Doi T, Molday R S, Khorana H G
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Proc Natl Acad Sci U S A. 1990 Jul;87(13):4991-5. doi: 10.1073/pnas.87.13.4991.
The role of the intradiscal polypeptide loops in bovine rhodopsin has been investigated by deletions in the N-terminal tail and in loops B-C, D-E, and E-F as well as by single amino acid substitutions in the D-E loop. Mutants with three types of phenotypes were observed. Type I mutants showed a rhodopsin-like chromophore and glycosylation. Type II mutants did not regenerate the chromophore and showed abnormal glycosylation. Type III mutants showed poor chromophore regeneration and abnormal glycosylation. Reduced transducin activation was shown by some type I and III mutants. Single amino acid substitutions in the D-E loop gave mostly type I mutants. Deletions in loops B-C, D-E, and F-G gave type II mutants, whereas deletions in the N-terminal tail produced type III mutants. Systematic deletions of two adjacent amino acids in loop D-E indicated that the amino acid sequences 171-182 and 189-192 were essential to rhodopsin structure. Immunofluorescence double-staining and transmission electron microscopy of one type II mutant (with residues 189 and 190 deleted) showed that it was mostly in the endoplasmic reticulum, whereas the wild-type protein was in the plasma membrane. We conclude that the first step in the assembly of the rhodopsin molecule is the formation of a three-dimensional structure in the intradiscal domain involving the bulk of the out-of-the-membrane polypeptide segments followed by the linkage of Cys-110 and Cys-187 through a disulfide bond.
通过对牛视紫红质N端尾巴以及B - C环、D - E环和E - F环进行缺失突变，以及对D - E环进行单个氨基酸替换，研究了盘内多肽环在牛视紫红质中的作用。观察到了三种表型的突变体。I型突变体表现出类似视紫红质的发色团和糖基化。II型突变体无法再生发色团且表现出异常糖基化。III型突变体表现出发色团再生不良和异常糖基化。一些I型和III型突变体显示出转导素激活减少。D - E环中的单个氨基酸替换大多产生I型突变体。B - C环、D - E环和F - G环的缺失产生II型突变体，而N端尾巴的缺失产生III型突变体。对D - E环中两个相邻氨基酸进行系统缺失表明，氨基酸序列171 - 182和189 - 192对视紫红质结构至关重要。对一个II型突变体（缺失189和190位残基）进行免疫荧光双重染色和透射电子显微镜观察表明，它主要存在于内质网中，而野生型蛋白存在于质膜中。我们得出结论，视紫红质分子组装的第一步是在盘内结构域形成三维结构（涉及大部分膜外多肽片段），随后通过二硫键连接Cys - 110和Cys - 187。
