Prithviraj Malavika, Freundlich Joel S, Morita Yasu S
Department of Microbiology, University of Massachusetts, Amherst, Massachusetts, USA.
Department of Pharmacology, Physiology, and Neuroscience, Rutgers University - New Jersey Medical School, Newark, New Jersey, USA.
J Biol Chem. 2025 Jun 11;301(7):110361. doi: 10.1016/j.jbc.2025.110361.
Mycobacterium smegmatis partitions its plasma membrane into two distinct regions: the inner membrane domain (IMD) and the conventional plasma membrane. IMD, enriched in the sub-polar regions of actively growing rod-shaped cells, contains many membrane proteins involved in cell envelope biosynthesis. Dibucaine, a membrane fluidizer, disrupts plasma membrane integrity and de-partitions the IMD from the subpolar regions. We do not know what governs the de-partitioning of the IMD in response to dibucaine stress. In this study, we investigated the stress response of the IMD under respiration defect. We first depleted MenG, a key enzyme in the menaquinone biosynthesis, by CRISPRi and observed that the IMD does not respond to dibucaine-induced membrane stress. CRISPRi-induced knockdown of qcrC, a gene encoding a component of an electron transport chain cytochrome, corroborated the results of menG knockdown. In contrast, neither CRISPRi knockdown of atpD, a gene encoding a component of the ATP synthase nor inhibition of ATP synthase by bedaquiline inhibited the dibucaine-induced de-partitioning of sub-polar IMD as robustly as CRISPRi knockdowns of menG and qcrC. Pretreatment with the protonophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP) prevented dibucaine-induced IMD de-partitioning. Furthermore, pretreatment with nigericin, which acts as an H/K antiporter and disrupts the proton gradient without affecting membrane potential, also inhibited the IMD de-partitioning in a way similar to CCCP. Taken together, our findings suggest that membrane stress-induced IMD delocalization is not a passive lipid dispersion but an active membrane rearrangement dependent on an electrochemical gradient of the proton.
内膜结构域（IMD）和传统的质膜。IMD富集于活跃生长的杆状细胞的亚极区，包含许多参与细胞壁生物合成的膜蛋白。膜流化剂地布卡因会破坏质膜完整性，并使IMD与亚极区分离。我们不知道是什么因素控制着IMD在应对地布卡因胁迫时的分离。在本研究中，我们调查了呼吸缺陷情况下IMD的应激反应。我们首先通过CRISPRi技术敲除了甲萘醌生物合成中的关键酶MenG，观察到IMD对地布卡因诱导的膜应激没有反应。CRISPRi诱导的qcrC基因敲除（qcrC基因编码电子传递链细胞色素的一个组分）证实了MenG基因敲除的结果。相比之下，CRISPRi对编码ATP合酶一个组分的atpD基因的敲除，以及贝达喹啉对ATP合酶的抑制，均未像CRISPRi对MenG和qcrC的敲除那样强烈抑制地布卡因诱导的亚极IMD分离。用质子载体羰基氰化物间氯苯腙（CCCP）预处理可防止地布卡因诱导的IMD分离。此外，尼日利亚菌素（一种作为H/K反向转运体并在不影响膜电位的情况下破坏质子梯度的物质）预处理也以类似于CCCP的方式抑制了IMD分离。综上所述，我们的研究结果表明，膜应激诱导的IMD去定位不是被动的脂质分散，而是依赖于质子电化学梯度的主动膜重排。
