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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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tER localization of a dimerized UCR. (A) Replacement of the CCD with monomeric or dimeric FKBP. A GFP-tagged UCR-CCD construct was modified to replace the CCD with either wild-type monomeric FKBP or the dimeric FKBP(F36M) mutant. Localization to Sec13-DsRed-labeled tER sites was then evaluated as in Figure 2. 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 3: tER localization of a dimerized UCR. (A) Replacement of the CCD with monomeric or dimeric FKBP. A GFP-tagged UCR-CCD construct was modified to replace the CCD with either wild-type monomeric FKBP or the dimeric FKBP(F36M) mutant. Localization to Sec13-DsRed-labeled tER sites was then evaluated as in Figure 2. 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: The role of the CCD in promoting tER localization has been mysterious. We speculated that dimerization of the CCD might be relevant. The CCD forms an antiparallel dimer (Whittle and Schwartz, 2010), so fusion of the CCD to the UCR could enhance the functional affinity of the UCR for tER sites. To test this model, we replaced the CCD in a UCR-CCD construct with either wild-type FK506-binding protein (FKBP), which is monomeric, or the FKBP(F36M) mutant, which forms an antiparallel dimer (Rollins et al., 2000). The UCR-FKBP construct was mostly cytosolic, whereas the UCR-FKBP(F36M) construct showed robust tER localization (Figure 3). The combined data suggest that the UCR is the main tER localization determinant of Sec16 but that the isolated UCR has only a weak affinity for tER sites. Stronger tER localization can be obtained by dimerizing the UCR via linkage to the CCD. However, as described later, the CCD is not actually needed for tER localization in the context of full-length Sec16, implying that other interactions of Sec16 can supplement the weak tER affinity of the UCR. We conclude that specific tER localization activity resides in the UCR but not the CCD.


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)

tER localization of a dimerized UCR. (A) Replacement of the CCD with monomeric or dimeric FKBP. A GFP-tagged UCR-CCD construct was modified to replace the CCD with either wild-type monomeric FKBP or the dimeric FKBP(F36M) mutant. Localization to Sec13-DsRed-labeled tER sites was then evaluated as in Figure 2. 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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 3: tER localization of a dimerized UCR. (A) Replacement of the CCD with monomeric or dimeric FKBP. A GFP-tagged UCR-CCD construct was modified to replace the CCD with either wild-type monomeric FKBP or the dimeric FKBP(F36M) mutant. Localization to Sec13-DsRed-labeled tER sites was then evaluated as in Figure 2. 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: The role of the CCD in promoting tER localization has been mysterious. We speculated that dimerization of the CCD might be relevant. The CCD forms an antiparallel dimer (Whittle and Schwartz, 2010), so fusion of the CCD to the UCR could enhance the functional affinity of the UCR for tER sites. To test this model, we replaced the CCD in a UCR-CCD construct with either wild-type FK506-binding protein (FKBP), which is monomeric, or the FKBP(F36M) mutant, which forms an antiparallel dimer (Rollins et al., 2000). The UCR-FKBP construct was mostly cytosolic, whereas the UCR-FKBP(F36M) construct showed robust tER localization (Figure 3). The combined data suggest that the UCR is the main tER localization determinant of Sec16 but that the isolated UCR has only a weak affinity for tER sites. Stronger tER localization can be obtained by dimerizing the UCR via linkage to the CCD. However, as described later, the CCD is not actually needed for tER localization in the context of full-length Sec16, implying that other interactions of Sec16 can supplement the weak tER affinity of the UCR. We conclude that specific tER localization activity resides in the UCR but not the CCD.

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