REGULATION OF SPERM FUNCTION BY PROTEIN PRENYLATION

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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.