Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada.
Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, N1G 2W1, Canada.
Plant Sci. 2021 Feb;303:110773. doi: 10.1016/j.plantsci.2020.110773. Epub 2020 Nov 26.
Dolichol is an essential polyisoprenoid within the endoplasmic reticulum of all eukaryotes. It serves as a membrane bound anchor onto which N-glycans are assembled prior to being transferred to nascent polypeptides, many of which enter the secretory pathway. Historically, it has been posited that the accumulation of dolichol represents the 'rate-limiting' step in the evolutionary conserved process of N-glycosylation, which ultimately affects the efficacy of approximately one fifth of the entire eukaryotic proteome. Therefore, this study aimed to enhance dolichol accumulation by manipulating the enzymes involved in its biosynthesis using an established Nicotiana benthamiana platform. Co-expression of a Solanum lycopersicum (tomato) cis-prenyltransferase (CPT) and its cognate partner protein, CPT binding protein (CPTBP), that catalyze the antepenultimate step in dolichol biosynthesis led to a 400-fold increase in the levels of long-chain polyprenols but resulted in only modest increases in dolichol accumulation. However, when combined with a newly characterized tomato polyprenol reductase, dolichol biosynthesis was enhanced by approximately 20-fold. We provide further evidence that in the aquatic macrophyte, Lemna gibba, dolichol is derived exclusively from the mevalonic acid (MVA) pathway with little participation from the evolutionary co-adopted non-MVA pathway. Taken together these results indicate that to effectively enhance the in planta accumulation of dolichol, coordinated synthesis and reduction of polyprenol to dolichol, is strictly required.
Dolichol 是所有真核生物内质网膜上必需的多异戊烯。它作为一种膜结合锚，在 N-聚糖组装到新生多肽之前，将其组装到其上，其中许多多肽进入分泌途径。历史上，人们一直认为，多萜醇的积累代表了 N-糖基化这一进化保守过程中的“限速”步骤，这最终影响了大约五分之一的整个真核生物蛋白质组的功效。因此，本研究旨在通过操纵其生物合成中涉及的酶来增强多萜醇的积累，使用已建立的 Nicotiana benthamiana 平台。共表达 Solanum lycopersicum（番茄）顺式 prenyltransferase（CPT）及其同源伴侣蛋白 CPT binding protein（CPTBP），催化多萜醇生物合成的倒数第二步，导致长链多萜醇水平增加了 400 倍，但多萜醇的积累仅略有增加。然而，当与新鉴定的番茄多萜醇还原酶结合使用时，多萜醇的生物合成增强了约 20 倍。我们提供了进一步的证据表明，在水生植物浮萍 Lemna gibba 中，多萜醇完全来自甲羟戊酸（MVA）途径，很少参与进化共同采用的非 MVA 途径。这些结果表明，为了有效地增强植物体内多萜醇的积累，需要严格协调多萜醇向多萜醇的合成和还原。
