9631340 Lilly The overall goal of this research program is to understand the regulation of cellular autolysis in basidiomycete fungi. Under natural conditions, nitrogen is the limiting nutrient in the growth of wood-decaying fungi. The wood-decaying basidiomycete Schizophyllum commune is a tractable model system for the study of autolysis as a response to the limited supply of nitrogen found in wood. Autolysis of older mycelia and subsequent relocation of nutrients to the expanding mycelial margin occur during nitrogen starvation. Increased proteolysis is an integral part of the general autolytic activity. Proteolytically released amino adds have been shown to be incorporated into proteins in the growing mycelial margin. This research focuses on the regulation of proteolytic activity as a model for the regulation of autolysis in general. Two putative vacuolar proteases, ScPrA (a serine protease) and ScPrB (a metalloprotease), appear to be most important in general proteolysis. The research has two main objectives: the first objective is to develop means to bst rigorously the hypothesis that the metalloprotease ScPrB is critical to autolysis and amino acid recycling. ScPrB will be purified and partially sequenced. Using ths sequence inforrnation, the gene for ScPrB wiU be cloned and charactereed. Constructs containing the ScPrB gene inbrrupted by a phleomycin selectable marker gene will be prepared. Gene replacement via transformation of S. commune protoplasts will be used to generate ScPrB mutants. These mutants will be charactereed using molecular techniques. The second objective is to initiate experiments to assess the role of the serine protease ScPrA in nitrogen-limited growth and study its properties. ScPrA is the second most active mycelial protease during nitrogen starvation. ScPrA' will be pyrified and partially sequenced. The sequence data will be used to construct probes for screening S. commune DNA libraries for the ScPrA gene. The demonstration that these enzymes are critical fo r nitrogen-limitation induced proteolysis would provide a model system for studying the regulation of the transition to mycelial autolysis. In addition, creation of mutants lacking these proteases will provide a system for defining the role of the vacuole in many physiological and developrnental processes in filamentous fungi. %%% The wood-decaying fungus Schizophyllum commune is a model system for the study of mycelia growth and development. When nutrients are limited, the older cells in the center of the fungus break down, releasing nutrients that are transported to the cells at the growing margin. This mechanism allows for the continued expansion of the mycelium in the nitrogen-poor environment provided by wood. In this project two major enzymes re'sponsible for breaking down proteins in the older cells will be purified and studied. The gene for one of them will be isolated, and mutant fungi will be constructed with a modified fomm of this gene. These mutants will make it possible to study the role of this enzyme in nutrient recycling. Understanding mechanisms of fungal growth may provide insight into means for control of fungal decay of wood, which has a rnajor economic impact. This project was,submitted under the Research in Undergraduate Institutions program. In addition to its sdentific significance, the research will provide opportunities for experiential leaming and scientific training of undergraduate students. ***
