PROJECT SUMMARY Lysosomes are the primary catabolic site of cells, serving to degrade extracellular material internalized by en- docytosis and intracellular components earmarked for turnover. These items and the hydrolytic enzymes that degrade them are delivered to lysosomes by multiple membrane trafficking pathways, which are operated by cellular protein machineries that are evolutionarily conserved from yeast to man. Mutations that disrupt the ac- tivities of these machineries are linked to genetic diseases, and pathogens exploit these trafficking pathways to establish infection. Many of the molecular mechanisms that operate membrane trafficking machineries are un- known. My lab addresses fundamental questions about membrane trafficking to lysosomes using the budding yeast Saccharomyces cerevisiae as a genetically tractable model organism. The major questions we plan to address over the next five years include the following. 1) What are the physiological mechanisms that regulate the trafficking of cell-surface receptors to the hydrolytic interior of the lysosome? This trafficking pathway func- tions at endosomes to sort endocytosed receptors into membrane-enclosed transport vesicles that are deliv- ered into the lysosome lumen. Recent progress from my lab has revealed that the formation of these vesicles is coordinated with other processes that are vital to cellular physiology, including ubiquitin protein homeostasis and endocytic pH regulation. We plan to identify the machineries that interface these different cellular systems and determine how they exert control over the protein machinery that mediates the formation of vesicles trans- ported to the lysosome lumen. These results will define ways in which receptor degradation is coordinated with cellular physiology. 2) What are the mechanisms that mediate the delivery of newly synthesized transmem- brane proteins that are destined to function at the lysosomal membrane? Using novel genetic tools we created for discovery and diagnostics, we recently identified two specific cellular machineries and several additional candidate machineries that function in this trafficking pathway. We plan to define the mechanisms by which these machineries function in transport, which will establish fundamental principles that underly the biogenesis of lysosomes. Our work is bolstered by ongoing productive collaborations that employ diverse interdisciplinary techniques, including high-resolution microscopy, biophysical analyses of protein assemblies reconstituted in vitro, proteomics, and genetic screening. Moving forward, our overall vision is to continue exploiting yeast for discovering and mechanistically understanding membrane trafficking to lysosomes while also addressing the extent to which these mechanisms are conserved in human cells. The information gained from this work will provide an understanding of how membrane trafficking pathways to lysosomes operate under normal physio- logical conditions and how ...