Phase separation and clustering of an ABC transporter in Mycobacterium tuberculosis
Phase separation and clustering of an ABC transporter in Mycobacterium tuberculosis
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DOI：
10.1073/pnas.1820683116
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发表时间：
2019-08-13
影响因子：
11.1
通讯作者：
Gsponer, Jorg
中科院分区：
文献类型：
--
作者：
Heinkel, Florian;Abraham, Libin;Gsponer, Jorg
相分离驱动许多细胞过程，从无膜细胞器的形成到信号蛋白的协同组装。多价性和内在紊乱等使凝聚物形成的特征不仅存在于胞质和核蛋白中，而且存在于膜相关蛋白中。ABC转运蛋白Rv 1747对结核分枝杆菌（Mtb）在感染宿主中的生长非常重要，其具有由2个磷酸苏氨酸结合Forkhead相关结构域组成的细胞质调节模块，所述结构域通过具有多个磷酸受体苏氨酸的内在无序连接体连接。在这里，我们证明了Rv 1747及其同源物在耻垢分枝杆菌中的调节模块作为浓度和磷酸化的函数形成液体样冷凝物。Mtb的丝氨酸/苏氨酸激酶和唯一的磷酸酶调节磷酸化增强的相分离，并与所得的缩合物差异共定位。Rv 1747调节模块也在支持的脂质双层上相分离，并且当在活酵母和M.包皮垢细胞与这些观察结果一致，单分子定位显微镜显示，内源性结核分枝杆菌转运蛋白在分枝杆菌膜内形成高阶簇。总的来说，这些数据表明相分离在这些分枝杆菌ABC转运蛋白的功能及其通过细胞内信号传导的调节中起关键作用。
Phase separation drives numerous cellular processes, ranging from the formation of membrane-less organelles to the cooperative assembly of signaling proteins. Features such as multivalency and intrinsic disorder that enable condensate formation are found not only in cytosolic and nuclear proteins, but also in membrane-associated proteins. The ABC transporter Rv1747, which is important for Mycobacterium tuberculosis (Mtb) growth in infected hosts, has a cytoplasmic regulatory module consisting of 2 phosphothreonine-binding Forkhead-associated domains joined by an intrinsically disordered linker with multiple phospho-acceptor threonines. Here we demonstrate that the regulatory modules of Rv1747 and its homolog in Mycobacterium smegmatis form liquid-like condensates as a function of concentration and phosphorylation. The serine/threonine kinases and sole phosphatase of Mtb tune phosphorylation-enhanced phase separation and differentially colocalize with the resulting condensates. The Rv1747 regulatory module also phase-separates on supported lipid bilayers and forms dynamic foci when expressed heterologously in live yeast and M. smegmatis cells. Consistent with these observations, single-molecule localization microscopy reveals that the endogenous Mtb transporter forms higher-order clusters within the Mycobacterium membrane. Collectively, these data suggest a key role for phase separation in the function of these mycobacterial ABC transporters and their regulation via intracellular signaling.