Molecular Genetic Analysis of Drosophila Proteasomes

Syracuse University, Syracuse NY

Investigators

Abstract

Regulated protein degradation is important for normal cell function, playing key roles in such diverse processes as growth control, metabolic regulation, embryonic development, neurotransmission, long-term memory, programmed cell death, and cell cycle progression. Targeted protein breakdown is also important for protecting cells against the potentially harmful effect of accumulated damaged or malformed polypeptides. Thus, an understanding of how cells selectively identify unwanted proteins and target them for destruction has fundamental importance for the study of eukaryotic development, cellular metabolism, and cell fate determination. An essential component of this pathway is the proteasome, a large, multi-subunit complex that acts as the proteolytic "machine" that removes abnormal polypeptides and short-lived regulatory proteins from cells. Previous work, using the tools of biochemistry and cell biology, has revealed much about the physical and biochemical properties of proteasomes, but many aspects of their biological function remain unclear. As an approach toward learning more about their role during development, a genetic and molecular study of Drosophila proteasomes is being pursued. This project is focused on pursuing the recent identification of a number of proteasome subunit genes that are expressed exclusively in the testis during the mid- to late stages of spermatogenesis. This observation represents the most striking example of developmentally regulated proteasome gene expression in any system described to date. Of prime interest is determining the role of these testis-specific proteasomes during sperm differentiation and/or function. To address this question a mutational approach is being used. Mutations that disrupt a number of the testis-specific proteasome genes will be created using targeted gene replacement methods. In addition, P-element insertional mutations in some of the male-specific proteasome genes will be characterized. The mutant phenotypes will be analyzed to determine how the mutations affect spermatogenesis and/or sperm function. All features of an animal cell's structure and function are determined by the collection of proteins that it contains, and the composition of that collection is a consequence of the balance between protein synthesis and degradation. While much is known about the regulation of protein synthesis, knowledge about the mechanisms controlling protein destruction is much more limited. It is now clear that a remarkable molecular machine known as the proteasome is responsible for most regulated protein degradation that occurs during the life of a cell. This project uses the fruit fly, Drosophila melanogaster, as the model system to address questions about the proteasome's role during the development of a specialized cell-type, the sperm cell. The existence of a structurally distinct proteasome in developing sperm cells suggests that proteasome-mediated protein degradation plays an important role in these cells, and this research is aimed at using genetic mutations to investigate what that role is.