β-COP localizes mainly to the cis-Golgi side in exocrine pancreas

We examined the distribution of the non-clathrin-coated vesicle-associated coat protein β-COP in rat exocrine pancreatic cells by immunogold cytochemistry. Labeling for β-COP was found in the Golgi region (48%) where it was associated with vesicles and buds of ~50 nm, showing a characteristic ~10-nm-thick coat. The other half of the label was present in the cytoplasm, not associated with visible coats or membranes, with a minor fraction present on small clusters of tubules and vesicles. Clathrin-coated vesicles were typically located at the trans-side of the Golgi complex, and showed a thicker coat of ~18 nm. Of the total β-COP labeling over the Golgi region, 68% occurred on the cis-side, 6% on the cisternae, 17% on the rims of the cisternae, and only 9% on the trans-side. For clathrin these figures were 16, 2, 4, and 78%, respectively. At the cis-Golgi side β-COP was present in transitional areas (TA), on so-called peripheral elements (PE), consisting of tubules and vesicles located between the cup-shaped transitional elements (TE) of the RER and the cis-most Golgi cisternae. Label for Sec23p was also present in TA but was located closer to the TE, while β-COP labeled PE were located near the cis-Golgi cisternae. Upon energy depletion, Golgi associated β-COP was almost exclusively (86%) in spherical aggregates of 200-500 nm in diameter, whereas the cis-side (6%), the cisternae (1%), the rims (4%) and trans-side (3%) of the Golgi complex, were barely labeled; 50% of the total label remained in the cytoplasm. The aggregates were predominantly located at the cis-side of the Golgi stack, next to, but distinct from the Sec23p positive TA, that were devoid of β-COP and had only a few recognizable vesicles left. Incubation with aluminum fluoride resulted in fragmentation of the Golgi complex into large clusters of β-COP positive vesicles, while 50% of the label remained in the cytoplasm, as in control cells. After 10 min of Brefeldin A treatment 91% of β-COP was cytoplasmic and only 7% associated with membranes of the Golgi complex. The total label for β-COP over exocrine cells remained unchanged during the incubation with either of the drugs, indicating that the drugs induce reallocation of β-COP. Our data suggest that β-COP plays a role in membrane transport at the cis-side of the Golgi complex.