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Sec12 binds to Sec16 at transitional ER sites.

Montegna EA, Bhave M, Liu Y, Bhattacharyya D, Glick BS - PLoS ONE (2012)

Bottom Line: Previously, we found that the tER localization of P. pastoris Sec12 requires a saturable binding partner.Biochemical experiments confirm that this C-terminal fragment of Sec16 binds to the cytosolic domain of Sec12.These results suggest that a Sec12-Sec16 interaction has a conserved role in ER export.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois, United States of America.

ABSTRACT
COPII vesicles bud from an ER domain known as the transitional ER (tER). Assembly of the COPII coat is initiated by the transmembrane guanine nucleotide exchange factor Sec12. In the budding yeast Pichia pastoris, Sec12 is concentrated at tER sites. Previously, we found that the tER localization of P. pastoris Sec12 requires a saturable binding partner. We now show that this binding partner is Sec16, a peripheral membrane protein that functions in ER export and tER organization. One line of evidence is that overexpression of Sec12 delocalizes Sec12 to the general ER, but simultaneous overexpression of Sec16 retains overexpressed Sec12 at tER sites. Additionally, when P. pastoris Sec12 is expressed in S. cerevisiae, the exogenous Sec12 localizes to the general ER, but when P. pastoris Sec16 is expressed in the same cells, the exogenous Sec12 is recruited to tER sites. In both of these experimental systems, the ability of Sec16 to recruit Sec12 to tER sites is abolished by deleting a C-terminal fragment of Sec16. Biochemical experiments confirm that this C-terminal fragment of Sec16 binds to the cytosolic domain of Sec12. Similarly, we demonstrate that human Sec12 is concentrated at tER sites, likely due to association with a C-terminal fragment of Sec16A. These results suggest that a Sec12-Sec16 interaction has a conserved role in ER export.

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Requirement of the C-terminal portion of PpSec16 for recruiting overexpressed PpSec12 to tER sites.As in Fig. 1, PpSec16-GFP was overexpressed in P. pastoris cells overexpressing PpSec12, except that deletions were introduced as indicated near the C-terminus of PpSec16. Two hundred randomly chosen cells from each of the indicated P. pastoris strains were examined by immunofluorescence and scored for colocalization of PpSec12-GG with PpSec16-GFP. Cells in which nearly all of the PpSec12-GG overlapped with PpSec16-GFP were scored as having strong colocalization (+). Cells in which PpSec12-GG showed clear concentration in the PpSec16-GFP puncta but also showed prominent staining outside of these puncta were scored as having partial colocalization (+/−). Cells showing no visible concentration of PpSec12-GG in the PpSec16-GFP puncta were scored as having no colocalization (−). Colocalization was virtually abolished by deleting the entire C-terminal portion of PpSec16, and was strongly reduced by deleting only the C-terminal conserved region (CTR).
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pone-0031156-g005: Requirement of the C-terminal portion of PpSec16 for recruiting overexpressed PpSec12 to tER sites.As in Fig. 1, PpSec16-GFP was overexpressed in P. pastoris cells overexpressing PpSec12, except that deletions were introduced as indicated near the C-terminus of PpSec16. Two hundred randomly chosen cells from each of the indicated P. pastoris strains were examined by immunofluorescence and scored for colocalization of PpSec12-GG with PpSec16-GFP. Cells in which nearly all of the PpSec12-GG overlapped with PpSec16-GFP were scored as having strong colocalization (+). Cells in which PpSec12-GG showed clear concentration in the PpSec16-GFP puncta but also showed prominent staining outside of these puncta were scored as having partial colocalization (+/−). Cells showing no visible concentration of PpSec12-GG in the PpSec16-GFP puncta were scored as having no colocalization (−). Colocalization was virtually abolished by deleting the entire C-terminal portion of PpSec16, and was strongly reduced by deleting only the C-terminal conserved region (CTR).

Mentions: To determine which parts of the C-terminal fragment of PpSec16 interact with PpSec12, we made deletions within the C-terminal fragment, and then simultaneously overexpressed PpSec12 and a mutant PpSec16 in P. pastoris. Two hundred cells from each strain were scored to determine if PpSec12 was strongly colocalized with PpSec16 at tER sites, or partially colocalized, or not colocalized (Fig. 5). In the control strain overexpressing full-length PpSec16, ∼90% of the cells showed strong colocalization of PpSec12 with PpSec16. Deletion of the entire C-terminal fragment of PpSec16 resulted in <1% of the cells showing strong colocalization. Deletion of the nonconserved stretch (residues 1967–2340) had a mild effect, with ∼80% of the cells showing strong colocalization, indicating that the nonconserved stretch within the C-terminal fragment plays some role in recruiting PpSec12 to tER sites. Deletion of the CTR (residues 2340–2550) had a dramatic effect, with only ∼5% of the cells showing strong colocalization, indicating that the CTR plays a major role in recruiting PpSec12 to tER sites. Smaller deletions that truncated the CTR had intermediate effects. These results indicate that recruitment of PpSec12 to tER sites requires a C-terminal fragment of PpSec16, and that the bulk of this interaction is mediated by the CTR.


Sec12 binds to Sec16 at transitional ER sites.

Montegna EA, Bhave M, Liu Y, Bhattacharyya D, Glick BS - PLoS ONE (2012)

Requirement of the C-terminal portion of PpSec16 for recruiting overexpressed PpSec12 to tER sites.As in Fig. 1, PpSec16-GFP was overexpressed in P. pastoris cells overexpressing PpSec12, except that deletions were introduced as indicated near the C-terminus of PpSec16. Two hundred randomly chosen cells from each of the indicated P. pastoris strains were examined by immunofluorescence and scored for colocalization of PpSec12-GG with PpSec16-GFP. Cells in which nearly all of the PpSec12-GG overlapped with PpSec16-GFP were scored as having strong colocalization (+). Cells in which PpSec12-GG showed clear concentration in the PpSec16-GFP puncta but also showed prominent staining outside of these puncta were scored as having partial colocalization (+/−). Cells showing no visible concentration of PpSec12-GG in the PpSec16-GFP puncta were scored as having no colocalization (−). Colocalization was virtually abolished by deleting the entire C-terminal portion of PpSec16, and was strongly reduced by deleting only the C-terminal conserved region (CTR).
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Related In: Results  -  Collection

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pone-0031156-g005: Requirement of the C-terminal portion of PpSec16 for recruiting overexpressed PpSec12 to tER sites.As in Fig. 1, PpSec16-GFP was overexpressed in P. pastoris cells overexpressing PpSec12, except that deletions were introduced as indicated near the C-terminus of PpSec16. Two hundred randomly chosen cells from each of the indicated P. pastoris strains were examined by immunofluorescence and scored for colocalization of PpSec12-GG with PpSec16-GFP. Cells in which nearly all of the PpSec12-GG overlapped with PpSec16-GFP were scored as having strong colocalization (+). Cells in which PpSec12-GG showed clear concentration in the PpSec16-GFP puncta but also showed prominent staining outside of these puncta were scored as having partial colocalization (+/−). Cells showing no visible concentration of PpSec12-GG in the PpSec16-GFP puncta were scored as having no colocalization (−). Colocalization was virtually abolished by deleting the entire C-terminal portion of PpSec16, and was strongly reduced by deleting only the C-terminal conserved region (CTR).
Mentions: To determine which parts of the C-terminal fragment of PpSec16 interact with PpSec12, we made deletions within the C-terminal fragment, and then simultaneously overexpressed PpSec12 and a mutant PpSec16 in P. pastoris. Two hundred cells from each strain were scored to determine if PpSec12 was strongly colocalized with PpSec16 at tER sites, or partially colocalized, or not colocalized (Fig. 5). In the control strain overexpressing full-length PpSec16, ∼90% of the cells showed strong colocalization of PpSec12 with PpSec16. Deletion of the entire C-terminal fragment of PpSec16 resulted in <1% of the cells showing strong colocalization. Deletion of the nonconserved stretch (residues 1967–2340) had a mild effect, with ∼80% of the cells showing strong colocalization, indicating that the nonconserved stretch within the C-terminal fragment plays some role in recruiting PpSec12 to tER sites. Deletion of the CTR (residues 2340–2550) had a dramatic effect, with only ∼5% of the cells showing strong colocalization, indicating that the CTR plays a major role in recruiting PpSec12 to tER sites. Smaller deletions that truncated the CTR had intermediate effects. These results indicate that recruitment of PpSec12 to tER sites requires a C-terminal fragment of PpSec16, and that the bulk of this interaction is mediated by the CTR.

Bottom Line: Previously, we found that the tER localization of P. pastoris Sec12 requires a saturable binding partner.Biochemical experiments confirm that this C-terminal fragment of Sec16 binds to the cytosolic domain of Sec12.These results suggest that a Sec12-Sec16 interaction has a conserved role in ER export.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois, United States of America.

ABSTRACT
COPII vesicles bud from an ER domain known as the transitional ER (tER). Assembly of the COPII coat is initiated by the transmembrane guanine nucleotide exchange factor Sec12. In the budding yeast Pichia pastoris, Sec12 is concentrated at tER sites. Previously, we found that the tER localization of P. pastoris Sec12 requires a saturable binding partner. We now show that this binding partner is Sec16, a peripheral membrane protein that functions in ER export and tER organization. One line of evidence is that overexpression of Sec12 delocalizes Sec12 to the general ER, but simultaneous overexpression of Sec16 retains overexpressed Sec12 at tER sites. Additionally, when P. pastoris Sec12 is expressed in S. cerevisiae, the exogenous Sec12 localizes to the general ER, but when P. pastoris Sec16 is expressed in the same cells, the exogenous Sec12 is recruited to tER sites. In both of these experimental systems, the ability of Sec16 to recruit Sec12 to tER sites is abolished by deleting a C-terminal fragment of Sec16. Biochemical experiments confirm that this C-terminal fragment of Sec16 binds to the cytosolic domain of Sec12. Similarly, we demonstrate that human Sec12 is concentrated at tER sites, likely due to association with a C-terminal fragment of Sec16A. These results suggest that a Sec12-Sec16 interaction has a conserved role in ER export.

Show MeSH
Related in: MedlinePlus