REGULATION OF SPERM FUNCTION BY PROTEIN PRENYLATION

描述：（改编自申请人的描述）精浆 膜被划分成不同分子组成的域， 在受精中的作用。 不同膜结构域的组装是 在精子发生期间开始，并在睾丸后 附睾的发育。 精子细胞和精子同时具有 法尼基转移酶（Ftase），其在翻译后蛋白中起作用 脂化和信号蛋白Ras，一种FTase底物， 异戊烯化以表达功能。 这一长期目标 他们的建议是确定蛋白质异戊二烯化在精子发育中的作用 并确定异戊二烯化的蛋白质是否被募集到特定的膜 域和调节精子功能。 有三个目标涉及这些目标。 要求1 是确定螯合法尼基转移酶的机制， 精子细胞的特定细胞质区域。 共免疫沉淀 分析将用于确定FTase是否与特异性锚定结合 蛋白质，免疫电子显微镜将用于识别结构 产生限制性FTase分布模式的机制。 目的2 是确定FTase是否在异戊烯化和膜靶向中起作用 精子细胞和附睾精子中的不同蛋白质。 法尼基化的 代谢标记的精子细胞和附睾精子的蛋白质将 通过N-末端微测序和免疫印迹鉴定， 已知异戊二烯化信号蛋白的抗体。 目标3：确定 Ras被募集到结构域特异性信号通路中， 睾丸后精子成熟 免疫定位和免疫印迹 将用于确定Ras是否整合到特定膜中 结构域在精子发育和免疫共沉淀实验 以确定Ras是否与特定于域的 效应蛋白 Ras蛋白-蛋白相互作用的抑制剂将是 测试对特定精子功能的影响。 这些实验将 为精浆的组装机制提供了新的见解， 膜，并可应用于生育调节策略和/或 改进.

DESCRIPTION: (Adapted from applicant's description) The sperm plasma membrane is partitioned into domains of distinct molecular composition and function in fertilization. The assembly of different membrane domains is initiated during spermiogenesis and continues during post-testicular development in the epididymis. Spermatids and spermatozoa possess both farnesyltransferase (Ftase), which functions in post-translational protein lipidation, and the signaling protein Ras, a FTase substrate, which requires prenylation for expression of function. The long range goals of this proposal are to define the role of protein prenylation in sperm development and to determine if prenylated proteins are recruited to specific membrane domains and regulate sperm function. Three aims address these goals. Aim 1 is to determine the mechanisms which sequester farnesyltransferase to specific cytoplasmic regions of spermatids. Co- immunoprecipitation analysis will be used to determine if FTase is bound to specific anchoring proteins, and immunoelectron microscopy will be used to identify structural mechanisms which generate the restricted FTase distribution pattern. Aim 2 is to determine if FTase functions in the prenylation and membrane targeting of distinct proteins in spermatids and epididymal spermatozoa. Farnesylated proteins of metabolically labeled spermatids and epididymal spermatozoa will be identified by N-terminus microsequencing and by immunoblotting with antibodies to known prenylated signaling proteins. Aim 3 is to determine if Ras is recruited into domain specific signaling pathways during post-testicular sperm maturation. Immunolocalization and immunoblotting will be utilized to determine if Ras is integrated into specific membrane domains during sperm development, and co-immunoprecipitation experiments will be performed to determine if Ras interacts with domain-specific effector proteins. Inhibitors of Ras protein-protein interactions will be tested for effects on specific sperm functions. These experiments will provide new insights into mechanisms which assemble the sperm plasma membrane and have application to strategies for fertility regulation and/or improvement.