Sec12 Binds to Sec16 at Transitional ER Sites

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囊泡（COPII vesicles）从被称为转运内质网（transitional ER，tER）的内质网（Endoplasmic Reticulum, ER）结构域出芽。COPII包被的组装由跨膜鸟苷酸交换因子（transmembrane guanine nucleotide exchange factor）Sec12启动。在出芽酵母巴斯德毕赤酵母（Pichia pastoris）中，Sec12富集于tER位点。此前我们发现，巴斯德毕赤酵母Sec12的tER定位需要一种可饱和结合的伴侣蛋白。本研究证实，该结合伴侣即为Sec16——一种参与内质网输出与tER组织的外周膜蛋白（peripheral membrane protein）。第一条实验证据为：过表达Sec12会使Sec12弥散定位至整体内质网，但同时过表达Sec16可将过表达的Sec12保留于tER位点。此外，当在酿酒酵母（Saccharomyces cerevisiae, S. cerevisiae）中表达巴斯德毕赤酵母Sec12时，外源性Sec12会定位于整体内质网；但若在同一细胞中表达巴斯德毕赤酵母Sec16，则可将外源性Sec12招募至tER位点。在这两个实验体系中，删除Sec16的C端片段会破坏Sec16将Sec12招募至tER位点的能力。生化实验证实，Sec16的该C端片段可与Sec12的胞质结构域（cytosolic domain）结合。同理，我们证实人类Sec12同样富集于tER位点，这一现象可能源于其与Sec16A的C端片段的相互作用。上述结果表明，Sec12与Sec16的相互作用在内质网输出过程中具有保守的生物学功能。