Our previous work characterized two dominant temperature-sensitive lethal mutants, DTS5 and DTS7, that encode abnormal B-type subunits of the proteasome. The dominant and conditional property of these mutants make them useful tools for examining the role of proteasome-mediated protein degradation in specific cellular and developmental processes. Our previous work also identified and characterized three new a-type subunit genes, a3t, a4t1 and a4t2, that are expressed exclusively in the male germline. This current proposal focuses on investigating the extent of developmentally-regulated proteasome subunit isoforms, and examining in detail the role of these testes-specific a-type subunits in spermatogenesis. A combination of molecular, immunological, and mutational approaches will be used to address the following questions:
-How many proteasome subunits have testes-specific isoforms encoded by distinct genes, and are there other examples of developmentally-regulated 20S subunit genes? -Where and when during spermatogenesis are the testes-specific proteasome subunits expressed? -What is the importance of proteasome-mediated protein degradation in spermatogenesis, and what is the functional role of the testes-specific subunits in this process?
Regulated protein degradation is important for normal cell function, playing a key role in such diverse processes as growth control, metabolic regulation, embryonic development, and cell cycle progression. Targetted protein breakdown is also important for protecting cells against a variety of abnormal conditions. For example, selective proteolysis acts to destroy malformed polypeptides, and it is necessary for intracellular processing of viral proteins so that infected cells can be eliminated by the body's immune system. Thus, an understanding of how cells selectively identify unwanted proteins and target them for destruction has fundamental importance for the study of development, mechanisms of aging, processing of viral antigens, and tumorigenesis. An essential component of intracellular proteolysis is the proteasome, a large, multi-subunit complex that acts as the proteolytic core of the ubiquitin-dependent protein degrading machinery, responsible for the removal of abnormal polypeptides and short-lived regulatory proteins from eukaryotic cells. Recent work, using the tools of biochemistry and cell biology, have revealed much about the physical and biochemical properties of proteasomes, but many aspects of their biological function are unclear. For example, among the fundamental questions that remain to be answered are, "Are there different types of proteasomes, and if so what is the functional significance of this heterogeneity?"; and "What role do proteasomes play in specific cellular or developmental events?" To address these questions, a genetic and molecular analysis of proteasomes in Drosophila melanogaster has been undertaken, and this research is aimed at continuing and extending these studies.
