Department of Biochemistry and Molecular Biology McGovern Medical School at the University of Texas Health Science Center, Houston, Texas, 77030, USA.
Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ, 08540, USA.
Sci Rep. 2020 Apr 14;10(1):6296. doi: 10.1038/s41598-020-63280-5.
Translocation of preproteins across the Escherichia coli inner membrane requires anionic lipids by virtue of their negative head-group charge either in vivo or in situ. However, available results do not differentiate between the roles of monoanionic phosphatidylglycerol and dianionic cardiolipin (CL) in this essential membrane-related process. To define in vivo the molecular steps affected by the absence of CL in protein translocation and insertion, we analyzed translocon activity, SecYEG stability and its interaction with SecA in an E. coli mutant devoid of CL. Although no growth defects were observed, co- and post-translational translocation of α-helical proteins across inner membrane and the assembly of outer membrane β-barrel precursors were severely compromised in CL-lacking cells. Components of proton-motive force which could impair protein insertion into and translocation across the inner membrane, were unaffected. However, stability of the dimeric SecYEG complex and oligomerization properties of SecA were strongly compromised while the levels of individual SecYEG translocon components, SecA and insertase YidC were largely unaffected. These results demonstrate that CL is required in vivo for the stability of the bacterial translocon and its efficient function in co-translational insertion into and translocation across the inner membrane of E. coli.
跨大肠杆菌内膜的前蛋白易位需要带负电荷的头基的阴离子脂质，无论是在体内还是在原位。然而，现有结果不能区分单阴离子磷脂酰甘油和二阴离子心磷脂 (CL) 在这一基本的膜相关过程中的作用。为了在体内定义 CL 缺失对蛋白质易位和插入的分子步骤的影响，我们分析了缺乏 CL 的大肠杆菌突变体中转录器活性、SecYEG 的稳定性及其与 SecA 的相互作用。尽管没有观察到生长缺陷，但在 CL 缺失的细胞中，α-螺旋蛋白的共翻译和翻译后易位穿过内膜以及外膜 β-桶前体的组装严重受损。可能损害蛋白质插入和跨内膜易位的质子动力势成分不受影响。然而，二聚体 SecYEG 复合物的稳定性和 SecA 的寡聚性质受到严重损害，而单个 SecYEG 转位器成分、SecA 和插入酶 YidC 的水平基本不受影响。这些结果表明，CL 在体内是细菌转位器稳定及其在大肠杆菌内膜共翻译插入和易位过程中的有效功能所必需的。
