The main purpose of this study is to understand how chitosomes (chitin synthetase microvesicles) operate in vivo and the mechanism by which cell wall chitin microfibrils are synthesized by a growing fungus. Our previous studies have demonstrated that a major part of the chitin synthetase in fungi is present in the form of chitosomes. These microvesicles are believed to move to the cell surface where their chitin synthetase zymogen becomes activated (probably by proteolysis) and where they synthesize chitin by a mechanism whose precise site and details are still unknown. A major point to investigate is whether or not chitosomes fuse with the plasma membrane and thereby chitin synthetase becomes an integral part of this membrane. We plan to trace the intracellular origin and fate of chitosomes and to explore the interaction of chitosomes and plasma membrane vesicles in vitro. For this reason, we will endeavor to develop critical procedures to isolate plasma membrane samples of highest purity from growing, walled cells of fungi. Two fungi will be compared. In addition to Mucor rouxii, our main test organism, we will also examine Neurospora crassa, mainly because of its proven suitability for plasma membrane research. The cellular localization of chitin synthetase will be traced by fractionation of cell-free extracts and by means of gold-or ferritin-labelled anti-chitin synthetase antibodies. Through these studies we hope to unravel the mechanism of chitin fibril assembly in fungal walls. Chitin is not the only polymer made by chitin synthetase in Mucor rouxii; we have found that this fungus makes chitosan through a coordinated glycosyl transfer and deacetylation (via chitin synthetase and chitin deacetylase.) Another major goal of this study will be to further understand details of the coordinated action that produces this important wall polymer. This study is part of a long-standing program to elucidate the biochemical bases of morphogenesis of Mucor.
