Role of endoplasmic reticulum-derived vesicles in the formation of Golgi elements in sec23 and sec18 Saccharomyces Cerevisiae mutants.

Background: In the yeast Saccharomyces cerevisiae, the Golgi apparatus consists of individual networks of membranous tubules interspersed throughout the cytoplasm. When sec23 and sec18 mutants are shifted from the permissive (20 degrees C) to the restrictive (37 degrees C) temperature, the secretory pathway is blocked between endoplasmic reticulum (ER) and Golgi elements. When examined with an electron microscope, sec23 displays an excess of ER membranes, whereas sec18 accumulates small vesicles. The present investigation describes the kinetics of the ultrastructural modifications of the Golgi and vesicular elements when sec23 and sec18 mutants are shifted for 10 min to restrictive temperature and then returned to permissive temperature for various time intervals.

Methods: S. cerevisiae sec23 and sec18 mutants from exponentially growing cultures at 20 degrees C were maintained for 10 min at the restrictive temperature of 37 degrees C and returned to the permissive temperature of 20 degrees C for different time intervals. Following fixation in glutaraldehyde and postfixation in potassium ferrocyanide reduce osmium, 80- to 200-nm-thick sections were prepared from Epon-embedded yeast cells. Using the thicker sections, stereopairs of electron microscopy photographs were prepared and used to visualize the three-dimensional configuration of the organelles. To follow the modifications of cell organelles, cell sections were selected at random in thinner sections and cell organelles were scored.

Results: At permissive temperature (20 degrees C), the Golgi apparatus consisted of individual networks of tubules dispersed in the cytoplasm, as in the wild type strain. When both mutants were shifted for 10 min at the restrictive temperature (37 degrees C), the main structural feature was the disappearance of all Golgi networks. In sec23 mutant cells, there was an increase in number of tubular, nonnodular networks corresponding to terminal portions of the endoplasmic reticulum; in sec18 cells, small 20- to 50-nm tubules and vesicles accumulated in the cytoplasm. Within minutes after the return of sec23 cells to permissive temperature (20 degrees C), small vesicles and tubules started to accumulate to reach a number similar or greater than that noted in sec18 cells observed under the same conditions. At later time intervals and in both mutants, the small tubules and vesicles decreased in number. This decrease was concomitant with the reappearance of fine nodular networks, followed later on by the reconstruction of networks of larger caliber and the formation of secretion granules.

Conclusions: It is concluded that a block of the secretory pathway upstream of the Golgi compartment leads to the disappearance of Golgi networks. The small vesicles and tubules that accumulate during the block in sec18 cells and within minutes after the shift at 20 degrees C in sec23 cells appear to fuse together to form new Golgi networks. Thus, the small vesicles would not fuse with a preexisting Golgi apparatus but would rather fuse together to produce new Golgi networks. Such networks appear as transitory structures continuously undergoing renewal.