A phospholipid acts as a chaperone in assembly of a membrane transport protein
A phospholipid acts as a chaperone in assembly of a membrane transport protein
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DOI：
10.1074/jbc.271.20.11615
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发表时间：
1996-05-17
影响因子：
4.8
通讯作者：
Dowhan, W
中科院分区：
文献类型：
--
作者：
Bogdanov, M;Sun, JZ;Dowhan, W
使用缺乏磷脂酰乙醇胺（PE）的大肠杆菌突变体和针对乳糖通透酶构象敏感表位（4 B1）的单克隆抗体（mAb 4 B1）来建立磷脂在膜蛋白组装中的新作用，表位4 B1在来自含PE的原生质球和右侧外膜囊泡中很容易检测到，但在来自含PE的原生质球和右侧外膜囊泡中不能检测到。表达乳糖通透酶的缺陷细胞。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和Western印迹分析，来自含PE膜的乳糖通透酶，而来自PE缺陷膜的乳糖通透酶，也被mAb 4 B1识别。大肠杆菌磷脂或PE（但不包括磷脂酰胆碱、磷脂酰甘油或心磷脂）在硝酸纤维素片上印迹（Western印迹）在从SDS-聚丙烯酰胺凝胶转移蛋白之前，来自PE缺陷细胞的通透酶重新获得mAb 4 B1的识别。因此，在组装形成表位4 B1期间需要PE，但是，一旦形成，在通透酶中保留足够的“构象记忆”，以在没有PE的情况下保留或改革该表位。如果部分变性，然后在有PE的情况下特异性地复性，则可以诱导缺乏表位4 B1的乳糖通透酶形成表位，这些结果首次确立了PE在乳糖通透酶组装中作为分子伴侣的作用。
A mutant of Escherichia coli lacking phosphatidylethanolamine (PE) and a monoclonal antibody (mAb 4B1) directed against a conformationally sensitive epitope (4B1) of lactose permease were used to establish a novel role for a phospholipid in the assembly of a membrane protein, Epitope 4B1 is readily detectable in spheroplasts and right-side-out membrane vesicles from PE-containing but not from PE-deficient cells expressing lactose permease. Lactose permease from PE-containing membranes, but not from PE-deficient membranes, subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and Western blot analysis is also recognized by mAb 4B1, If total E. coli phospholipids or PE (but not phosphatidylcholine, phosphatidylglycerol, or cardiolipin) are blotted on nitrocellulose sheets (Eastern blot) prior to transfer of proteins from SDS-polyacrylamide gels, the permease from PE-deficient cells regains its recognition by mAb 4B1, Therefore, PE is required during assembly to form epitope 4B1, but, once formed, sufficient ''conformational memory'' is retained in the permease to either retain or reform this epitope in the absence of PE, Lactose permease lacking epitope 4B1 can be induced to form the epitope if partially denatured and then renatured in the presence of PE specifically, These results establish for the first time a role for PE as a molecular chaperone in the assembly of the lactose permease.