Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada.
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, 5A Pawinskiego Street, 02-106, Warsaw, Poland.
Plant J. 2015 Jun;82(6):903-914. doi: 10.1111/tpj.12859. Epub 2015 May 26.
Dolichol plays an indispensable role in the N-glycosylation of eukaryotic proteins. As proteins enter the secretory pathway they are decorated by a 'glycan', which is preassembled onto a membrane-anchored dolichol molecule embedded within the endoplasmic reticulum (ER). Genetic and biochemical evidence in yeast and animals indicate that a cis-prenyltransferase (CPT) is required for dolichol synthesis, but also point to other factor(s) that could be involved. In this study, RNAi-mediated suppression of one member of the tomato CPT family (SlCPT3) resulted in a ~60% decrease in dolichol content. We further show that the involvement of SlCPT3 in dolichol biosynthesis requires the participation of a distantly related partner protein, designated as CPT-binding protein (SlCPTBP), which is a close homolog of the human Nogo-B receptor. Yeast two-hybrid and co-immunoprecipitation assays demonstrate that SlCPT3 and its partner protein interact in vivo and that both SlCPT3 and SlCPTBP are required to complement the growth defects and dolichol deficiency of the yeast dolichol mutant, rer2∆. Co-expression of SlCPT3 and SlCPTBP in yeast and in E. coli confirmed that dolichol synthase activity strictly requires both proteins. Finally, organelle isolation and in vivo localization of fluorescent protein fusions showed that both SlCPT3 and SlCPTBP localize to the ER, the site of dolichol accumulation and synthesis in eukaryotes.
多萜醇在真核蛋白质的 N-糖基化中起着不可或缺的作用。当蛋白质进入分泌途径时，它们会被一种“聚糖”所修饰，这种聚糖预先组装在嵌入内质网 (ER) 的膜锚定多萜醇分子上。酵母和动物的遗传和生化证据表明，顺式 prenyltransferase (CPT) 是多萜醇合成所必需的，但也指出可能涉及其他因素。在这项研究中，通过 RNAi 介导抑制番茄 CPT 家族的一个成员（SlCPT3）导致多萜醇含量下降约 60%。我们进一步表明，SlCPT3 参与多萜醇生物合成需要参与一个远缘相关的伴侣蛋白，称为 CPT 结合蛋白（SlCPTBP），它是人类 Nogo-B 受体的紧密同源物。酵母双杂交和共免疫沉淀实验表明 SlCPT3 和其伴侣蛋白在体内相互作用，并且 SlCPT3 和 SlCPTBP 都需要补充酵母多萜醇突变体 rer2∆的生长缺陷和多萜醇缺乏。SlCPT3 和 SlCPTBP 在酵母和大肠杆菌中的共表达证实了多萜醇合酶活性严格需要这两种蛋白质。最后，细胞器分离和荧光蛋白融合的体内定位表明，SlCPT3 和 SlCPTBP 都定位于内质网，这是真核生物中多萜醇积累和合成的部位。
