Proteomic analysis of secretory mechanisms in the rat hepatocyte and mammary epithelial cell.

摘要

Proteomics is the study of the protein complement of a defined biological structure. My focus has been to use a global proteomic approach to analyze and identify the total protein complement of the Golgi complex in two epithelial cell types, the hepatocyte and the mammary epithelial cell. The overall goal was to elucidate novel secretory mechanisms of the organelle. Initial proteomic studies were conducted in hepatocytes to optimize conditions for analyses and to identify unknowns (Taylor, R. S., Wu, C. C., Hays, L. G., Eng, J. K., Yates, J. R., K. E. Howell. 2000. Electrophoresis 21:3441–3459). Later, comparative studies were carried out in mammary epithelial cells during the developmental transition of Stage II lactogenesis to look at differential expression during the functional transition of the Golgi complex from basal secretion (late pregnancy) to maximal secretion (mid-lactation).; The proteomic analyses of rat liver Golgi fractions yielded numerous unknown proteins. One of these, GMx33α, was shown to be a novel highly regulated cytosolic protein of 33 kD localized mainly to the surface of the trans-face of the Golgi complex at steady state (Wu, C. C., Taylor, R. S., Lane, D. R., Ladinsky, M. S., Weisz, J. A., and K. E. Howell. 2000. Traffic 1:963–975).; Comparative proteomic analyses of enriched Golgi fractions from two distinct functional states of the Golgi complex from mammary epithelial cells isolated from pregnant (basal secretion) and lactating (maximal secretion) rats allowed for the identification of differentially expressed Golgi proteins (Wu, C. C., Yates, J. R., Neville, M. C., and K. E. Howell. 2000. Traffic 1 :769–782). Additional studies were conducted to determine the origin of the milk fat globule membrane by comparing the proteome of cytoplasmic lipid droplets fractionated from lactating mouse mammary epithelial cells to the proteome of the secreted milk fat globule membranes fractionated from expressed mouse milk (Wu, C. C., Howell, K. E., Neville, M. C., Yates, J. R., J. L. McManaman. 2000. Electrophoresis 21:3480–3482).; Together, these studies have (1) generated numerous preliminary observations to fuel future projects involving secretory mechanisms, (2) have provided data to support a novel mechanistic integration of Golgi function into the classical constitutive secretory pathway, and (3) demonstrated the power and applicability of proteomics as a cell biology tool.