ULTRASTRUCTURAL MODIFICATIONS OF VESICULAR AND GOLGI ELEMENTS IN THE SACCHAROMYCES-CEREVISIAE SEC21 MUTANT AT PERMISSIVE AND NONPERMISSIVE TEMPERATURES

Background: The secretory protein transit between cisternae of endopla smic reticulum (ER) and Golgi elements is blocked when the yeast Sacch aromyces cerevisiae sec21 mutant is shifted from the permissive (24 de grees C) to a non-permissive (37 degrees C) temperature, but 30-50 nm vesicles accumulate in the cytoplasm. At the semi-permissive temperatu re of 33 degrees C there is no complete block but rather a slowdown of the protein transport between ER and Golgi. The purpose of the presen t investigation is to analyze the structural expression of these event s. Methods: S. cerevisiae sec21 mutants were maintained for 90 min at semi-restrictive (33 degrees C) or restrictive (37 degrees C) temperat ures and then progressively returned to 24 degrees C. Following fixati on in glutaraldehyde and a postfixation in potassium ferrocyanide redu ced osmium, 0.08 to 0.2 mu m thick sections were cut from Epon embedde d yeasts. Using the thicker sections, stereopairs of electron microsco pe photographs were prepared and used to visualize the three-dimension al configuration of the organelles. Results: At permissive temperature , the Golgi elements appeared as isolated networks of membranous tubul es dispersed throughout the cytoplasm. The diameter of these membranou s tubules varied considerably from one Gels element to another. Larger tubules showed at their intersections distensions with size and stain ing intensity comparable with that of the secretory granules seen at p roximity of the Golgi networks or at the cell periphery. Small vesicle s in the 30-50 nm size range were rarely if ever observed in cells gro wn at permissive temperature. Gels networks and secretion granules wer e less conspicuous in mutant cells maintained at 33 degrees C and comp letely disappeared at 37 degrees C. In both cases, the main structural feature was the presence in the cytoplasm of numerous small vesicles and of short membranous tubules with a diameter identical to that of t he small vesicles. As soon as 5 minutes after shifting mutants from 33 degrees C to 24 degrees C, the small vesicles disappeared from the cy toplasm, while secretory granules were actively produced in extensivel y developed Golgi network. When mutants were returned from 37 degrees C to 24 degrees C, the disappearance of small vesicles was more progre ssive and concomitant with the progressive reconstruction of Golgi net works. Conclusions: It is thus postulated that, in the above mentioned conditions, the small vesicles of the sec21 mutant did not act as int ermediate carriers between the endoplasmic reticulum and a pre-existin g Golgi apparatus, but rather fused together to produce newly formed G olgi networks. (C) 1994 Wiley-Liss, Inc.