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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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Domain analysis of P. pastoris Sec16. (A) Summary of the effects of Sec16 partial deletions on cell viability. Top, the UCR (residues 500–868), CCD (residues 1030–1459), a nonconserved glutamine-rich region (Q; residues 1829–1958), and the conserved CTR (residues 2392–2550). The endogenous SEC16 gene was replaced with alleles containing the indicated deletions. Right, the ability of each mutant allele to support growth. Only the deletions marked in red caused a loss of viability. (B) Differential effects of a CCD point mutation and a CCD deletion. The indicated mutations were introduced at the SEC16 locus by gene replacement in a strain expressing Sec13-GFP. Cultures were grown at 23°C, and then half of each culture was shifted to 36.5°C for 1 h. Cells were imaged by differential interference contrast and fluorescence microscopy. Scale bar, 5 μm. (C) Yeast two-hybrid analysis of Sec16-COPII interactions. The “prey” vector encoded the indicated fragments of P. pastoris Sec16, and the “bait” vector encoded the indicated full-length P. pastoris COPII coat proteins. Growth on plates lacking histidine reflects an interaction. With this system, the UCR can self-activate when used as bait, so constructs containing the UCR were tested only as prey. The other constructs gave the same results when the Sec16 fragments were used as bait and the COPII coat proteins were used as prey (unpublished data).
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Figure 1: Domain analysis of P. pastoris Sec16. (A) Summary of the effects of Sec16 partial deletions on cell viability. Top, the UCR (residues 500–868), CCD (residues 1030–1459), a nonconserved glutamine-rich region (Q; residues 1829–1958), and the conserved CTR (residues 2392–2550). The endogenous SEC16 gene was replaced with alleles containing the indicated deletions. Right, the ability of each mutant allele to support growth. Only the deletions marked in red caused a loss of viability. (B) Differential effects of a CCD point mutation and a CCD deletion. The indicated mutations were introduced at the SEC16 locus by gene replacement in a strain expressing Sec13-GFP. Cultures were grown at 23°C, and then half of each culture was shifted to 36.5°C for 1 h. Cells were imaged by differential interference contrast and fluorescence microscopy. Scale bar, 5 μm. (C) Yeast two-hybrid analysis of Sec16-COPII interactions. The “prey” vector encoded the indicated fragments of P. pastoris Sec16, and the “bait” vector encoded the indicated full-length P. pastoris COPII coat proteins. Growth on plates lacking histidine reflects an interaction. With this system, the UCR can self-activate when used as bait, so constructs containing the UCR were tested only as prey. The other constructs gave the same results when the Sec16 fragments were used as bait and the COPII coat proteins were used as prey (unpublished data).

Mentions: In agreement with earlier studies of S. cerevisiae Sec16 (Espenshade et al., 1995), the CTR was essential (Figure 1A). A second sequence upstream of the CCD was also found to be essential (Figure 1A). Alignment of this upstream region of Sec16 from various yeast species revealed several conserved peptides (Supplemental Figure S1). We therefore designated the second essential sequence the upstream conserved region (UCR; residues 500–868). Although the UCR as a whole is essential, cells can tolerate separate deletions of two subregions of the UCR (residues 500–647 or 649–868), implying some redundancy of function within this region.


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)

Domain analysis of P. pastoris Sec16. (A) Summary of the effects of Sec16 partial deletions on cell viability. Top, the UCR (residues 500–868), CCD (residues 1030–1459), a nonconserved glutamine-rich region (Q; residues 1829–1958), and the conserved CTR (residues 2392–2550). The endogenous SEC16 gene was replaced with alleles containing the indicated deletions. Right, the ability of each mutant allele to support growth. Only the deletions marked in red caused a loss of viability. (B) Differential effects of a CCD point mutation and a CCD deletion. The indicated mutations were introduced at the SEC16 locus by gene replacement in a strain expressing Sec13-GFP. Cultures were grown at 23°C, and then half of each culture was shifted to 36.5°C for 1 h. Cells were imaged by differential interference contrast and fluorescence microscopy. Scale bar, 5 μm. (C) Yeast two-hybrid analysis of Sec16-COPII interactions. The “prey” vector encoded the indicated fragments of P. pastoris Sec16, and the “bait” vector encoded the indicated full-length P. pastoris COPII coat proteins. Growth on plates lacking histidine reflects an interaction. With this system, the UCR can self-activate when used as bait, so constructs containing the UCR were tested only as prey. The other constructs gave the same results when the Sec16 fragments were used as bait and the COPII coat proteins were used as prey (unpublished data).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: Domain analysis of P. pastoris Sec16. (A) Summary of the effects of Sec16 partial deletions on cell viability. Top, the UCR (residues 500–868), CCD (residues 1030–1459), a nonconserved glutamine-rich region (Q; residues 1829–1958), and the conserved CTR (residues 2392–2550). The endogenous SEC16 gene was replaced with alleles containing the indicated deletions. Right, the ability of each mutant allele to support growth. Only the deletions marked in red caused a loss of viability. (B) Differential effects of a CCD point mutation and a CCD deletion. The indicated mutations were introduced at the SEC16 locus by gene replacement in a strain expressing Sec13-GFP. Cultures were grown at 23°C, and then half of each culture was shifted to 36.5°C for 1 h. Cells were imaged by differential interference contrast and fluorescence microscopy. Scale bar, 5 μm. (C) Yeast two-hybrid analysis of Sec16-COPII interactions. The “prey” vector encoded the indicated fragments of P. pastoris Sec16, and the “bait” vector encoded the indicated full-length P. pastoris COPII coat proteins. Growth on plates lacking histidine reflects an interaction. With this system, the UCR can self-activate when used as bait, so constructs containing the UCR were tested only as prey. The other constructs gave the same results when the Sec16 fragments were used as bait and the COPII coat proteins were used as prey (unpublished data).
Mentions: In agreement with earlier studies of S. cerevisiae Sec16 (Espenshade et al., 1995), the CTR was essential (Figure 1A). A second sequence upstream of the CCD was also found to be essential (Figure 1A). Alignment of this upstream region of Sec16 from various yeast species revealed several conserved peptides (Supplemental Figure S1). We therefore designated the second essential sequence the upstream conserved region (UCR; residues 500–868). Although the UCR as a whole is essential, cells can tolerate separate deletions of two subregions of the UCR (residues 500–647 or 649–868), implying some redundancy of function within this region.

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