Background: The secretory protein transit between cisternae of endoplasmic reticulum (ER) and Golgi elements is blocked when the yeast Saccharomyces cerevisiae sec21 mutant is shifted from the permissive (24 degrees C) to a non-permissive (37 degrees C) temperature, but 30-50 nm vesicles accumulate in the cytoplasm. At the semi-permissive temperature 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 present investigation is to analyze the structural expression of these events.

Methods: S. cerevisiae sec21 mutants were maintained for 90 min at semi-restrictive (33 degrees C) or restrictive (37 degrees C) temperatures and then progressively returned to 24 degrees C. Following fixation in glutaraldehyde and a postfixation in potassium ferrocyanide reduced osmium, 0.08 to 0.2 microns thick sections were cut from Epon embedded yeasts. Using the thicker sections, stereopairs of electron microscope photographs were prepared and used to visualize the three-dimensional configuration of the organelles.

Results: At permissive temperature, the Golgi elements appeared as isolated networks of membranous tubules dispersed throughout the cytoplasm. The diameter of these membranous tubules varied considerably from one Golgi element to another. Larger tubules showed at their intersections distensions with size and staining intensity comparable with that of the secretory granules seen at proximity of the Golgi networks or at the cell periphery. Small vesicles in the 30-50 nm size range were rarely if ever observed in cells grown at permissive temperature. Golgi networks and secretion granules were less conspicuous in mutant cells maintained at 33 degrees C and completely 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 the small vesicles. As soon as 5 minutes after shifting mutants from 33 degrees C to 24 degrees C, the small vesicles disappeared from the cytoplasm, while secretory granules were actively produced in extensively developed Golgi network. When mutants were returned from 37 degrees C to 24 degrees C, the disappearance of small vesicles was more progressive and concomitant with the progressive reconstruction of Golgi networks.

Conclusions: It is thus postulated that, in the above mentioned conditions, the small vesicles of the sec21 mutant did not act as intermediate carriers between the endoplasmic reticulum and a pre-existing Golgi apparatus, but rather fused together to produce newly formed Golgi networks.

• PMID: 7810913
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Journal Article | Research Support, Non-U.S. Gov't

Authors

Rambourg A, Clermont Y, Jackson CL, Képès F

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