DS28-6, a temperature-sensitive mutant of Chinese hamster ovary cells, expresses key phenotypic changes associated with brefeldin A treatment.

Abstract

The temperature-sensitive Chinese hamster ovary (CHO) cell mutant DS28-6 has been previously shown to be pleiotropically defective in protein secretion. We have examined the mutant cells to determine the intracellular site of the block in secretion. By transmission electron microscopy a time-dependent disassembly of the Golgi apparatus was found under nonpermissive temperature, which resulted in the loss of the cisternal stack. Complete reorganization of the Golgi apparatus occurred after shift to permissive temperature. Under nonpermissive temperature, a microtubule-and energydependent redistribution of Golgi mannosidase I and galactosyltransferase into a pattern characteristic of the endoplasmic reticulum (ER) was observed. Inhibition of protein synthesis by cycloheximide had no influence on Golgi mannosidase H redistribution. Evidence for Golgi apparatus-associated processing of oligosaccharides in the ER was obtained by lectin-gold cytochemistry revealing the presence of the galactose (013-+4)N-acetylglucosamine sequence and sialic acid residues. Furthermore, 7-nitrobenz-2-oxa-1,3-diazol-4-yl-tagged ceramide, a lipidic trans-Golgi apparatus marker in CHO cells, exhibited an energy-dependent redistribution into the ER. These effects were fully reversible upon shift to permissive temperature. Thus, mutant DS28-6 cells exhibit key features of the brefeldin A phenotype, which suggests that the observed brefeldin A effects result from interference with a normally occurring cellular process.

Retention of secretory proteins in an intermediate compartment and disappearance of the Golgi complex in an END4 mutant of Chinese hamster ovary cells

“…It should be emphasized that the Golgi complex disappears at the resolution of fluorescence microscopy: ultrastructural studies are in progress and it is possible that Golgi remnants may be identified at high resolution. The absence of the Golgi agrees with the recent report of Zuber et al (59) that the Golgi complex disappeared at high temperature in mutant DS28-6, which is in the same complementation group as mutant V.24.1. Zuber et al (59) further reported several aspects of the disappearing Golgi in DS28-6 cells that were similar to the effects of BFA on the Golgi.…”

“…The absence of the Golgi agrees with the recent report of Zuber et al (59) that the Golgi complex disappeared at high temperature in mutant DS28-6, which is in the same complementation group as mutant V.24.1. Zuber et al (59) further reported several aspects of the disappearing Golgi in DS28-6 cells that were similar to the effects of BFA on the Golgi. (a) The redistribution of the Golgi enzymes mannosidase II and galactosyl transferase to the ER by immunofluorescence microscopy.…”

“…The complex carbohydrate within the ER observed by Zuber et al (59) using lectin-gold cytochemistry could have come from preexisting glycoproteins transported from the Golgi to the ER rather than by processing of oligosaccharide chains in the ER. However, the possibility that Golgi components are not returning to the ER cannot be dismissed and we can think of three explanations that might account for this~ First, Gotgi membrane might fragment into vesicles too small to be detected by immunofluorescence microscopy if vesicles involved in transport between Golgi cisternae formed but did not bind to or fuse with their target membranes.…”

Golgi-disturbing agents

“…A similar effect was seen when rab6 was overexpressed in its GTP-bound form; again resident Golgi proteins (such as galT) were redistributed to the ER (Martinez et al 1997). A similar phenomenon was found in a temperature-sensitive mutant of CHO cells in which redistribution of Golgi proteins to the ER occurred under non-permissive conditions, which was reversible after shift to a permissive temperature; in this report the presence of Golgi proteins and their products in the ER was directly shown by immunocytochemistry (Zuber et al 1991). In all three cases, the molecular mechanism remains to be elucidated; nevertheless, these effects show that anterograde and retrograde movements along the secretory pathway are in a dynamic equilibrium which can be profoundly disturbed.…”

A fluorescent lipid analogue can be used to monitor secretory activity and for isolation of mammalian secretion mutants.

“…In addition, because in mammalian cells the morphology of the secretory organelles is better visualized and described, questions involving organelle identity and biogenesis may be more easily answered there. As an outstanding example of this, three mutant CHO cell lines have been isolated in which the onset of a block in secretion is accompanied by a rapid and reversible loss of the Golgi complex (Zuber et al, 1991;Kao and Draper, 1992;Guo et al, 1994). This phenotype, which is reminiscent of the effects of BFA on secretion and on the morphology of the Golgi complex, strongly suggests a coupling of secretory activity and organelle identity (Hurtley, 1992).…”