EFFECTS OF BREFELDIN-A ON THE 3-DIMENSIONAL STRUCTURE OF THE GOLGI-APPARATUS IN A SENSITIVE STRAIN OF SACCHAROMYCES-CEREVISIAE

Background: Brefeldin A (BFA), when added to the medium of cultured ma mmalian cells, induces a reversible block of secretion and disrupts th e Golgi apparatus whereas Golgi enzyme markers appear to redistribute into the endoplasmic reticulum (ER). It has been shown in addition tha t in mammalian cells, BFA would prevent the assembly of coatomer prote ins (COP) onto membranes by inhibiting the GTP-dependent interaction o f the ADP-ribosylation factor (ARF) with such membranes. The purpose o f the present study is to analyze, by stereoelectron microscopy, the s tructural modifications of Golgi elements and of the ER-Golgi relation ship in a BFA-sensitive yeast mutant, S. cerevisiae erg6. Methods: S. cerevisiae erg6 cells were placed in a medium containing 100 mu g/ml B FA dissolved in 1% alcohol and collected after exposures of 0.5, 1.5, 5, 10, 15, 20, 30, and 70 min to the drug. Yeasts placed in a BFA-free medium but containing 1% alcohol served as controls. After fixation i n 2% glutaraldehyde, the cells were postfixed in reduced osmium and em bedded in Epon. Then 0.08-0.2 mu m thick sections stained with lead ci trate were examined with the electron microscope. Photographs of the t hicker sections, tilted at +/-15 degrees from the 0 degrees position o f the goniometric stage, were used to prepare stereopairs from which t he three-dimensional configuration of the organelles was visualized. S ince BFA is known to prevent the interaction of ARF with membranes, th e phenotype of the arf1 mutant deficient in this protein was also exam ined for comparative purposes. Results: In control cells, as in wild-t ype strains, two types of Golgi elements were observed: small networks of fine tubules seen close and occasionally connected to ER cisternae and coarser tubular networks showing nodular distensions having a siz e comparable to that of secretion granules. The latter networks were c onsidered as trans-Golgi elements and the former as cis-Golgi elements . Several networks of both types were distributed throughout the cytop lasm. At short time intervals (0.5-5 min) of BFA treatment, the trans- Golgi elements disappeared from the cytoplasm, while the ER-connected cis-Golgi elements developed and formed large spheroidal masses freque ntly showing concentrically arranged fine tubular networks. Such spher oidal, cage-like structures later disappeared, and after 30 min Golgi elements were no longer identifiable, while ER cisternae assumed pleom orphic configurations as the cells showed signs of degeneration. S. ce revisiae arf1 mutants presented a phenotype similar to that of BFA-tre ated S. cerevisiae erg6. Conclusions: It is therefore concluded that s oon after exposure to BFA there is, in this sensitive yeast mutant, a transitory hypertrophy of the ER-connected cis-Golgi network presumabl y resulting from a block at the exit end of this compartment. At longe r time intervals (i.e., after 30 min) the Golgi elements are no longer formed, and the cells present signs of cell degeneration. (C) 1995 Wi ley-Liss, Inc.