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Abstract：
In steroid-synthesizing cells, like the MA-10 mouse tumor Leydig cells, the peripheral-type benzodiazepine receptor (PBR) is an outer mitochondrial membrane protein involved in the regulation of cholesterol transport from the outer to the inner mitochondrial membrane, the rate-determining step in steroid biosynthesis. Expression of PBR in Escherichia coli DE3 cells, which have no PBR, no cholesterol, and do not make steroids, induced the ability to take up cholesterol in a time-dependent, temperature-sensitive, and energy-independent manner. These cells took up no other steroids tested. Addition of the high affinity PBR ligand PK 11195 to cholesterol-loaded membranes, obtained from cells transfected with PBR, resulted in the release of the uptaken cholesterol. Expression in DE3 cells of mutant PBRs demonstrated that deletions in the cytoplasmic carboxy-terminus dramatically reduced the cholesterol uptake function of PBR, although it retained full capacity to bind PK 11195. Site-directed mutagenesis in the carboxy-terminal region of PBR demonstrated that bacteria expressing the mutant PBR proteins PBR(Y153S) and PBR(R156L) do not accumulate cholesterol, suggesting that amino acids Y153 and R156 are involved in the interaction of the receptor with cholesterol. Considering these results, we postulate the existence of a common cholesterol recognition/interaction amino acid consensus pattern (-L/V-(X)(1-5)-Y-(X)(1-5)-R/K-). Indeed, we found this amino acid consensus pattern in all proteins shown to interact with cholesterol. In conclusion, these data suggest that the expression of PBR confers the ability to take up and release, upon ligand activation, cholesterol. Considering the widespread occurrence of this protein and its tissue and cell specific subcellular localization, these results suggest a more general role of PBR in intracellular cholesterol transport and compartmentalization.
摘要：
在类固醇合成细胞中，像MA-10小鼠肿瘤Leydig细胞，外周型苯并二氮杂受体（PBR）是一种外线粒体膜蛋白，参与调节胆固醇从线粒体外膜到内膜的运输，类固醇生物合成中的速率决定步骤。PBR在大肠杆菌DE3细胞中的表达,没有PBR，没有胆固醇,不要制造类固醇，诱导吸收胆固醇的能力是时间依赖性的，温度敏感，和能源独立的方式。这些细胞不吸收其他测试的类固醇。将高亲和力PBR配体PK11195添加到负载胆固醇的膜上，从用PBR转染的细胞中获得，导致摄入的胆固醇的释放。突变型PBR在DE3细胞中的表达表明，细胞质羧基末端的缺失显着降低了PBR的胆固醇摄取功能，尽管它保留了结合PK11195的全部能力。PBR羧基末端区域的定点诱变表明，表达突变PBR蛋白PBR（Y153S）和PBR（R156L）的细菌不会积累胆固醇，提示氨基酸Y153和R156参与受体与胆固醇的相互作用。考虑到这些结果,我们假设存在常见的胆固醇识别/相互作用氨基酸共识模式（-L/V-（X）（1-5）-Y-（X）（1-5）-R/K-）。的确,我们在所有显示与胆固醇相互作用的蛋白质中发现了这种氨基酸共有模式.总之,这些数据表明，PBR的表达赋予了吸收和释放的能力，在配体激活后，胆固醇。考虑到该蛋白的广泛存在及其组织和细胞特异性亚细胞定位，这些结果表明,PBR在细胞内胆固醇转运和区室化中的作用更为普遍.
