SYNMIKRO, LOEWE Center for Synthetic Microbiology, Marburg, Germany.
Fachbereich Chemie, Philipps-Universität Marburg, Marburg, Germany.
BMC Biol. 2018 Sep 3;16(1):97. doi: 10.1186/s12915-018-0561-0.
Proteins within the cytoplasmic membrane display distinct localization patterns and arrangements. While multiple models exist describing the dynamics of membrane proteins, to date, there have been few systematic studies, particularly in bacteria, to evaluate how protein size, number of transmembrane domains, and temperature affect their diffusion, and if conserved localization patterns exist.
We have used fluorescence microscopy, single-molecule tracking (SMT), and computer-aided visualization methods to obtain a better understanding of the three-dimensional organization of bacterial membrane proteins, using the model bacterium Bacillus subtilis. First, we carried out a systematic study of the localization of over 200 B. subtilis membrane proteins, tagged with monomeric mVenus-YFP at their original gene locus. Their subcellular localization could be discriminated in polar, septal, patchy, and punctate patterns. Almost 20% of membrane proteins specifically localized to the cell poles, and a vast majority of all proteins localized in distinct structures, which we term microdomains. Dynamics were analyzed for selected membrane proteins, using SMT. Diffusion coefficients of the analyzed transmembrane proteins did not correlate with protein molecular weight, but correlated inversely with the number of transmembrane helices, i.e., transmembrane radius. We observed that temperature can strongly influence diffusion on the membrane, in that upon growth temperature upshift, diffusion coefficients of membrane proteins increased and still correlated inversely to the number of transmembrane domains, following the Saffman-Delbrück relation.
The vast majority of membrane proteins localized to distinct multimeric assemblies. Diffusion of membrane proteins can be suitably described by discriminating diffusion coefficients into two protein populations, one mobile and one immobile, the latter likely constituting microdomains. Our results show there is high heterogeneity and yet structural order in the cell membrane, and provide a roadmap for our understanding of membrane organization in prokaryotes.
细胞质膜内的蛋白质具有独特的定位模式和排列方式。虽然有多个模型描述了膜蛋白的动力学，但迄今为止，很少有系统的研究，特别是在细菌中，来评估蛋白质大小、跨膜结构域数量和温度如何影响它们的扩散，以及是否存在保守的定位模式。
我们使用荧光显微镜、单分子跟踪（SMT）和计算机辅助可视化方法，以模型细菌枯草芽孢杆菌为研究对象，更好地了解细菌膜蛋白的三维组织。首先，我们对 200 多个枯草芽孢杆菌膜蛋白进行了系统研究，这些蛋白在其原始基因座处被标记为单体 mVenus-YFP。它们的亚细胞定位可以区分极性、隔室、点状和点状模式。近 20%的膜蛋白特异性定位于细胞极，绝大多数蛋白定位于不同的结构，我们称之为微域。我们使用 SMT 对选定的膜蛋白进行了动力学分析。分析的跨膜蛋白的扩散系数与蛋白质分子量无关，但与跨膜螺旋数（即跨膜半径）成反比。我们观察到温度可以强烈影响膜上的扩散，即在生长温度升高时，膜蛋白的扩散系数增加，并且仍然与跨膜结构域的数量成反比，符合 Saffman-Delbrück 关系。
绝大多数膜蛋白定位于不同的多聚体组装体。可以通过将扩散系数区分成两个蛋白质群体来适当地描述膜蛋白的扩散，一个是移动的，一个是不移动的，后者可能构成微域。我们的结果表明，细胞膜具有高度的异质性和结构有序性，并为我们理解原核生物的膜组织提供了路线图。
