Dempwolff Felix, Schmidt Felix K, Hervás Ana B, Stroh Alex, Rösch Thomas C, Riese Cornelius N, Dersch Simon, Heimerl Thomas, Lucena Daniella, Hülsbusch Nikola, Stuermer Claudia A O, Takeshita Norio, Fischer Reinhard, Eckhardt Bruno, Graumann Peter L
SYNMIKRO, LOEWE Center for Synthetic Microbiology, Philipps Universität Marburg, Marburg, Germany.
Department of Chemistry, Philipps Universität Marburg, Marburg, Germany.
PLoS Genet. 2016 Jun 30;12(6):e1006116. doi: 10.1371/journal.pgen.1006116. eCollection 2016 Jun.
Biological membranes have been proposed to contain microdomains of a specific lipid composition, in which distinct groups of proteins are clustered. Flotillin-like proteins are conserved between pro-and eukaryotes, play an important function in several eukaryotic and bacterial cells, and define in vertebrates a type of so-called detergent-resistant microdomains. Using STED microscopy, we show that two bacterial flotillins, FloA and FloT, form defined assemblies with an average diameter of 85 to 110 nm in the model bacterium Bacillus subtilis. Interestingly, flotillin microdomains are of similar size in eukaryotic cells. The soluble domains of FloA form higher order oligomers of up to several hundred kDa in vitro, showing that like eukaryotic flotillins, bacterial assemblies are based in part on their ability to self-oligomerize. However, B. subtilis paralogs show significantly different diffusion rates, and consequently do not colocalize into a common microdomain. Dual colour time lapse experiments of flotillins together with other detergent-resistant proteins in bacteria show that proteins colocalize for no longer than a few hundred milliseconds, and do not move together. Our data reveal that the bacterial membrane contains defined-sized protein domains rather than functional microdomains dependent on flotillins. Based on their distinct dynamics, FloA and FloT confer spatially distinguishable activities, but do not serve as molecular scaffolds.
有人提出生物膜含有特定脂质组成的微结构域，其中不同的蛋白质群聚集在一起。类弗洛蒂林蛋白在原核生物和真核生物之间保守，在几种真核细胞和细菌细胞中发挥重要作用，并在脊椎动物中定义了一种所谓的抗去污剂微结构域。使用受激发射损耗显微镜（STED显微镜），我们发现在模式细菌枯草芽孢杆菌中，两种细菌弗洛蒂林蛋白FloA和FloT形成了平均直径为85至110纳米的特定聚集体。有趣的是，弗洛蒂林微结构域在真核细胞中的大小相似。FloA的可溶性结构域在体外形成高达数百千道尔顿的高阶寡聚体，表明与真核弗洛蒂林蛋白一样，细菌聚集体部分基于它们自我寡聚的能力。然而，枯草芽孢杆菌的旁系同源物显示出显著不同的扩散速率，因此不会共定位到一个共同的微结构域中。在细菌中对弗洛蒂林蛋白与其他抗去污剂蛋白进行的双色延时实验表明，蛋白质共定位不超过几百毫秒，并且不会一起移动。我们的数据表明，细菌膜含有特定大小的蛋白质结构域，而不是依赖于弗洛蒂林蛋白的功能性微结构域。基于它们不同的动力学，FloA和FloT赋予空间上可区分的活性，但不作为分子支架。
