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

Background: In early descriptions of ultrastructural alter- ations of secretory (sec) mutants of the yeast Saccharomyces cereuisiae, two mutants, sec7 and secl4, were shown to produce cell structures, the so- called Berkeley bodies thought at first to correspond to Golgi structures. In sec7 mutants grown at restrictive temperature, secretion granules soon dis- appeared, whereas networks of Golgi tubules increased in size and trans- formed into stacks of seven to eight flattened elements. At these time inter- vals, structures resembling Berkeley bodies appeared to be extensions of the endoplasmic reticulum (Rambourg et al., 1993). It is the purpose of the present study to examine by electron microscopy S. cereuisiae secl4 mu- tants and to compare the modifications along their secretory pathway with those occurring in a homologous mutant of Yarrowia lipolytica. Methods: S. cereuisiae sec 14 mutant cells coming from exponentially growing cultures were examined either at 24°C or after shifting at 37°C for 0,2, 5, 10, 15,20, 30, 45, 60, 90, and 120 min. Y. lipolytica mutant cells were first cultured in 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 differentiate from yeast to mycelian form. In both cases, cells were fixed in 2% glutaraldehyde, treated for 15 min in 1% sodium metaperiodate, post- fixed in reduced osmium, and embedded in Epon. To visualize the three- dimensional configuration of cell organelles, stereopairs were prepared from sections stained with lead citrate and tilted at 2 15" from the 0" posi- tion of the goniometric stage of the electron microscope. Results: In S. cereuisiae mutant cells shifted for 2 min at the restrictive temperature, faintly stained networks of thin anastomosed tubules were located at close proximity and often continuous with faintly stained ER cisternae. More intensely stained tubular networks with nodular dilations having the size of secretion granules were dispersed throughout the cyto- plasm. Later on, the faintly stained ER elements and related tubular net- works decreased in number, whereas the intensely stained nodular tubular networks increased in frequency. The incidence of secretion granules also increased and were distributed at random throughout the cytoplasm. Wide- meshed, intensely stained fenestrated spheres were often encountered and increased in number, in parallel to the increase in the number of nodular tubular networks. At late time intervals, the fenestrated spheres decreased in number as they seemingly transformed into spherical bodies identical to vacuoles. In contrast to what occurred in S. cereuisiae secl4 mutant, the main ultrastructural modification observed in Y. lipolytica transferred to the YPD medium was the formation of deep plasma membrane invagina- tions. Conclusions: It appears that two functionally homologous PIPC transfer