PHOSPHATIDYLETHANOLAMINE IS REQUIRED FOR IN-VIVO FUNCTION OF THE MEMBRANE-ASSOCIATED LACTOSE PERMEASE OF ESCHERICHIA-COLI
PHOSPHATIDYLETHANOLAMINE IS REQUIRED FOR IN-VIVO FUNCTION OF THE MEMBRANE-ASSOCIATED LACTOSE PERMEASE OF ESCHERICHIA-COLI
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DOI：
10.1074/jbc.270.2.732
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发表时间：
1995-01-13
影响因子：
4.8
通讯作者：
DOWHAN, W
中科院分区：
文献类型：
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作者：
BOGDANOV, M;DOWHAN, W
用缺乏磷脂酰乙醇胺（PE）作为膜组分的突变大肠杆菌细胞进行的实验（DeCoggny，A.，希科克，P. N.，和Dowhan，W.（1991）J.Biol.Chem.266，5323-5332），以确定PE是否是乳糖通透酶在体内的完全功能所必需的。两种乳糖主动转运的V-max（在缺乏β-半乳糖苷酶，lacZ的细胞中）和不可水解的乳糖类似物，甲基-β-D-吡喃半乳糖苷（TMG），被缺乏PE的突变细胞相对于含有PE的细胞减少5-10倍，而两种底物的摄取的Km在两种类型的细胞中是相同的，低速率的TMG和乳糖摄取的PE缺陷细胞是不受质子载体的存在（解偶联剂）和TMG摄取的数量级为解偶联剂处理的含有PE的细胞中摄取速率大大降低，乳糖进入两种类型细胞的lacZ（+）衍生物的速率（作为易化扩散的量度）几乎相同。解偶联剂的存在对乳糖（lacZ细胞）和TMG运输在PE缺陷细胞的Km值没有影响，解偶联剂的存在对V-max有很小的影响。在含PE的细胞中，TMG运输的这些动力学参数被解偶联剂降低到与PE缺陷细胞发现的水平，而解偶联剂降低含PE的（lacZ）细胞的乳糖摄取到低于可测量的水平。由两种细胞制成的倒置膜囊泡均能负载TMG，但ATP激活负载TMG的囊泡仅诱导TMG从含PE的囊泡中快速、上坡、依赖于渗透酶的外排。无PE的细胞表观主动转运活性的降低不是由于膜通透性的改变，而是由于Δ（μ H+）降低。（质子电化学梯度）穿过细胞膜，或降低膜相关lac通透酶蛋白的水平，这些结果表明，在没有PE的情况下，Iac通透酶不能将底物摄取偶联到Delta（mu H+）。以便实现底物的积累，结果仅进行促进扩散。
Experiments with mutant Escherichia coli cells lacking phosphatidylethanolamine (PE) as a membrane component (DeChavigny, A., Heacock, P. N., and Dowhan, W. (1991) J. Biol. Chem. 266, 5323-5332) were carried out to establish whether or not PE is necessary for full function of the lac permease in vivo. The V-max for active transport of both lactose (in cells lacking beta-galactosidase, lacZ) and the unhydrolyzable lactose analog, methyl-beta-D-galactopyranoside (TMG), by mutant cells lacking PE was reduced 5-10-fold relative to cells containing PE, while the K-m for the uptake of both substrates was the same in both types of cells, The low rate of TMG and lactose uptake by PE-deficient cells was unaffected by the presence of a protonophore (uncoupler) and for TMG uptake was on the order of the greatly reduced rate of uptake in uncoupler-treated cells containing PE, The rate of entry of lactose into lacZ(+) derivatives of both types of cells, as a measure of facilitated diffusion, was nearly the same. The K-m for lactose (lacZ cells) and TMG transport in PE-deficient cells was unaffected by the presence of an uncoupler which had a small effect on V-max. In PE-containing cells these kinetic parameters for TMG transport were reduced by an uncoupler to the level found with PE-deficient cells while an uncoupler reduced lactose uptake by PE-containing (lacZ) cells to below measureable levels. Inverted membrane vesicles made from both types of cells could be loaded with TMG, but energizing TMG-loaded vesicles by ATP only induced rapid, uphill, permease-dependent efflux of TMG from PE-containing vesicles, The decrease in apparent active transport activity of cells with no PE was not due to a change in membrane permeability, to a reduced Delta(mu H+) (proton electrochemical gradient) across the cell membrane, or to a reduced level of membrane-associated lac permease protein, These results suggest that in the absence of PE the Iac permease cannot couple substrate uptake to Delta(mu H+) in order to effect accumulation of substrate and as a result only carries out facilitated diffusion.