Alveolar macrophages play a major role in pulmonary homeostasis by clearing injurious agents from the lung. Macrophages removes these agents by internalizing them and directing them to lysosomes where they are sequestered and degraded. Derangement's in vesicle traffic or lysosome functions result in a compromise of normal lung function. It is proposed to examine the mechanisms involved in intracellular vesicle traffic and lysosome function. The investigators demonstrated extensive lysosome-lysosome-fusion using both in vivo and in vitro assays. They demonstrated that a heterotrimeric G-protein, tentatively identified as a G-alpha-i is required from homotypic lysosome fusion. They now propose to determine the role of this G-protein plays in both the specificity and mechanism of lysosome fusion. In addition they will determine the factors that allow an endosome to gain the ability to fuse with lysosomes. This will be accomplished by examining the properties of endosomes that do or do not show fusion with lysosome. These endosomes are obtained using a unique procedure that synchronizes endosome formation and movement. The investigators have cloned the gene responsible of the beige/Chediak-Higashi Syndrome. This autosomal recessive diseases results in a phenotype which affects lysosomes, cytolytic granules, melanosomes, and platelet dense granules. Antisera against the Beige/Chediak protein will be used to probe the function of the gene product and its subcellular location. They developed procedures to measure lysosome size. Using these procedures and in vitro fusion assay they will determine whether the CHS/beige protein affects fusion or fission. These assays will also be used to define the biochemical function of this protein. The investigators have also identified a gene, VPS 41, in yeast that affects vacuole biogenesis, mutations give rise to many small vacuoles. They cloned this gene and its human and plant homologues. The yeast protein is involved in transfer of lysosomal enzymes from the Golgi to the late endosome. Antibodies prepared to the yeast or human gene will be used to defines its location and function. These studies will yield insight into the mechanisms of lysosome biogenesis and the molecules that regulate endocytic traffic and vesicle size.
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