Structural Insights into Triglyceride Storage Mediated by Fat Storage-Inducing Transmembrane (FIT) Protein 2

Fat storage-Inducing Transmembrane proteins 1 & 2 (FIT1/FITM1 and FIT2/FITM2) belong to a unique family of evolutionarily conserved proteins localized to the endoplasmic reticulum that are involved in triglyceride lipid droplet formation. FIT proteins have been shown to mediate the partitioning of cellular triglyceride into lipid droplets, but not triglyceride biosynthesis. FIT proteins do not share primary sequence homology with known proteins and no structural information is available to inform on the mechanism by which FIT proteins function. Here, we present the experimentally-solved topological models for FIT1 and FIT2 using N-glycosylation site mapping and indirect immunofluorescence techniques. These methods indicate that both proteins have six-transmembrane-domains with both N- and C-termini localized to the cytosol. Utilizing this model for structure-function analysis, we identified and characterized a gain-of-function mutant of FIT2 (FLL(157-9)AAA) in transmembrane domain 4 that markedly augmented the total number and mean size of lipid droplets. Using limited-trypsin proteolysis we determined that the FLL(157-9)AAA mutant has enhanced trypsin cleavage at K86 relative to wild-type FIT2, indicating a conformational change. Taken together, these studies indicate that FIT2 is a 6 transmembrane domain-containing protein whose conformation likely regulates its activity in mediating lipid droplet formation.

脂肪储存诱导跨膜蛋白1与2（FIT1/FITM1和FIT2/FITM2）属于一类进化保守的独特蛋白家族，定位于内质网，参与甘油三酯脂滴的形成过程。已有研究证实，FIT蛋白可介导细胞内甘油三酯向脂滴的分配，但不参与甘油三酯的生物合成；FIT蛋白与已知蛋白不存在一级序列同源性，且目前尚无可用的结构信息来阐明其发挥功能的分子机制。本研究通过N-糖基化位点作图（N-glycosylation site mapping）与间接免疫荧光技术（indirect immunofluorescence techniques），解析了FIT1与FIT2的实验拓扑模型，结果显示这两种蛋白均具有6个跨膜结构域，其N端与C端均定位于细胞质基质中。基于该拓扑模型开展结构-功能分析，我们鉴定并表征了FIT2的一个功能获得性突变体FLL(157-9)AAA，该突变位于第4跨膜结构域，可显著增加脂滴的总数量与平均粒径；通过有限胰蛋白酶水解实验（limited-trypsin proteolysis），我们发现相较于野生型FIT2，FLL(157-9)AAA突变体在K86位点的胰蛋白酶切割效率更高，提示其发生了构象变化。综上，本研究结果表明，FIT2是一类含有6个跨膜结构域的蛋白，其构象或可调控其介导脂滴形成的生物学活性。