bharucha N, Liu Y, Papanikou E, McMahon C, Esaki M, Jeffrey PD, Hughson FM, Glick BS - Mol. Biol. Cell (2013)

Removal of Sec16 from tER sites by anchoring on cytosolic ribosomes. (A) Drug-induced displacement of Sec16 from tER sites to ribosomes. Endogenous Sec16 was modified with an FRB-GFP dual tag to visualize Sec16 at tER sites in the absence of rapamycin (–Rap). Rapamycin was then added to 1 μg/ml, and cells were imaged after 10 min of incubation with the drug (+Rap). Fluorescence and differential interference contrast images (DIC) were combined, with the same exposure times for both panels. (B) tER dispersal induced by anchoring away Sec16. Gene replacement was used to tag Sec16 with two tandem copies of FRB and to tag Sec31 with GFP. tER sites were then visualized in the absence of rapamycin or after incubation for 10 min in the presence of 1 μg/ml rapamycin. Fluorescence and DIC images were combined, with the same exposure times for both panels. The tER sites per cell were counted in ∼50 cells from each sample. Plotted are mean and SEM. Scale bars, 5 μm.
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Related In: Results  -  Collection

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Figure 7: Removal of Sec16 from tER sites by anchoring on cytosolic ribosomes. (A) Drug-induced displacement of Sec16 from tER sites to ribosomes. Endogenous Sec16 was modified with an FRB-GFP dual tag to visualize Sec16 at tER sites in the absence of rapamycin (–Rap). Rapamycin was then added to 1 μg/ml, and cells were imaged after 10 min of incubation with the drug (+Rap). Fluorescence and differential interference contrast images (DIC) were combined, with the same exposure times for both panels. (B) tER dispersal induced by anchoring away Sec16. Gene replacement was used to tag Sec16 with two tandem copies of FRB and to tag Sec31 with GFP. tER sites were then visualized in the absence of rapamycin or after incubation for 10 min in the presence of 1 μg/ml rapamycin. Fluorescence and DIC images were combined, with the same exposure times for both panels. The tER sites per cell were counted in ∼50 cells from each sample. Plotted are mean and SEM. Scale bars, 5 μm.
Mentions: As a control to visualize ribosomal anchoring, we tagged Sec16 by gene replacement with an FRB-GFP dual tag. Within 5–10 min after rapamycin addition, most of the Sec16-FRB-GFP molecules redistributed from tER sites to a diffuse cytosolic localization (Figure 7A). Similar results were obtained when Sec16 was tagged with two copies of FRB followed by GFP (FRBx2-GFP; unpublished data). Addition of rapamycin inhibited growth of the Sec16-FRB-GFP strain but not of the parental strain (Supplemental Figure S6A). These results confirm that FRB-tagged Sec16 can be functionally inactivated by anchoring on ribosomes.

Bottom Line: An upstream conserved region (UCR) localizes Sec16 to tER sites.We propose that Sec16 does not in fact organize COPII.Instead, regulation of COPII turnover can account for the influence of Sec16 on tER sites.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637 Department of Molecular Biology, Princeton University, Princeton, NJ 08544.

ABSTRACT
During the budding of coat protein complex II (COPII) vesicles from transitional endoplasmic reticulum (tER) sites, Sec16 has been proposed to play two distinct roles: negatively regulating COPII turnover and organizing COPII assembly at tER sites. We tested these ideas using the yeast Pichia pastoris. Redistribution of Sec16 to the cytosol accelerates tER dynamics, supporting a negative regulatory role for Sec16. To evaluate a possible COPII organization role, we dissected the functional regions of Sec16. The central conserved domain, which had been implicated in coordinating COPII assembly, is actually dispensable for normal tER structure. An upstream conserved region (UCR) localizes Sec16 to tER sites. The UCR binds COPII components, and removal of COPII from tER sites also removes Sec16, indicating that COPII recruits Sec16 rather than the other way around. We propose that Sec16 does not in fact organize COPII. Instead, regulation of COPII turnover can account for the influence of Sec16 on tER sites.

Show MeSH
Related in: MedlinePlus