THE PATHWAY OF GOLGI CLUSTER FORMATION IN OKADAIC ACID-TREATED CELLS

Using stereology and immunoelectron microscopy we examined the pathway of Gels cluster formation during treatment with the phosphatase inhib itor okadaic acid, During the first hour the Golgi stack of suspension HeLa cells lost 90% of its membrane without appreciable reduction in the number of cisternae. During this time clusters of tubules and vesi cles (Golgi clusters) appeared and these contained only a fraction of the Golgi membrane present in untreated cells. Despite the overall red uction in membrane the fetal amount of immunolabeling for galactosyltr ansferase over the Gels clusters of a typical cell was maintained, ind icating that galactosyltransferase had been retained in Golgi membrane s. The observation that, after 40 min okadaic acid treatment, labeling density for galactosyltransferase within trans Gels cisternae increas ed 1.6-fold (n = 3, CE 10%) suggests that membrane loss from trans cis ternae was selective. Careful evaluation of immunolabeled clusters sho wed that most of the galactosyltransferase labeling was located over c omplex tubular profiles and not vesicular profiles. Tubular structures were also observed during disassembly and these were found both conne cted to disassembling cisternae and within forming Golgi clusters, ind icating that they were intermediates in cluster formation. We also inv estigated the role of vesicular transport in cluster formation, During disassembly we found no accumulation of COP-coated buds and vesicles over Golgi membrane. However, aluminium fluoride, previously found to arrest transport in the Golgi stack, completely inhibited membrane dep letion and stack disassembly, Taken together, our results indicate tha t during Gels cluster formation, membrane leaves the Golgi but galacto syltransferase is retained within a tubular reticulum which is a direc t descendant of trans-Golgi cisternae. Membrane depletion may require ongoing vesicular transport and we postulate that it arises because of an imbalance in membrane traffic into and out of the Golgi apparatus, (C) 1995 Academic Press, Inc.