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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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Viability of S. cerevisiae cells carrying PpSEC12 as the only gene from the SEC12 family.A plasmid shuffle was performed in sed4Δ sec12Δ cells, with SED4 in a URA3 plasmid plus either ScSEC12 (top row) or PpSEC12 (middle row) in a LEU2 plasmid. Both strains grew on rich media (YPD) and also on media containing 5-FOA, indicating that PpSEC12 could replace ScSEC12 even in the absence of SED4. As a control, sec12Δ cells carrying ScSEC12 on a URA3 plasmid were plated on the same media, and no growth was seen in the presence of 5-FOA.
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pone-0031156-g007: Viability of S. cerevisiae cells carrying PpSEC12 as the only gene from the SEC12 family.A plasmid shuffle was performed in sed4Δ sec12Δ cells, with SED4 in a URA3 plasmid plus either ScSEC12 (top row) or PpSEC12 (middle row) in a LEU2 plasmid. Both strains grew on rich media (YPD) and also on media containing 5-FOA, indicating that PpSEC12 could replace ScSEC12 even in the absence of SED4. As a control, sec12Δ cells carrying ScSEC12 on a URA3 plasmid were plated on the same media, and no growth was seen in the presence of 5-FOA.

Mentions: This experiment was performed using a plasmid shuffle approach. The chromosomal SED4 and SEC12 genes were deleted, and were replaced using two plasmids: a URA3 plasmid carrying SED4, and a LEU2 plasmid carrying either ScSEC12 or PpSEC12. 5-fluoroorotic acid (5-FOA) was then used to select for cells that had lost the URA3 SED4 plasmid. The cells that had lost the URA3 SED4 plasmid were viable regardless of whether the LEU2 plasmid carried ScSEC12 or PpSEC12 (Fig. 7). Thus, PpSec12 can replace ScSec12 even in a sed4Δ strain of S. cerevisiae, indicating that a strong interaction of a Sec12 family member with Sec16 is not essential for life in this yeast.


Sec12 binds to Sec16 at transitional ER sites.

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

Viability of S. cerevisiae cells carrying PpSEC12 as the only gene from the SEC12 family.A plasmid shuffle was performed in sed4Δ sec12Δ cells, with SED4 in a URA3 plasmid plus either ScSEC12 (top row) or PpSEC12 (middle row) in a LEU2 plasmid. Both strains grew on rich media (YPD) and also on media containing 5-FOA, indicating that PpSEC12 could replace ScSEC12 even in the absence of SED4. As a control, sec12Δ cells carrying ScSEC12 on a URA3 plasmid were plated on the same media, and no growth was seen in the presence of 5-FOA.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3275590&req=5

pone-0031156-g007: Viability of S. cerevisiae cells carrying PpSEC12 as the only gene from the SEC12 family.A plasmid shuffle was performed in sed4Δ sec12Δ cells, with SED4 in a URA3 plasmid plus either ScSEC12 (top row) or PpSEC12 (middle row) in a LEU2 plasmid. Both strains grew on rich media (YPD) and also on media containing 5-FOA, indicating that PpSEC12 could replace ScSEC12 even in the absence of SED4. As a control, sec12Δ cells carrying ScSEC12 on a URA3 plasmid were plated on the same media, and no growth was seen in the presence of 5-FOA.
Mentions: This experiment was performed using a plasmid shuffle approach. The chromosomal SED4 and SEC12 genes were deleted, and were replaced using two plasmids: a URA3 plasmid carrying SED4, and a LEU2 plasmid carrying either ScSEC12 or PpSEC12. 5-fluoroorotic acid (5-FOA) was then used to select for cells that had lost the URA3 SED4 plasmid. The cells that had lost the URA3 SED4 plasmid were viable regardless of whether the LEU2 plasmid carried ScSEC12 or PpSEC12 (Fig. 7). Thus, PpSec12 can replace ScSec12 even in a sed4Δ strain of S. cerevisiae, indicating that a strong interaction of a Sec12 family member with Sec16 is not essential for life in this yeast.

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