Loss of epithelial polarity is accompanied by differential association of proteins with intracellular membranes

Cellular membranes play an important role in the formation and maintenance of epithelial polarity, which is lost early during carcinogenesis. We set o ut to identify membrane proteins which are altered during loss of cell pola rity in mammary epithelium. As a model system we used murine mammary epithe lial cells expressing the conditional oncoprotein c-JunER, which induces a reversible loss of polarity upon P-estradiol-driven activation [1]. When gr own either in the absence or presence of hormone, these cells exhibit a pol arized or unpolarized phenotype, respectively. Different membrane fractions of polarized or unpolarized cells were analyzed by two-dimensional electro phoresis (2-DE) and differentially expressed membrane proteins were identif ied. To distinguish between transmembrane orientation and peripheral attach ment of these proteins, were performed extractions with carbonate at high p H or with Triton X-114. In addition, cytosolic proteins of both states were analyzed to investigate their differential association with distinct membr ane fractions. We found ten protein spots preferentially or exclusively in polarized cells and 17 other proteins as being upregulated during loss of p olarity. Some of the peripheral membrane proteins were identified by micros equencing. The resident Golgi protein nucleobindin and fructose-bisphosphat e aldolase were preferentially associated with membranes of polarized cells , whereas alpha B crystallin was detected exclusively and in high amounts i n unpolarized cells.