Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas 77030, USA.
J Biol Chem. 2013 Feb 22;288(8):5873-85. doi: 10.1074/jbc.M112.442988. Epub 2013 Jan 15.
Energy-dependent uphill transport but not energy-independent downhill transport by lactose permease (LacY) is impaired when expressed in Escherichia coli cells or reconstituted in liposomes lacking phosphatidylethanolamine (PE) and containing only anionic phospholipids. The absence of PE results in inversion of the N-terminal half and misfolding of periplasmic domain P7, which are required for uphill transport of substrates. Replacement of PE in vitro by lipids with no net charge (phosphatidylcholine (PC), monoglucosyl diacylglycerol (GlcDAG), or diglucosyl diacylglycerol (GlcGlcDAG)) supported wild type transmembrane topology of the N-terminal half of LacY. The restoration of uphill transport in vitro was dependent on LacY native topology and proper folding of P7. Support of uphill transport by net neutral lipids in vitro (PE > PC ≫ GlcDAG ≠ GlcGlcDAG provided that PE or PC contained one saturated fatty acid) paralleled the results observed previously in vivo (PE = PC > GlcDAG ≠ GlcGlcDAG). Therefore, a free amino group is not required for uphill transport as previously concluded based on the lack of in vitro uphill transport when fully unsaturated PC replaced E. coli-derived PE. A close correlation was observed in vivo and in vitro between the ability of LacY to carry out uphill transport, the native conformation of P7, and the lipid headgroup and fatty acid composition. Therefore, the headgroup and the fatty acid composition of lipids are important for defining LacY topological organization and catalytically important structural features, further illustrating the direct role of lipids, independent of other cellular factors, in defining membrane protein structure/function.
当乳糖透过酶（LacY）在缺乏磷脂酰乙醇胺（PE）并仅含有阴离子磷脂的大肠杆菌细胞中表达或在缺乏 PE 的脂质体中再构成时，能量依赖的向上转运但不是能量独立的向下转运都会受损。PE 的缺失导致 N 端半部分的反转和周质域 P7 的错误折叠，这是底物向上转运所必需的。体外用不带净电荷的脂质（磷脂酰胆碱（PC）、单葡萄糖基二酰基甘油（GlcDAG）或双葡萄糖基二酰基甘油（GlcGlcDAG））替代 PE 支持 LacY 的 N 端半部分的野生型跨膜拓扑结构。体外恢复向上转运依赖于 LacY 天然拓扑结构和 P7 的正确折叠。体外带净中性脂质（PE > PC > GlcDAG ≠ GlcGlcDAG，前提是 PE 或 PC 含有一个饱和脂肪酸）对向上转运的支持与之前体内观察到的结果一致（PE = PC > GlcDAG ≠ GlcGlcDAG）。因此，正如之前基于完全不饱和 PC 替代大肠杆菌衍生的 PE 时体外缺乏向上转运所推断的那样，一个游离氨基基团对于向上转运不是必需的。体内和体外之间观察到 LacY 进行向上转运的能力、P7 的天然构象以及脂质头部基团和脂肪酸组成之间存在密切相关性。因此，脂质的头部基团和脂肪酸组成对于确定 LacY 的拓扑结构组织和催化上重要的结构特征很重要，进一步说明了脂质在独立于其他细胞因素的情况下直接决定膜蛋白结构/功能的作用。
