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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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Localization of GFP-tagged fragments of P. pastoris Sec16. These fusions were expressed from the AOX1 promoter as second copies in a P. pastoris strain expressing both wild-type Sec16 and Sec13-DsRed. GFP was fused to the N‑terminus of full-length Sec16, Sec16 lacking residues 500–868 (ΔUCR), residues 500–868 only (UCR alone), or residues 500–1459 only (UCR‑CCD). Expression of the GFP fusions was induced by shifting to methanol medium for 3 h, and then the cells were imaged by fluorescence microscopy. Right, merged fluorescence and differential interference contrast images. Arrows show examples of full-length GFP-Sec16 puncta that do not entirely colocalize with Sec13-DsRed puncta. Scale bar, 5 μm.
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Figure 2: Localization of GFP-tagged fragments of P. pastoris Sec16. These fusions were expressed from the AOX1 promoter as second copies in a P. pastoris strain expressing both wild-type Sec16 and Sec13-DsRed. GFP was fused to the N‑terminus of full-length Sec16, Sec16 lacking residues 500–868 (ΔUCR), residues 500–868 only (UCR alone), or residues 500–1459 only (UCR‑CCD). Expression of the GFP fusions was induced by shifting to methanol medium for 3 h, and then the cells were imaged by fluorescence microscopy. Right, merged fluorescence and differential interference contrast images. Arrows show examples of full-length GFP-Sec16 puncta that do not entirely colocalize with Sec13-DsRed puncta. Scale bar, 5 μm.

Mentions: For mammalian and Drosophila Sec16 homologues, a region upstream of the CCD is important for tER localization (Bhattacharyya and Glick, 2007; Ivan et al., 2008; Hughes et al., 2009). To determine whether the UCR of P. pastoris Sec16 has the same function, various portions of Sec16 were fused to green fluorescent protein (GFP) and expressed in cells that also contained untagged wild-type Sec16 and DsRed-tagged Sec13 (Figure 2). As a control, full-length Sec16 localized to tER sites, although some of the Sec16 puncta were significantly displaced from the COPII puncta (arrows in Figure 2). A construct lacking the UCR was primarily in the cytosol. The UCR alone conferred clear tER localization but also yielded substantial cytosolic fluorescence. A construct consisting of the UCR plus the CCD localized to tER sites as efficiently as full-length Sec16. Similar results were obtained with mammalian Sec16A and Sec16B (Hughes et al., 2009; Budnik et al., 2011; D. Bhattacharyya, personal communication) and with Drosophila Sec16 (Ivan et al., 2008). Thus a conserved property of Sec16 is that a region upstream of the CCD is necessary for full tER localization but sufficient only for partial tER localization. Full tER localization can be obtained by fusing this upstream region to 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)

Localization of GFP-tagged fragments of P. pastoris Sec16. These fusions were expressed from the AOX1 promoter as second copies in a P. pastoris strain expressing both wild-type Sec16 and Sec13-DsRed. GFP was fused to the N‑terminus of full-length Sec16, Sec16 lacking residues 500–868 (ΔUCR), residues 500–868 only (UCR alone), or residues 500–1459 only (UCR‑CCD). Expression of the GFP fusions was induced by shifting to methanol medium for 3 h, and then the cells were imaged by fluorescence microscopy. Right, merged fluorescence and differential interference contrast images. Arrows show examples of full-length GFP-Sec16 puncta that do not entirely colocalize with Sec13-DsRed puncta. Scale bar, 5 μm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814151&req=5

Figure 2: Localization of GFP-tagged fragments of P. pastoris Sec16. These fusions were expressed from the AOX1 promoter as second copies in a P. pastoris strain expressing both wild-type Sec16 and Sec13-DsRed. GFP was fused to the N‑terminus of full-length Sec16, Sec16 lacking residues 500–868 (ΔUCR), residues 500–868 only (UCR alone), or residues 500–1459 only (UCR‑CCD). Expression of the GFP fusions was induced by shifting to methanol medium for 3 h, and then the cells were imaged by fluorescence microscopy. Right, merged fluorescence and differential interference contrast images. Arrows show examples of full-length GFP-Sec16 puncta that do not entirely colocalize with Sec13-DsRed puncta. Scale bar, 5 μm.
Mentions: For mammalian and Drosophila Sec16 homologues, a region upstream of the CCD is important for tER localization (Bhattacharyya and Glick, 2007; Ivan et al., 2008; Hughes et al., 2009). To determine whether the UCR of P. pastoris Sec16 has the same function, various portions of Sec16 were fused to green fluorescent protein (GFP) and expressed in cells that also contained untagged wild-type Sec16 and DsRed-tagged Sec13 (Figure 2). As a control, full-length Sec16 localized to tER sites, although some of the Sec16 puncta were significantly displaced from the COPII puncta (arrows in Figure 2). A construct lacking the UCR was primarily in the cytosol. The UCR alone conferred clear tER localization but also yielded substantial cytosolic fluorescence. A construct consisting of the UCR plus the CCD localized to tER sites as efficiently as full-length Sec16. Similar results were obtained with mammalian Sec16A and Sec16B (Hughes et al., 2009; Budnik et al., 2011; D. Bhattacharyya, personal communication) and with Drosophila Sec16 (Ivan et al., 2008). Thus a conserved property of Sec16 is that a region upstream of the CCD is necessary for full tER localization but sufficient only for partial tER localization. Full tER localization can be obtained by fusing this upstream region to 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