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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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Displacement of Sec16 by removal of COPII coat proteins from tER sites. (A) Drug-induced displacement of Sec23 from tER sites to ribosomes. The procedure was the same as in Figure 7A, except that Sec23 was tagged with FRB-GFP. (B) Drug-induced displacement of Sec31 from tER sites. Gene replacement was used to tag Sec23 and Shl23 with FRB and to tag Sec31 with GFP. The Sec31-GFP pattern was then visualized in the absence of rapamycin (–Rap) or after incubation for 10 min in the presence of 1 μg/ml rapamycin (+Rap). Fluorescence and differential interference contrast images were combined, with the same exposure times for both panels. (C) Drug-induced displacement of Sec16 from tER sites. The procedure was the same as in B, except that Sec16 was tagged with GFP. Scale bars, 5 μm.
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Figure 8: Displacement of Sec16 by removal of COPII coat proteins from tER sites. (A) Drug-induced displacement of Sec23 from tER sites to ribosomes. The procedure was the same as in Figure 7A, except that Sec23 was tagged with FRB-GFP. (B) Drug-induced displacement of Sec31 from tER sites. Gene replacement was used to tag Sec23 and Shl23 with FRB and to tag Sec31 with GFP. The Sec31-GFP pattern was then visualized in the absence of rapamycin (–Rap) or after incubation for 10 min in the presence of 1 μg/ml rapamycin (+Rap). Fluorescence and differential interference contrast images were combined, with the same exposure times for both panels. (C) Drug-induced displacement of Sec16 from tER sites. The procedure was the same as in B, except that Sec16 was tagged with GFP. Scale bars, 5 μm.

Mentions: The complementary experiment was to anchor away COPII and ask whether Sec16 remained at tER sites. For this purpose, we tagged the inner COPII coat subunit Sec23 with FRB. As a control to visualize ribosomal anchoring, we tagged Sec23 by gene replacement with FRB-GFP. Within 5–10 min after rapamycin addition, most of the Sec23-FRB-GFP molecules redistributed from tER sites to a diffuse cytosolic location, although a weak punctate signal was still visible in some cells (Figure 8A). P. pastoris also contains a nonessential, tER-localized Sec23 homologue called Shl23 (Esaki et al., 2006), so we tagged Shl23 with FRB as well. As expected, anchoring away Sec23-FRB and Shl23-FRB strongly inhibited cell growth (Supplemental Figure S6B). In a strain containing Sec23-FRB and Shl23-FRB, addition of rapamycin should trap the inner COPII coat subunits on ribosomes, thereby displacing both layers of the COPII coat from tER sites. When the outer COPII coat protein Sec31-GFP was visualized in a strain containing Sec23-FRB and Shl23-FRB, most of the Sec31‑GFP molecules were displaced from tER sites within 5–10 min after rapamycin addition (Figure 8B). These results indicate that rapamycin-induced anchoring removed both layers of the COPII coat from tER sites.


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)

Displacement of Sec16 by removal of COPII coat proteins from tER sites. (A) Drug-induced displacement of Sec23 from tER sites to ribosomes. The procedure was the same as in Figure 7A, except that Sec23 was tagged with FRB-GFP. (B) Drug-induced displacement of Sec31 from tER sites. Gene replacement was used to tag Sec23 and Shl23 with FRB and to tag Sec31 with GFP. The Sec31-GFP pattern was then visualized in the absence of rapamycin (–Rap) or after incubation for 10 min in the presence of 1 μg/ml rapamycin (+Rap). Fluorescence and differential interference contrast images were combined, with the same exposure times for both panels. (C) Drug-induced displacement of Sec16 from tER sites. The procedure was the same as in B, except that Sec16 was tagged with GFP. Scale bars, 5 μm.
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Related In: Results  -  Collection

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Figure 8: Displacement of Sec16 by removal of COPII coat proteins from tER sites. (A) Drug-induced displacement of Sec23 from tER sites to ribosomes. The procedure was the same as in Figure 7A, except that Sec23 was tagged with FRB-GFP. (B) Drug-induced displacement of Sec31 from tER sites. Gene replacement was used to tag Sec23 and Shl23 with FRB and to tag Sec31 with GFP. The Sec31-GFP pattern was then visualized in the absence of rapamycin (–Rap) or after incubation for 10 min in the presence of 1 μg/ml rapamycin (+Rap). Fluorescence and differential interference contrast images were combined, with the same exposure times for both panels. (C) Drug-induced displacement of Sec16 from tER sites. The procedure was the same as in B, except that Sec16 was tagged with GFP. Scale bars, 5 μm.
Mentions: The complementary experiment was to anchor away COPII and ask whether Sec16 remained at tER sites. For this purpose, we tagged the inner COPII coat subunit Sec23 with FRB. As a control to visualize ribosomal anchoring, we tagged Sec23 by gene replacement with FRB-GFP. Within 5–10 min after rapamycin addition, most of the Sec23-FRB-GFP molecules redistributed from tER sites to a diffuse cytosolic location, although a weak punctate signal was still visible in some cells (Figure 8A). P. pastoris also contains a nonessential, tER-localized Sec23 homologue called Shl23 (Esaki et al., 2006), so we tagged Shl23 with FRB as well. As expected, anchoring away Sec23-FRB and Shl23-FRB strongly inhibited cell growth (Supplemental Figure S6B). In a strain containing Sec23-FRB and Shl23-FRB, addition of rapamycin should trap the inner COPII coat subunits on ribosomes, thereby displacing both layers of the COPII coat from tER sites. When the outer COPII coat protein Sec31-GFP was visualized in a strain containing Sec23-FRB and Shl23-FRB, most of the Sec31‑GFP molecules were displaced from tER sites within 5–10 min after rapamycin addition (Figure 8B). These results indicate that rapamycin-induced anchoring removed both layers of the COPII coat from tER sites.

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