This proposal addresses the mechanism, at the molecular level, underlying the secretion of milk-fat droplets from mammary epithelial cells during lactation. Fat droplets assembled in the cytoplasm are released from the surface of secretory cells coated with an outer layer of membrane. This layer gives rise to the fat- globule membrane (FGM) on the surface of fat droplets in milk. The proposed experiments will test the hypothesis that butyrophilin, the major protein of FGM, functions as a specific receptor for cytoplasmic fat droplets, and that the budding of fat droplets from the apical surface occurs through the progressive cross-linking of butyrophilin molecules to each other and to other FGM proteins via disulfide bonds. It will be determined if butyrophilin is enriched in the FGM relative to the apical plasma membrane and becomes cross-linked by disulfide bonds in the cytoplasmic domain during formation of FGM. The butyrophilin gene will be inserted into cells in culture, and into mice, to see whether butyrophilin associates with fat droplets in cells other than mammary secretory cells. Butyrophilin gene expression will be eliminated in transgenic mice to test whether milk-fat secretion is inhibited in lactating animals. %%% Exocrine secretion (cellular secretion which proceeds via exocytosis of secretory content) has become an extremely well studied aspect of cell biology, and tremendous breakthroughs in our understanding of exocytotic secretion have been and continue to take place. In contrast, apocrine secretion (secretion which proceeds by the "budding off" of apical cytoplasmic content), as typified by milk fat globule secretion, has received relatively little attention in recent years. This proposal promises to increase our understanding of the molecular mechanisms involved in milk fat secretion. In addition to contributing to fundamental knowledge of cellular processes, a better understanding of milk fat secretion will have great significance for potential biotechnological applications, such as the use of transgenic bovine milk as a source of commercially useful membrane proteins.
