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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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Destabilization of P. pastoris Sec16 by mutations that weaken Sec13 binding. (A) Two-hybrid analysis of CCD-Sec13 interactions. The wild-type (WT) CCD of P. pastoris Sec16, or a mutant CCD carrying the P1092L or I1075D mutation was tested against P. pastoris Sec13 in a yeast two-hybrid screen (James, 2001). Where indicated, Sec13 was used as the “bait” and the CCD as the “prey” or vice versa. Interactions were detected by growth on a plate lacking histidine (–His) or, in a more stringent test, a plate lacking histidine and adenine (–His –Ade). (B) Solubility of wild-type and mutant CCD variants. In E. coli, a C-terminally FLAG-tagged wild-type or mutant CCD was either expressed alone or coexpressed with C‑terminally S peptide–tagged Sec13. The cells were lysed in detergent and centrifuged, and equivalent amounts of the pellet (P) and supernatant (S) fractions were analyzed by SDS–PAGE, followed by immunoblotting with anti-FLAG antibody. (C) Effects of CCD mutations on tER site number. For strains expressing Sec13‑­GFP plus either WT or a mutant Sec16, cultures were grown at 23°C, and then a portion of each culture was shifted for 2 h to 36.5°C. The tER sites were counted in ∼100 cells from each sample. Plotted are mean and SEM.
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Figure 4: Destabilization of P. pastoris Sec16 by mutations that weaken Sec13 binding. (A) Two-hybrid analysis of CCD-Sec13 interactions. The wild-type (WT) CCD of P. pastoris Sec16, or a mutant CCD carrying the P1092L or I1075D mutation was tested against P. pastoris Sec13 in a yeast two-hybrid screen (James, 2001). Where indicated, Sec13 was used as the “bait” and the CCD as the “prey” or vice versa. Interactions were detected by growth on a plate lacking histidine (–His) or, in a more stringent test, a plate lacking histidine and adenine (–His –Ade). (B) Solubility of wild-type and mutant CCD variants. In E. coli, a C-terminally FLAG-tagged wild-type or mutant CCD was either expressed alone or coexpressed with C‑terminally S peptide–tagged Sec13. The cells were lysed in detergent and centrifuged, and equivalent amounts of the pellet (P) and supernatant (S) fractions were analyzed by SDS–PAGE, followed by immunoblotting with anti-FLAG antibody. (C) Effects of CCD mutations on tER site number. For strains expressing Sec13‑­GFP plus either WT or a mutant Sec16, cultures were grown at 23°C, and then a portion of each culture was shifted for 2 h to 36.5°C. The tER sites were counted in ∼100 cells from each sample. Plotted are mean and SEM.

Mentions: The P1092L point mutation lies within the CCD. Based on a structural comparison with the S. cerevisiae CCD‑Sec13 complex, the P1092L mutation is unlikely to affect dimerization of the CCD, but could potentially disrupt the CCD-Sec13 interface (see Figure S3 of Whittle and Schwartz, 2010). Indeed, a yeast two-hybrid analysis suggested that binding of Sec13 to the CCD is weakened by the P1092L mutation (Figure 4A).


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)

Destabilization of P. pastoris Sec16 by mutations that weaken Sec13 binding. (A) Two-hybrid analysis of CCD-Sec13 interactions. The wild-type (WT) CCD of P. pastoris Sec16, or a mutant CCD carrying the P1092L or I1075D mutation was tested against P. pastoris Sec13 in a yeast two-hybrid screen (James, 2001). Where indicated, Sec13 was used as the “bait” and the CCD as the “prey” or vice versa. Interactions were detected by growth on a plate lacking histidine (–His) or, in a more stringent test, a plate lacking histidine and adenine (–His –Ade). (B) Solubility of wild-type and mutant CCD variants. In E. coli, a C-terminally FLAG-tagged wild-type or mutant CCD was either expressed alone or coexpressed with C‑terminally S peptide–tagged Sec13. The cells were lysed in detergent and centrifuged, and equivalent amounts of the pellet (P) and supernatant (S) fractions were analyzed by SDS–PAGE, followed by immunoblotting with anti-FLAG antibody. (C) Effects of CCD mutations on tER site number. For strains expressing Sec13‑­GFP plus either WT or a mutant Sec16, cultures were grown at 23°C, and then a portion of each culture was shifted for 2 h to 36.5°C. The tER sites were counted in ∼100 cells from each sample. Plotted are mean and SEM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814151&req=5

Figure 4: Destabilization of P. pastoris Sec16 by mutations that weaken Sec13 binding. (A) Two-hybrid analysis of CCD-Sec13 interactions. The wild-type (WT) CCD of P. pastoris Sec16, or a mutant CCD carrying the P1092L or I1075D mutation was tested against P. pastoris Sec13 in a yeast two-hybrid screen (James, 2001). Where indicated, Sec13 was used as the “bait” and the CCD as the “prey” or vice versa. Interactions were detected by growth on a plate lacking histidine (–His) or, in a more stringent test, a plate lacking histidine and adenine (–His –Ade). (B) Solubility of wild-type and mutant CCD variants. In E. coli, a C-terminally FLAG-tagged wild-type or mutant CCD was either expressed alone or coexpressed with C‑terminally S peptide–tagged Sec13. The cells were lysed in detergent and centrifuged, and equivalent amounts of the pellet (P) and supernatant (S) fractions were analyzed by SDS–PAGE, followed by immunoblotting with anti-FLAG antibody. (C) Effects of CCD mutations on tER site number. For strains expressing Sec13‑­GFP plus either WT or a mutant Sec16, cultures were grown at 23°C, and then a portion of each culture was shifted for 2 h to 36.5°C. The tER sites were counted in ∼100 cells from each sample. Plotted are mean and SEM.
Mentions: The P1092L point mutation lies within the CCD. Based on a structural comparison with the S. cerevisiae CCD‑Sec13 complex, the P1092L mutation is unlikely to affect dimerization of the CCD, but could potentially disrupt the CCD-Sec13 interface (see Figure S3 of Whittle and Schwartz, 2010). Indeed, a yeast two-hybrid analysis suggested that binding of Sec13 to the CCD is weakened by the P1092L mutation (Figure 4A).

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