Department of Biological Sciences, Biodiscovery Institute, University of North Texas, 1155 Union Circle #305220, Denton, TX, 76203-5017, USA.
Plant Biology Program and Department of Pharmaceutical Sciences, University of Kentucky, Lexington, KY, USA.
Planta. 2019 Jul;250(1):79-94. doi: 10.1007/s00425-019-03148-9. Epub 2019 Mar 27.
Mouse FIT2 protein redirects the cytoplasmic terpene biosynthetic machinery to lipid-droplet-forming domains in the ER and this relocalization supports the efficient compartmentalization and accumulation of sesquiterpenes in plant cells. Mouse (Mus musculus) fat storage-inducing transmembrane protein 2 (MmFIT2), an endoplasmic reticulum (ER)-resident protein with an important role in lipid droplet (LD) biogenesis in mammals, can function in plant cells to promote neutral lipid compartmentalization. Surprisingly, in affinity capture experiments, the Nicotiana benthamiana 5-epi-aristolochene synthase (NbEAS), a soluble cytoplasm-localized sesquiterpene synthase, was one of the most abundant proteins that co-precipitated with GFP-tagged MmFIT2 in transient expression assays in N. benthamiana leaves. Consistent with results of pull-down experiments, the subcellular location of mCherry-tagged NbEAS was changed from the cytoplasm to the LD-forming domains in the ER, only when co-expressed with MmFIT2. Ectopic co-expression of NbEAS and MmFIT2 together with mouse diacylglycerol:acyl-CoA acyltransferase 2 (MmDGAT2) in N. benthamiana leaves substantially increased the numbers of cytoplasmic LDs and supported the accumulation of the sesquiterpenes, 5-epi-aristolochene and capsidiol, up to tenfold over levels elicited by Agrobacterium infection alone. Taken together, our results suggest that MmFIT2 recruits sesquiterpene synthetic machinery to ER subdomains involved in LD formation and that this process can enhance the efficiency of sesquiterpene biosynthesis and compartmentalization in plant cells. Further, MmFIT2 and MmDGAT2 represent cross-kingdom lipogenic protein factors that may be used to engineer terpene accumulation more broadly in the cytoplasm of plant vegetative tissues.
小鼠 FIT2 蛋白将细胞质萜类生物合成机械重新定位到内质网中的脂滴形成域，这种重定位支持倍半萜在植物细胞中的有效区室化和积累。小鼠（Mus musculus）脂肪储存诱导跨膜蛋白 2（MmFIT2）是一种内质网（ER）驻留蛋白，在哺乳动物的脂滴（LD）生物发生中起重要作用，可在植物细胞中发挥作用，促进中性脂质区室化。令人惊讶的是，在亲和捕获实验中，Nicotiana benthamiana 5-epi-aristolochene 合酶（NbEAS），一种可溶性细胞质定位的倍半萜合酶，是与 GFP 标记的 MmFIT2 在 N. benthamiana 叶片中的瞬时表达测定中最丰富的共沉淀蛋白之一。与下拉实验的结果一致，当与 MmFIT2 共表达时，mCherry 标记的 NbEAS 的亚细胞定位从细胞质改变为内质网中的 LD 形成域。在 N. benthamiana 叶片中外源共表达 NbEAS 和 MmFIT2 以及小鼠二酰基甘油：酰基辅酶 A 酰基转移酶 2（MmDGAT2），大大增加了细胞质 LD 的数量，并支持倍半萜 5-epi-aristolochene 和capsidiol 的积累，比单独用农杆菌感染引起的水平增加了十倍。总之，我们的结果表明，MmFIT2 将倍半萜合成机械募集到参与 LD 形成的内质网亚域，并且该过程可以提高植物细胞中倍半萜生物合成和区室化的效率。此外，MmFIT2 和 MmDGAT2 代表跨领域的脂生成蛋白因子，可用于更广泛地在植物营养组织的细胞质中工程萜类化合物的积累。
