IDENTIFICATION OF A 200-KD, BREFELDIN-SENSITIVE PROTEIN ON GOLGI MEMBRANES

A mAb AD7, raised against canine liver Golgi membranes, recognizes a n ovel, 200-kD protein (p200) which is found in a wide variety of cultur ed cell lines. Immunofluorescence staining of cultured cells with the AD7 antibody produced intense staining of p200 in the juxtanuclear Gol gi complex and more diffuse staining of p200 in the cytoplasm. The p20 0 protein in the Golgi complex was colocalized with other Golgi protei ns, including mannosidase II and beta-COP, a coatomer protein. Localiz ation of p200 by immunoperoxidase staining at the electron microscopic level revealed concentrations of p200 at the dilated rims of Golgi ci sternae. Biochemical studies showed that p200 is a peripheral membrane protein which partitions to the aqueous phase of Triton X-114 solutio ns and is phosphorylated. The p200 protein is located on the cytoplasm ic face of membranes, since it was accessible to trypsin digestion in microsomal preparations. and is recovered in approximately equal amoun ts in membrane pellets and in the cytosol of homogenized cells. Immuno fluorescence staining of normal rat kidney cells exposed to the toxin brefeldin A (BFA), showed that there was very rapid redistribution of p200, which was dissociated from Golgi membranes in the presence of th is drug. The effect of BFA was reversible, since upon removal of the t oxin, AD7 rapidly reassociated with the Golgi complex. In the BFA-resi stant cell line PtK1, BFA failed to cause redistribution of p200 from Golgi membranes. Taken together, these results indicate that the p200 Golgi membrane-associated protein has many properties in common with t he coatomer protein, beta-COP.