Abstract
The fungal metabolite brefeldin A (BFA) causes the inhibition of protein secretion and the disruption of the structure and function of the Golgi complex in mammalian cells. Here we show that BFA has identical effects in the fission yeast Schizosaccharomyces pombe which normally contains a Golgi complex of stacked cisternae similar to the Golgi complexes in animal cells. After treatment with BFA, secretion was inhibited, Golgi complexes disappeared, and there was an accumulation of endoplasmic reticulum. These results indicate that the effects of BFA in fungi are very similar to those in mammalian cells and provide direct evidence for an effect of BFA on Golgi morphology in fungi. Five spontaneous BFA-resistant mutants were isolated. Genetic analysis showed that the mutations conferring BFA resistance were dominant and in two separate linkage groups. One of the BFA-resistant mutations was found to be allelic to crm1, a gene affecting chromatin structure. All BFA-resistant mutants overexpressed a 20-kDa protein, and the corresponding gene obr1 was isolated and sequenced. However, obr1 overexpression was not sufficient to confer BFA resistance. Plasmids capable of conferring BFA resistance to wild type cells were isolated from libraries constructed from the two BFA-resistant mutants. These plasmids contain six different genes capable of conferring resistance when present in high copy. One of these genes encoded the transcription factor pap1, a homolog of the mammalian AP1 protein. The overexpression of pap1 probably confers BFA resistance indirectly by inducing expression of one or more other proteins. The isolation of several genes conferring BFA resistance suggests several mechanisms are involved.
Highlights
The fungal metabolite brefeldin A (BFA) causes the membranes into the endoplasmic reticulum (ER) (Lippincottinhibition of protein secretion and thedisruption of the Schwartz etal. 1989; Doms et al, 1989; Fujiwara et al, 1988)
Understanding the mechanism of BFA acmally contains a Golgicomplex of stacked cisternae tion through identification of the protein(s) that interact with similar to the Golgicomplexes in animal cells.After this drug may reveal the identitoifems olecules that treatment with BFA, secretion was inhibited, Golgi com- are critical in controlling thestructureand function of orplexes disappeared, and there was an accumulation of endoplasmic reticulum
Each culture was genetic system for identifying a protein(s) whose function is affected by BFA, we examined BFA sensitivity in wild type S. cereuisiae and S. pombe using a growth inhibition assay
Summary
Yeast Strains, and Genetic MethodsS. pombe was grown in YES or in EMM (Mitchinson, 1970) and Saccharomyces cerevisiae in YEPD (Sherman et al, 1986). Pombe was grown in YES or in EMM (Mitchinson, 1970) and Saccharomyces cerevisiae in YEPD (Sherman et al, 1986). The antibiotics brefeldin A, cycloheximide, and nystatin were purchased from Sigma. Genotype sequence NVSDDELEIA was synthesized (5' AAC GT(TC)TC(TC) GA(TC) GA(TC) CAI (CT)TI GAI AT(TC)GC 3'). Sp2h2' 4 BAPl BAP2 BAP3 BAP4 BAP5 S. cerevisiae 8S1 leul-32, ade216, urn h- ura, burl-1 h- ura, barl-2 h- ura, bur h ura, bar h- ura, bar a lehui2s4, , ura, ade
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
