Establishment of plasma membrane polarity in mammary epithelial cells correlates with changes in prolactin trafficking and in annexin VI recruitment to membranes

Mammary epithelial cells (MEC) of lactating animals ferry large amounts of milk constituents in vesicular structures which have mostly been characteri zed by morphological approaches (Ollivier-Bousquet, 1998). Recently, we hav e shown that under conditions of lipid deprivation, perturbed prolactin tra ffic paralleled changes in the membrane phospholipid composition and in the cytosol versus membrane distribution of annexin VI (Ollivier-Bousquet et a l., 1997). To obtain additional information on the membrane events involved in the vesicular transport of the hormone to the apical pole of the cell, we conducted a biochemical study on prolactin-containing vesicles in MEC at two different stages of differentiation. We first showed that MEC of pregn ant and lactating rabbits exhibited membrane characteristics of non-polariz ed and polarized cells respectively, using annexin IV and the alpha-6 subun it of integrin as membrane markers. Incubation of both cell types with biot inylated prolactin for 1 h at 15 degrees C, followed by a 10-min chase at 3 7 degrees C revealed that prolactin transport was activated upon MEC membra ne polarization. This was confirmed by subcellular fractionation of prolact in-containing vesicles on discontinuous density gradients. In non-polarized MEG, I-125-prolactin was mainly recovered in gradient fractions enriched w ith endocytotic Vesicles either after incubation at 15 degrees C or after a 10-min chase at 37 degrees C. In contrast, in polarized MEG, the hormone s witched from endocytotic compartments to a fraction enriched in exocytotic clathrin-coated vesicles during the 10-min chase at 37 degrees C. Associati on of annexin VI to prolactin carriers was next studied in both non-polariz ed and polarized cells. Membrane compartments collected at each gradient in terface were solubilized under mild conditions by Triton X-100 (TX100) and the distribution of annexin VI in TX100-insoluble and TX100-soluble fractio ns was analyzed by Western blotting. Upon MEC polarization, the amount of a nnexin VI recovered in TX100-insoluble fractions changed. Quite interesting ly, it increased in a membrane fraction enriched with endocytotic clathrin- coated vesicles, suggesting that annexin VI may act as a sorting signal in prolactin transport. (C) 2000 Elsevier Science B.V. All rights reserved.