TRANSFORMATIONS OF MEMBRANE-BOUND ORGANELLES IN SEC14 MUTANTS OF THE YEASTS SACCHAROMYCES-CEREVISIAE AND YARROWIA-LIPOLYTICA

Background: In early descriptions of ultrastructural alterations of se cretory (sec) mutants of the yeast Saccharomyces cerevisiae, two mutan ts, sec7 and sec14, were shown to produce cell structures, the so-call ed Berkeley bodies thought at first to correspond to Golgi structures. In sec7 mutants grown at restrictive temperature, secretion granules soon disappeared, whereas networks of Golgi tubules increased in size and transformed into stacks of seven to eight flattened elements. At t hese time intervals, structures resembling Berkeley bodies appeared to be extensions of the endoplasmic reticulum (Rambourg et al., 1993). I t is the purpose of the present study to examine by electron microscop y S. cerevisiae sec14 mutants and to compare the modifications along t heir secretory pathway with those occurring in a homologous mutant of Yarrowia lipolytica. Methods: S. cerevisiae sec 14 mutant cells coming from exponentially growing cultures were examined either at 24 degree s C or after shifting at 37 degrees C for 0, 2, 5, 10, 15, 20, 30, 45, 60, 90, and 120 min. Y. lipolytica mutant cells were first cultured i n YNB in 5000 medium and then transferred for 0, 6, 8, 12, 20, and 24 hr, in a phosphate-buffered YPD medium, which allows wild cells to dif ferentiate from yeast to mycelian form. In both cases, cells were fixe d in 2% glutaraldehyde, treated for 15 min in 1% sodium metaperiodate, postfixed in reduced osmium, and embedded in Epon. To visualize the t hree-dimensional configuration of cell organelles, stereopairs were pr epared from sections stained with lead citrate and tilted at +/-15 deg rees from the 0 degrees position of the goniometric stage of the elect ron microscope. Results: In S. cerevisiae mutant cells shifted for 2 m in at the restrictive temperature, faintly stained networks of thin an astomosed tubules were located at close proximity and often continuous with faintly stained ER cisternae. More intensely stained tubular net works with nodular dilations having the size of secretion granules wer e dispersed throughout the cytoplasm. Later on, the faintly stained ER elements and related tubular networks decreased in number, whereas th e intensely stained nodular tubular networks increased in frequency. T he incidence of secretion granules also increased and were distributed at random throughout the cytoplasm. Wide-meshed, intensely stained fe nestrated spheres were often encountered and increased in number, in p arallel to the increase in the number of nodular tubular networks. At late time intervals, the fenestrated spheres decreased in number as th ey seemingly transformed into spherical bodies identical to vacuoles. In contrast to what occurred in S. cerevisiae sec14 mutant, the main u ltrastructural modification observed in Y. lipolytica transferred to t he YPD medium was the formation of deep plasma membrane invaginations. Conclusions: It appears that two functionally homologous PI/PC transf er proteins (Sec14p(sc) and Sec14p(yl)) control distinct physiological processes in the two sec14 mutants examined. Such differences are per haps related to the regulatory role of these proteins in different tar get organelles, i.e., the Golgi apparatus in S. cerevisiae or the plas ma membrane in Y. lipolytica. (C) 1996 Wiley-Liss, Inc.