Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois, United States of America.
PLoS One. 2012;7(2):e31156. doi: 10.1371/journal.pone.0031156. Epub 2012 Feb 8.
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.
COPII 小泡从一种称为内质网（ER）的过渡内质网（tER）域中出芽。COPII 外壳的组装由跨膜鸟嘌呤核苷酸交换因子 Sec12 启动。在毕赤酵母 Pichia pastoris 中，Sec12 集中在 tER 位点。此前，我们发现 P. pastoris Sec12 的 tER 定位需要一个可饱和的结合伴侣。我们现在表明，这种结合伴侣是 Sec16，一种在外质网输出和 tER 组织中起作用的外周膜蛋白。有一条证据线表明，Sec12 的过表达使 Sec12 去定位到一般 ER，但同时过表达 Sec16 使过表达的 Sec12 保留在 tER 位点。此外，当 P. pastoris Sec12 在 S. cerevisiae 中表达时，外源性 Sec12 定位于一般 ER，但当 P. pastoris Sec16 在相同的细胞中表达时，外源性 Sec12 被招募到 tER 位点。在这两个实验系统中，Sec16 使 Sec12 募集到 tER 位点的能力通过删除 Sec16 的 C 末端片段而被废除。生化实验证实，Sec16 的这个 C 末端片段与 Sec12 的胞质域结合。同样，我们证明人 Sec12 集中在 tER 位点，可能是由于与 Sec16A 的 C 末端片段的关联。这些结果表明，Sec12-Sec16 相互作用在 ER 输出中具有保守作用。
