Alveolar macrophages play a major role in pulmonary homeostasis by clearing injurious agents from the lung. Macrophages remove these agents by binding and internalizing them. Derangements in the ability of macrophages to mediate disposal of these agents results in a compromise of normal lung function. We propose to examine the mechanisms involved in internalization and in the intracellular pathway taken by internalized molecules and bacteria. Conjugation of the enzyme horseradish peroxidase to specific ligands provides a biochemical tool to probe the intracellular endocytic pathway. Using this tool to identify specific intracellular compartments, and antibodies in conjunction with western blots to identify specific enzymes and ligands, we will determine if early endocytic vesicles contain proteases capable of degrading ligands, and if the selective removal of the Na+K+ ATPase from late endocytic compartments is responsible for endosomal pH regulation. To determine if early endosomes transfer material to late endosomes by shuttle vesicles or by a developmental maturation of the early endosome, we have devised a procedure by which vesicle development can be synchronized and the properties of the intermediates examined. We will use a monoclonal antibody approach and an in vitro endosome fusion assay to define molecules present on endosomes which are responsible for selective vesicle targeting. To examine what membrane proteins recycle from late endocytic compartments and the routes taken by these proteins we will label membrane proteins in vivo using a special class of identifiable photoaffinity reagents. We propose procedures designed to examine the behavior of plasma membrane proteins that get trapped in late endosomal compartments. This will allow us to test the hypothesis that the rate limiting step in plasma membrane protein turnover is not internalization but entry of the internalized molecule into late endosomes. A major class of agents internalized by macrophages are bacteria, some of which have managed to subvert the normal intracellular traffic and live within cells as intracellular parasites. We propose the hypothesis that Chlamydia resides as an intracellular parasite because they are internalized by receptors which, while cap-able of binding actin, lack lysosomal targeting sequences on their cytosolic domains. To examine this hypothesis we propose to isolate cellular proteins responsible for Chlamydial binding and examine their properties. We will also determine the source of membrane comprising the growing Chlamydial inclusion body. Our hypothesis is that proteins in this membrane are derived from the bacteria rather than the host. These studies will yield insight into the normal mechanisms of macrophage activities and into derangements which may compromise lung function.
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