Departments of Pharmacology, Cleveland, Ohio 44106.
Ophthalmology and Visual Sciences, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
J Biol Chem. 2010 Mar 26;285(13):9667-9682. doi: 10.1074/jbc.M109.063941. Epub 2010 Jan 25.
Regeneration of visual chromophore in the vertebrate visual cycle involves the retinal pigment epithelium-specific protein RPE65, the key enzyme catalyzing the cleavage and isomerization of all-trans-retinyl fatty acid esters to 11-cis-retinol. Although RPE65 has no predicted membrane spanning domains, this protein predominantly associates with microsomal fractions isolated from bovine retinal pigment epithelium (RPE). We have re-examined the nature of RPE65 interactions with native microsomal membranes by using extraction and phase separation experiments. We observe that hydrophobic interactions are the dominant forces that promote RPE65 association with these membranes. These results are consistent with the crystallographic model of RPE65, which features a large lipophilic surface that surrounds the entrance to the catalytic site of this enzyme and likely interacts with the hydrophobic core of the endoplasmic reticulum membrane. Moreover, we report a critical role for phospholipid membranes in preserving the retinoid isomerization activity and physical properties of RPE65. Isomerase activity measured in bovine RPE was highly sensitive to phospholipase A(2) treatment, but the observed decline in 11-cis-retinol production did not directly reflect inhibition by products of lipid hydrolysis. Instead, a direct correlation between the kinetics of phospholipid hydrolysis and retinoid isomerization suggests that the lipid membrane structure is critical for RPE65 enzymatic activity. We also provide evidence that RPE65 operates in a multiprotein complex with retinol dehydrogenase 5 and retinal G protein-coupled receptor in RPE microsomes. Modifications in the phospholipid environment affecting interactions with these protein components may be responsible for the alterations in retinoid metabolism observed in phospholipid-depleted RPE microsomes. Thus, our results indicate that the enzymatic activity of native RPE65 strongly depends on its membrane binding and phospholipid environment.
脊椎动物视觉循环中的视觉色素再生涉及视网膜色素上皮特异性蛋白 RPE65，这是催化全反式视黄基脂肪酸酯裂解和异构化为 11-顺式视黄醇的关键酶。尽管 RPE65 没有预测的跨膜结构域，但这种蛋白质主要与从牛视网膜色素上皮（RPE）分离的微粒体部分相关联。我们通过使用提取和相分离实验重新检查了 RPE65 与天然微粒体膜相互作用的性质。我们观察到疏水相互作用是促进 RPE65 与这些膜结合的主要力。这些结果与 RPE65 的晶体结构模型一致，该模型具有一个大的疏水面，环绕着该酶催化位点的入口，并且可能与内质网膜的疏水区相互作用。此外，我们报告了磷脂膜在保持 RPE65 的视黄醇异构化活性和物理性质方面的关键作用。在牛 RPE 中测量的异构酶活性对磷脂酶 A2 的处理非常敏感，但观察到的 11-顺式视黄醇产量下降并没有直接反映脂质水解产物的抑制作用。相反，磷脂水解动力学与视黄醇异构化之间的直接相关性表明，脂质膜结构对于 RPE65 的酶活性至关重要。我们还提供了证据表明，RPE65 在 RPE 微粒体中与视黄醇脱氢酶 5 和视网膜 G 蛋白偶联受体一起作为一个多蛋白复合物发挥作用。影响与这些蛋白质成分相互作用的磷脂环境的修饰可能是导致在磷脂耗尽的 RPE 微粒体中观察到的视黄醇代谢改变的原因。因此，我们的结果表明，天然 RPE65 的酶活性强烈依赖于其膜结合和磷脂环境。
