MOLECULAR BASIS OF PROTEIN TRANSPORT IN PHOTORECEPTORS

描述（改编自申请人的摘要）：一个新的独立的 申请人计划继续研究 视紫红质突变申请人的早期工作建立了两个 不同类型的缺陷，在视紫红质突变体牵连在ADRP。一 类是有缺陷的蛋白质退出ER/高尔基体复合体;第二个 类无法进行极化排序，并且无法将 视紫红质到视杆外节。在本建议书中，申请人将 继续观察第二类突变体，希望 破译视紫红质定位的机制。 在第一个具体目标中，将使用三个互补系统， 识别负责视紫红质靶向的信号， 合适的膜。狗肾的极化上皮细胞， MDCK细胞，将用于分析大量的视紫红质突变体。 确定该系统对反射光感受器的适用性 行为，蝾螈原代视网膜文化将被检查， 适合作为实验系统。最后，转基因小鼠将 用于确定两个实验系统的可靠性， 反映体内情况。这项工作将建立 C-末端结构域的要求和充分性以及 搜索排序过程中的其他重要序列。 在第二个具体目标中，其他分子组分的分选 将通过识别与视紫红质结合的蛋白质来寻求这一过程 排序序列。酵母双杂交系统和直接 将使用蛋白质/蛋白质过滤器结合测定。被识别基因 将进行测序，以确定编码基因的性质及其 将确定亚细胞位置。

DESCRIPTION (Adapted from applicant's abstract): A newly independent applicant plans to continue work on mechanisms underlying the defects in rhodopsin mutations. Earlier work by the applicant established two different types of defects in rhodopsin mutants implicated in ADRP. One class is defective in protein exit from the ER/Golgi complex; the second class is incapable of polarized sorting and fails to targeting the rhodopsin to the rod outer segment. In this proposal, the applicant will follow up the observations on this second class of mutants in hopes of deciphering the mechanisms involved in rhodopsin localization. In the first specific aim, three complementary systems will be used to identify the signals responsible for the targeting of rhodopsin to the appropriate membrane. A polarized epithelial cell from dog kidney, the MDCK cell, will be used to analyze large numbers of rhodopsin mutants. To determine the suitability of this system for reflecting photoreceptor behavior, a salamander primary retinal culture will be examined for suitability as an experimental system. Finally, transgenic mice will be used to determine the reliability of both experimental systems in reflecting the in vivo situation. The work will establish the requirement and the sufficiency of the C-terminus domain as well as search for other important sequences in the sorting process. In the second specific aim, other molecular components of the sorting process will be sought by identifying proteins that bind to rhodopsin sorting sequences. Both the yeast two hybrid system and a direct protein/protein filter binding assays will be used. Identified genes will be sequenced to determine the nature of the encoded gene and their subcellular location will be determined.