Dissecting the Molecular Properties of Prokaryotic Flotillins

Flotillins are universally conserved proteins that are present in all kingdoms of life. Recently it was demonstrated that the B. subtilis flotillin YuaG (FloT) has a direct influence on membrane domain formation by orchestrating lipid domains. Thereby it allocates a proper environment for diverse cellular machineries. YuaG creates platforms for signal transduction, processes crucial for biofilm formation, sporulation, competence, secretion, and others. Even though, flotillins are an emerging topic of research in the field of microbiology little is known about the molecular architecture of prokaryotic flotillins. All flotillins share common structural elements and are tethered to the membrane N’- terminally, followed by a so called PHB domain and a flotillin domain. We show here that prokaryotic flotillins are, similarly to eukaryotic flotillins, tethered to the membrane via a hairpin loop. Further it is demonstrated by sedimentation assays that B. subtilis flotillins do not bind to the membrane via their PHB domain contrary to eukaryotic flotillins. Size exclusion chromatography experiments, blue native PAGE and cross linking experiments revealed that B. subtilis YuaG can oligomerize into large clusters via the PHB domain. This illustrates an important difference in the setup of prokaryotic flotillins compared to the organization of eukaryotic flotillins.

弗洛蒂林蛋白（flotillin）是一类普遍保守的蛋白质，广泛存在于所有生命界中。近期研究证实，枯草芽孢杆菌（B. subtilis）的弗洛蒂林蛋白YuaG（FloT）可通过调控脂质结构域，直接影响膜结构域的形成，借此为多种细胞复合物营造适宜的微环境。YuaG可构建信号转导平台，参与诸多对生物膜形成、芽孢形成、感受态形成、分泌等过程至关重要的生命活动。尽管弗洛蒂林蛋白是微生物学领域新兴的研究热点，但目前学界对原核弗洛蒂林蛋白的分子架构仍知之甚少。所有弗洛蒂林蛋白均具有共同的结构元件：它们通过N端锚定在细胞膜上，随后依次包含一个PHB结构域（PHB domain）以及一个弗洛蒂林结构域。本研究证实，原核弗洛蒂林蛋白与真核弗洛蒂林蛋白类似，均通过发夹环锚定在细胞膜上。进一步的沉降试验证实，与真核弗洛蒂林蛋白不同，枯草芽孢杆菌的弗洛蒂林蛋白并非通过PHB结构域结合细胞膜。尺寸排阻色谱试验、蓝色非变性聚丙烯酰胺凝胶电泳（Blue Native PAGE）以及交联试验结果显示，枯草芽孢杆菌的YuaG可通过PHB结构域寡聚化形成大型簇聚体。这一发现揭示了原核弗洛蒂林蛋白与真核弗洛蒂林蛋白在组装机制上的重要差异。