MODIFICATIONS OF THE GOLGI-APPARATUS IN SACCHAROMYCES-CEREVISIAE LACKING MICROTUBULES

Background: Disassembly of cytoplasmic microtubules by nocodazole in c ultured mammalian cells leads to the disruption of the continuous ribb onlike Golgi apparatus and dispersal of the Gels elements from their n ormal juxtanuclear location, close to the microtubule-organizing cente r (MTOC), toward the cell periphery. Clearing of the drug induces reas sembly of the microtubules from the MTOC and reorganization of the Gol gi elements into a continuous ribbonlike juxtanuclear structure. In th e yeast Saccharomyces cerevisiae, the Golgi apparatus does not form a continuous structure as in mammalian cells but instead constitutes ind ependent units dispersed throughout the cytoplasm. It is the purpose o f this article to investigate the role of microtubules in the structur e and distribution of the Golgi elements in S. cerevisiae by studying the ultrastructure of cell organelles either in mutant cells deficient in beta-tubulin or in wild-type cells treated with the microtubule-de polymerizing drug nocodazole. Methods: Two S. cerevisiae yeast strains were used in this study: a control wild-type strain, CUY226 (ade2-101 , his3-Delta 200, leu2-Delta 1, lys2-801, ura3-52 Mat alpha), and a mu tant strain, CUY66 (tub2-401, ade2-101, ura3-52, Mat alpha). Nocodazol e was added to the wild-type cells cultivated at 30 degrees C, and cel ls were fixed 5 min, 20 min, and 60 min, respectively, after adding th e drug to the culture. Both strains were fixed and examined 5 min, 20 min, and 60 min after shifting the cultures from the permissive temper ature of 30 degrees C to the restrictive temperature of 14 degrees C. Cells were fixed in 2% glutaraldehyde, treated for 15 min in 1% sodium metaperiodate, postfixed in reduced osmium, and embedded in Epon. To visualize the three-dimensional configuration of cell organelles, ster eopairs were prepared from sections stained with lead citrate and tilt ed at +/-15 degrees from the 0 degrees position of the goniometric sta ge of the electron microscope. Results: In mutant cells shifted to res trictive temperature and wild-type cells treated with nocodazole, the main ultrastructural modification was a fragmentation of networks of m embranous tubules, which probably correspond to the yeast Gels apparat us. Secretion granules were still present in growing buds, and they we re dispersed in the cytoplasm, which contained in addition numerous sm all vesicles in the 30-60-nm diameter range. Conclusions: In normal ce lls, small vesicles may originate from the endoplasmic reticulum and f use together to give rise to Gels networks (Rambourg et al. 1994. Anat . Rec., 240:32-41). If this hypothesis is correct, the observations re ported might indicate that intact microtubules orient the flow of smal l vesicles and favor their fusion into Golgi networks. (C) 1996 Wiley- Liss, Inc.