Eukaryotic cells contain a variety of discrete membrane-enclosed organelles. This highly compartmentalized organization is essential to the normal functioning of the cell. Each of these subcellular compartments has unique structural and functional characteristics which are conferred by particular proteins, lipids and/or carbohydrates that comprise them. The lysosome is the major organelle responsible for intracellular degradation in mammalian cells. It contains numerous soluble hydrolases as well as a variety of membrane-associated proteins. Tay Sachs disease, pseudo-Hurler polydystrophy and I cell disease are three of over thirty human genetic disorders which are known to result from the absence of certain proteins in the lysosome. Many of these diseases specifically result from the improper sorting of these proteins. This correlation between sorting defects and disease states underscores the importance of correct protein sorting in cell physiology. The investigators long-term goal is to develop a precise understanding of the molecular events involved in the recognition, sorting and transport of proteins to the lysosome-like vacuole in yeast cells. The investigator will use yeast as a model system to study these problems since the pathways used for protein transport appear to be very similar to those in animal cells, and in addition yeast are amenable to useful genetic approaches which are not as applicable to higher eukaryotes. In this proposal, the investigators will focus on analyzing factors involved in delivery of membrane-associated vacuolar proteins. Initial studies of alkaline phosphatase (ALP) indicate that it is a membrane protein that is delivered to the vacuole by a mechanism that is at least in part different from that used by soluble vacuolar hydrolases. Furthermore, the spatial location of the ALP targeting signal suggests that differences in sorting may reflect an interaction with unique sorting components such as a specific receptor. The investigator will further characterize the vacuolar sorting signal in ALP and will attempt to identify the ALP receptor through a combination of biochemical and genetic approaches. In addition, the investigator will utilize a gene fusion approach to select mutants which are defective in the localization of this protein. These mutants should allow the investigator to define components of the sorting and transport apparatus that recognize and target this protein to the vacuole membrane. These new mutants will be compared with previously identified mutants which missort soluble vacuolar hydrolases. An identification of the sorting determinant in ALP coupled with a characterization of the genes involved in its recognition and delivery should further our understanding of the transport process.
