MOLECULAR BASIS OF PROTEIN TRANSPORT IN PHOTORECEPTORS

描述(改编自申请人的摘要)：一个新的独立 申请者计划继续研究潜在缺陷的机制 视紫红质突变。申请人的早期工作建立了两个 与ADRP有关的视紫红质突变体的不同类型的缺陷。一 第二类是内质网/高尔基复合体退出的蛋白质有缺陷； 类不能进行两极排序，并且无法将 视紫红质至视杆外段。在本建议书中，申请人将 对这第二类突变体的后续观察，希望 破译视紫红质定位的机制。 在第一个具体目标中，将使用三个互补的系统来 识别负责视紫红质靶向的信号 合适的膜。来自狗肾的极化上皮细胞， MDCK细胞，将用于分析大量视紫红质突变体。 以确定该系统对光感受器反射的适用性 行为，火蜥蜴的视网膜原代培养将被检查 作为实验系统的适用性。最后，转基因小鼠将被 用于确定两个实验系统的可靠性 反映了体内的情况。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，对其他分子成分进行了分类 将通过识别与视紫红质结合的蛋白质来寻求这一过程 对序列进行排序。酵母双杂交系统和直接 将使用蛋白质/蛋白质过滤器结合分析。已识别的基因 将被测序以确定编码基因的性质和它们的 亚蜂窝位置将被确定。

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.