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 th roughout the cytoplasm. When sec23 and sec18 mutants are shifted from the permissive (20 degrees C) to the restrictive (37 degrees C) temper ature, the secretory pathway is blocked between endoplasmic reticulum (ER) and Golgi elements. When examined with an electron microscope, se c23 displays an excess of ER membranes, whereas sec18 accumulates smal l vesicles. The present investigation describes the kinetics of the ul trastructural modifications of the Golgi and vesicular elements when s ec23 and sec18 mutants are shifted for 10 min to restrictive temperatu re and then returned to permissive temperature for various time interv als. Methods: S. cerevisiae sec23 and sec18 mutants from exponentially growing cultures at 20 degrees C were maintained for 10 min at the re strictive temperature of 37 degrees C and returned to the permissive t emperature of 20 degrees C for different time intervals. Following fix ation in glutaraldehyde and postfixation in potassium ferrocyanide red uced osmium, 80- to 200-nm-thick sections were prepared from Epon-embe dded yeast cells. Using the thicker sections, stereopairs of electron microscopy photographs were prepared and used to visualize the three-d imensional configuration of the organelles. To follow the modification s of cell organelles, cell sections were selected at random in thinner sections and cell organelles were scored. Results: At permissive temp erature (20 degrees C), the Golgi apparatus consisted of individual ne tworks of tubules dispersed in the cytoplasm, as in the wild type stra in. When both mutants were shifted for 10 min at the restrictive tempe rature (37 degrees C), the main structural feature was the disappearan ce of all Golgi networks. In sec23 mutant cells, there was an increase in number of tubular, nonnodular networks corresponding to terminal p ortions of the endoplasmic reticulum; in sec18 cells, small 20- to 50- nm tubules and vesicles accumulated in the cytoplasm. Within minutes a fter 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 s ame conditions. At later time intervals and in both mutants, the small tubules and vesicles decreased in number This decrease was concomitan t 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 disap pearance of Golgi networks. The small Vesicles and tubules that accumu late during the block in sec18 cells and within minutes after the shif t at 20 degrees C in sec23 cells appear to fuse together to form new G olgi networks. Thus, the small vesicles would not fuse with a preexist ing Golgi apparatus but would rather fuse together to produce new Golg i networks. Such networks appear as transitory structures continuously undergoing renewal. (C) 1998 Wiley-Liss, Inc.