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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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Redistribution of Sec16 to the cytosol by the P1092L mutation but not by deletion of the entire CCD. (A) Localization of Sec16 mutants. GFP-tagged Sec16‑P1092L or Sec16-ΔCCD was expressed from the SEC16 promoter in cells that also expressed wild-type Sec16 as well as Sec13-DsRed. Cells were grown at 30°C, then imaged by differential interference contrast (DIC) and fluorescence microscopy. Right, merged fluorescence and DIC images. Scale bar, 5 μm. (B) Quantitation of the data from A. The percentage of the total GFP signal at punctate tER sites was measured. Plotted are mean and SEM.
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Figure 5: Redistribution of Sec16 to the cytosol by the P1092L mutation but not by deletion of the entire CCD. (A) Localization of Sec16 mutants. GFP-tagged Sec16‑P1092L or Sec16-ΔCCD was expressed from the SEC16 promoter in cells that also expressed wild-type Sec16 as well as Sec13-DsRed. Cells were grown at 30°C, then imaged by differential interference contrast (DIC) and fluorescence microscopy. Right, merged fluorescence and DIC images. Scale bar, 5 μm. (B) Quantitation of the data from A. The percentage of the total GFP signal at punctate tER sites was measured. Plotted are mean and SEM.

Mentions: How does a destabilized CCD affect Sec16 activity? The sec16‑P1092L mutation does not reduce cellular levels of the mutant Sec16 (Connerly et al., 2005). Instead, fluorescence microscopy revealed that the Sec16-P1092L protein was largely displaced from tER sites to the cytosol (Connerly et al., 2005; Figure 5). This effect was not due to disrupted CCD function because a mutant Sec16 protein lacking the CCD showed strong tER localization (Figure 5). We conclude that the P1092L mutation destabilizes the CCD and leads to mislocalization of Sec16, thereby causing thermosensitive growth even though the CCD is nonessential. Thus, in the sec16‑P1092L mutant, the altered tER structure is due to loss of Sec16 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)

Redistribution of Sec16 to the cytosol by the P1092L mutation but not by deletion of the entire CCD. (A) Localization of Sec16 mutants. GFP-tagged Sec16‑P1092L or Sec16-ΔCCD was expressed from the SEC16 promoter in cells that also expressed wild-type Sec16 as well as Sec13-DsRed. Cells were grown at 30°C, then imaged by differential interference contrast (DIC) and fluorescence microscopy. Right, merged fluorescence and DIC images. Scale bar, 5 μm. (B) Quantitation of the data from A. The percentage of the total GFP signal at punctate tER sites was measured. Plotted are mean and SEM.
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Related In: Results  -  Collection

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Figure 5: Redistribution of Sec16 to the cytosol by the P1092L mutation but not by deletion of the entire CCD. (A) Localization of Sec16 mutants. GFP-tagged Sec16‑P1092L or Sec16-ΔCCD was expressed from the SEC16 promoter in cells that also expressed wild-type Sec16 as well as Sec13-DsRed. Cells were grown at 30°C, then imaged by differential interference contrast (DIC) and fluorescence microscopy. Right, merged fluorescence and DIC images. Scale bar, 5 μm. (B) Quantitation of the data from A. The percentage of the total GFP signal at punctate tER sites was measured. Plotted are mean and SEM.
Mentions: How does a destabilized CCD affect Sec16 activity? The sec16‑P1092L mutation does not reduce cellular levels of the mutant Sec16 (Connerly et al., 2005). Instead, fluorescence microscopy revealed that the Sec16-P1092L protein was largely displaced from tER sites to the cytosol (Connerly et al., 2005; Figure 5). This effect was not due to disrupted CCD function because a mutant Sec16 protein lacking the CCD showed strong tER localization (Figure 5). We conclude that the P1092L mutation destabilizes the CCD and leads to mislocalization of Sec16, thereby causing thermosensitive growth even though the CCD is nonessential. Thus, in the sec16‑P1092L mutant, the altered tER structure is due to loss of Sec16 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