Mechanisms Controlling Organelle Acidification Along the Regulated Secretory Pathway

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The eukaryotic cell is exquisitely compartmentalized into membrane-enclosed spaces that provide sequestered microenvironments particularly suited for various metabolic functions. In particular, one aspect of these microenvironments within the subcellular compartments is the pH: for example, some compartments, such as lysosomes, are extremely acidic, while others, such as the endoplasmic reticulum, provide a pH environment close to neutral. Some of these compartments are part of the dynamic membrane trafficking system of the inter-related secretory and endocytic pathways, which are involved in the processing and secretion of proteins and other macromolecules synthesized by the cell and the uptake of proteins and hormones from the cell's surface and its environment. A key feature of this inter-related set of trafficking pathways is the differences in pH among the various compartments. Acidification of the exocytic and endocytic pathways controls a variety of cellular processes, including viral entry, protein sorting, and proteolytic processing. A specific example of this generalization, which forms the model system on which this project is based, is the regulated secretory pathway for the peptide hormone ACTH (adrenocorticotropic hormone). Here, the establishment of distinct ionic milieus within individual compartments is essential for proper sorting and processing of the various peptide hormones and their precursor molecules. For example, the trans-Golgi network (TGN) maintains a lumen that is acidic enough for sorting of regulated secretory products, and yet alkaline enough to prevent premature activation of prohormones and lysosomal enzymes. Upon budding from the TGN, secretory granules (SG's) acidify further to allow processing of the packaged prohormones. Thus, molecular mechanisms must be in place to ensure that the TGN and SG each maintains its unique level of acidity. How this is accomplished remains mysterious. A major difficulty has been the lack of a systematic method to study acidification of individual organelles along the regulated secretory path. The goal of this project is to use a novel fluorescence targeting method to study the regulatory mechanisms that control acidification of the secretory pathway. This elegant but technically challenging method, which involves genetic engineering and novel pH-sensitive fluorescent dyes, is expected to allow the systematic introduction of pH-sensitive dyes into specific compartments such that the pH dynamics of each organelle can be measured in living cells. The mouse pituitary cell line AtT-20 will be used as a model system because of its well characterized regulated secretory pathway and the role of organelle acidification in prohormone processing. The fluorescence targeting method will be used to determine the pH values of individual compartments along the regulated secretory pathway. The mechanisms that maintain individual organelles at their respective pH's will be elucidated. In particular, Drs. Moore and Machen will test the hypothesis that progressive acidification along the secretory pathway is generated by iterative retention of proton leaks within the earlier compartments.