Stamer W D, Perkumas K M, Hoffman E A, Roberts B C, Epstein D L, McKay B S
Department of Ophthalmology and Vision Science, The University of Arizona, Tucson, AZ 85711, USA.
Exp Eye Res. 2006 Dec;83(6):1386-95. doi: 10.1016/j.exer.2006.07.018. Epub 2006 Sep 12.
Mutations in myocilin (MYOC) associate with glaucoma and ocular hypertension. Unfortunately, the specific role of MYOC, a widely expressed protein of unknown function, in ocular hypertension is unknown. Since MYOC localizes both to intracellular membranes and to the cytosol, we tested the hypothesis that MYOC is a cytosolic protein that associates with cellular membranes via its coiled-coil domain. Using green fluorescent protein (GFP) chimeras in expression and metabolic labeling studies, we observed that MYOC's putative signal peptide failed to traffic GFP into the secretory machinery and out of transfected cells. Next, we tested which of MYOC's three folding domains were responsible for targeting. In cell fractionation and immunofluorescence microscopy studies, the coiled-coil, but not the helix-turn-helix or olfactomedin domains, was necessary and sufficient to target GFP chimeras to cell membranes. Interestingly, a vesicular phenotype required sequential addition of the helix-turn-helix and olfactomedin domains to the coiled-coil. Taken together, these data indicate that the coiled-coil domain, not the putative signal sequence, is responsible for the targeting of MYOC to the secretory machinery.
肌纤蛋白（MYOC）的突变与青光眼和高眼压症相关。遗憾的是，作为一种功能未知但广泛表达的蛋白质，MYOC在高眼压症中的具体作用尚不清楚。由于MYOC既定位于细胞内膜又定位于胞质溶胶，我们测试了这样一个假设，即MYOC是一种通过其卷曲螺旋结构域与细胞膜相关联的胞质蛋白。在表达和代谢标记研究中使用绿色荧光蛋白（GFP）嵌合体，我们观察到MYOC的推定信号肽未能将GFP转运到分泌机制并转运出转染细胞。接下来，我们测试了MYOC的三个折叠结构域中哪一个负责靶向作用。在细胞分级分离和免疫荧光显微镜研究中，卷曲螺旋结构域而非螺旋-转角-螺旋结构域或嗅觉介质结构域，对于将GFP嵌合体靶向细胞膜是必要且充分的。有趣的是，一种囊泡表型需要将螺旋-转角-螺旋结构域和嗅觉介质结构域依次添加到卷曲螺旋结构域上。综上所述，这些数据表明，负责将MYOC靶向分泌机制的是卷曲螺旋结构域，而非推定的信号序列。
