Zhou B K, Kobayashi T, Donatien P D, Bennett D C, Hearing V J, Orlow S J
Ronald O. Perelman Department of Dermatology, New York University School of Medicine, New York 10016.
Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7076-80. doi: 10.1073/pnas.91.15.7076.
To identify a broad spectrum of melanosomal proteins, antisera were raised in rabbits against melanosomal protein fractions separated on the basis of their solubility in the nonionic detergent Triton X-114. Antisera against the different fractions each recognized a distinct set of bands when used for immunoblotting analysis with extracts of melanocytes cultured from wild-type black mice. Immunoblotting with antisera to whole melanosomes or to Triton X-114-soluble melanosomal proteins that segregated with the detergent phase gave identical patterns with protein extracts from melanocytes from wild-type mice and from mice homozygous for the si (silver) coat color mutation. By contrast, an antiserum against Triton X-114 soluble melanosomal proteins that segregated in the aqueous phase recognized an 85-kDa protein that was present in extracts from wild-type melanocytes but was absent from si melanocytes. This suggested that the protein was encoded at the si (silver) locus. This was confirmed by employing an antiserum directed against the carboxyl terminus of the predicted murine silver protein sequence. The detergent solubility, biochemical characteristics, and immunologic properties of the 85-kDa protein and of the authentic si gene product were identical. Further analysis demonstrated that this protein corresponds to a melanosomal matrix glycoprotein that we recently described. Our results suggest that employing polyclonal antisera to fractionated organelles such as melanosomes, to screen tissues from mutant mice, a technique that we call "organelle scanning", can serve as a powerful means of identifying new organellar proteins and their respective genes.
为了鉴定广泛的黑素小体蛋白，用兔制备了抗血清，该抗血清针对根据其在非离子去污剂Triton X-114中的溶解性分离的黑素小体蛋白组分。当用从野生型黑色小鼠培养的黑素细胞提取物进行免疫印迹分析时，针对不同组分的抗血清各自识别出一组独特的条带。用抗全黑素小体或抗与去污剂相分离的Triton X-114可溶性黑素小体蛋白的抗血清进行免疫印迹，野生型小鼠和si（银色）毛色突变纯合小鼠的黑素细胞蛋白提取物呈现相同的模式。相比之下，针对在水相中分离的Triton X-114可溶性黑素小体蛋白的抗血清识别出一种85 kDa的蛋白，该蛋白存在于野生型黑素细胞提取物中，但在si黑素细胞中不存在。这表明该蛋白是由si（银色）基因座编码的。通过使用针对预测的小鼠银色蛋白序列羧基末端的抗血清证实了这一点。85 kDa蛋白和真实的si基因产物的去污剂溶解性、生化特性和免疫学特性是相同的。进一步分析表明，该蛋白对应于我们最近描述的一种黑素小体基质糖蛋白。我们的结果表明，使用针对诸如黑素小体等分级细胞器的多克隆抗血清来筛选突变小鼠的组织，我们称之为“细胞器扫描”的技术，可以作为鉴定新的细胞器蛋白及其各自基因的有力手段。
