The long term goal of my laboratory is to define the molecular mechanisms which regulate the biosynthesis and function of lysosomes, intracellular organelles which contain hydrolytic enzymes. These organelles which normally function to degrade extracellular and intracellular material have been implicated in human disease. Over 30 human lysosomal genetic diseases have been described, many resulting from the deficiency of one or more lysosomal enzyme. Other diseases involve general lysosomal dysfunction (e.g. Chediak-Higashi Syndrome). Secretion of lysosomal hydrolases into the external milieu also may be partially responsible for inflammatory diseases (including arthritis) as well as tumor metastasis. In previous studies we have extensively characterized the biosynthesis, transport, modification and proteolytic processing of 3 lysosomal enzymes in the simple eukaryote Dictyostelium discoideum, an organism amenable to these studies because of the ease in which it can be manipulated genetically and biochemically. This organism also lacks the targeting receptors (man-6- phosphate receptors) for lysosomal enzymes found in many mammalian cells, thus it has been a useful system to investigate alternative sorting mechanisms. We now propose to use recombinant DNA techniques to determine the location of the protein domains necessary and sufficient for the targeting of the hydrolases to lysosomes. This will also involve site directed mutagenesis to alter proteinase cleavage sites to determine if the proteolytic processing of lysosomal enzyme precursors is required for proper sorting. We will also use monoclonal antibodies to characterize the biosynthesis and transport of lysosomal membrane proteins. Finally we propose to biochemically and genetically analyze the process of induced lysosomal enzyme secretion which will include dissecting the signal transduction pathway responsible for secretion as well as attempting to define the biochemical defect(s) in a collection of mutants we have isolated which undersecrete enzymes. These combined approaches will increase our knowledge concerning the mechanisms of lysosome biosynthesis and function in all cells.
