y复合物结构重访:超分辨率显微镜研究y复合物组分在膜定位上的差异。

IF 1.2 4区 生物学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Maximilian Greger, Paul Klemm, Felix Dempwolff, Marcus Lechner, Rebecca Hinrichs
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引用次数: 0

摘要

生物体在其栖息地生存的一个关键因素是其在蛋白质水平上迅速适应环境变化的能力。影响蛋白质丰度的一种快速有效的机制是核糖核酸酶对mRNA稳定性的调节。在原核模式生物枯草芽孢杆菌中,膜锚定的RNase Y通过降解和成熟mRNA发挥重要的调节作用。先前的研究表明,RNase Y与形成Y复合物的三种蛋白质协同作用。除了作为RNase Y的特异性因子外,已经提出了Y复合物在自然能力,生物膜形成以及孢子形成过程中的非核糖核酸酶独立功能。在之前的工作中,使用单分子跟踪,我们发现y复合物是高度动态的,并且存在于体内的多种成分中。通过生化分析,Y复合物募集到RNase Y被证明是由YaaT介导的,而YlbF和YmcA没有表现出任何直接的相互作用。在这里,我们使用三维结构照明显微镜(SIM)超分辨率和双分子荧光互补(BiFC)来进一步表征RNase Y/ Y复合物的定位和相互作用。通过使用结构照明显微镜(SIM)观察Y复合物蛋白和RNase Y,我们提供了额外的证据,表明YaaT的定位不同于YmcA和YlbF,因为YaaT在膜近端定位的比例似乎高于YmcA和YlbF。我们还发现YaaT膜结合的强度与培养基有关。通过使用双分子荧光互补(BiFC)提供了Y-复合物蛋白与RNase Y膜近端相互作用的证据。综上所述,我们的数据支持一个模型,其中Y复合物通过YaaT专门与RNase Y连接,并且Y复合物的组成以功能依赖的方式潜在地波动(图1)。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Y-complex architecture revisited: Super resolution microscopy to investigate differences in membrane localization for components in Y-complex.

A key factor for the survival of an organism in its habitat is its ability to quickly adapt to changes in its environment on the protein level. One fast and efficient mechanism to influence protein abundance is the regulation of mRNA stability by ribonucleases. In the prokaryotic model organism Bacillus subtilis, the membrane-anchored RNase Y performs a crucial regulatory role by degrading and maturating mRNA. Previous studies have shown that RNase Y acts in concert with three proteins forming the Y-complex. In addition to its role as specificity factor for RNase Y, ribonuclease-independent functions have been proposed for the Y-complex during natural competence, biofilm formation, as well as sporulation. In previous work, using single-molecule tracking, we showed that the Y-complex is highly dynamic and present in multiple compositions in vivo. Using biochemical analysis, recruitment of the Y-complex to RNase Y was shown to be mediated by YaaT whereas YlbF and YmcA did not display any direct interactions. Here we employ 3D- structured illumination microscopy (SIM) super resolution and bimolecular fluorescence complementation (BiFC) to further characterize RNase Y/ Y-complex localizations and interactions. By visualizing the Y-complex proteins and RNase Y using structured illumination microscopy (SIM), we provide additional evidence that YaaT localizes differently than YmcA and YlbF, in that the fraction of YaaT is localized membrane-proximal appears to be higher than the one observed for YmcA and YlbF. We also show that the strength of YaaT membrane association is culture medium dependent. Evidence for membrane-proximal interaction of the Y-complex proteins with RNase Y is provided through the use of bimolecular fluorescence complementation (BiFC). Taken together, our data support a model where the Y-complex is exclusively tethered to RNase Y by YaaT and where the composition of the Y-complex is fluctuating potentially in a function-dependent manner (Figure1).

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来源期刊
CiteScore
5.20
自引率
2.60%
发文量
13
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