Effects of brefeldin A on the three-dimensional structure of the Golgi apparatus in a sensitive strain of Saccharomyces cerevisiae.

BACKGROUND

Brefeldin A (BFA), when added to the medium of cultured mammalian cells, induces a reversible block of secretion and disrupts the Golgi apparatus whereas Golgi enzyme markers appear to redistribute into the endoplasmic reticulum (ER). It has been shown in addition that in mammalian cells, BFA would prevent the assembly of coatomer proteins (COP) onto membranes by inhibiting the GTP-dependent interaction of the ADP-ribosylation factor (ARF) with such membranes. The purpose of the present study is to analyze, by stereoelectron microscopy, the structural modifications of Golgi elements and of the ER-Golgi relationship in a BFA-sensitive yeast mutant, S. cerevisiae erg6.

METHODS

S. cerevisiae erg6 cells were placed in a medium containing 100 micrograms/ml BFA 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 in 2% glutaraldehyde, the cells were postfixed in reduced osmium and embedded in Epon. Then 0.08-0.2 microns thick sections stained with lead citrate were examined with the electron microscope. Photographs of the thicker sections, tilted at +/- 15 degrees from the 0 degree position of the goniometric stage, were used to prepare stereopairs from which the three-dimensional configuration of the organelles was visualized. Since BFA is known to prevent the interaction of ARF with membranes, the phenotype of the arf1 mutant deficient in this protein was also examined for comparative purposes.

RESULTS

In control cells, as in wild-type 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 size comparable to that of secretion granules. The latter networks were considered as trans-Golgi elements and the former as cis-Golgi elements. Several networks of both types were distributed throughout the cytoplasm. 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 frequently showing concentrically arranged fine tubular networks. Such spheroidal, cage-like structures later disappeared, and after 30 min Golgi elements were no longer identifiable, while ER cisternae assumed pleomorphic configurations as the cells showed signs of degeneration. S. cerevisiae arf1 mutants presented a phenotype similar to that of BFA-treated S. cerevisiae erg6.

CONCLUSIONS

It is therefore concluded that soon after exposure to BFA there is, in this sensitive yeast mutant, a transitory hypertrophy of the ER-connected cis-Golgi network presumably resulting from a block at the exit end of this compartment. At longer time intervals (i.e., after 30 min) the Golgi elements are no longer formed, and the cells present signs of cell degeneration.