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Brefeldin A sensitivity and resistance in Schizosaccharomyces pombe. Isolation of multiple genes conferring
T G Turi1, P Webster, J K Rose
1Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510.
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.
Insights
Brefeldin A (BFA) inhibits protein secretion and disrupts Golgi in yeast, similar to mammalian cells. Researchers identified BFA-resistant yeast mutants, revealing new insights into Golgi complex regulation.
Area of Science:
- Cell Biology
- Mycology
- Genetics
Background:
- Brefeldin A (BFA) is a fungal metabolite known to inhibit protein secretion and disrupt the Golgi complex in mammalian cells.
- The fission yeast Schizosaccharomyces pombe possesses a Golgi complex structurally similar to that of animal cells.
Purpose of the Study:
- To investigate the effects of BFA on the Golgi complex in the fission yeast S. pombe.
- To identify and characterize genes involved in BFA resistance in yeast.
Main Methods:
- Treatment of S. pombe with BFA.
- Isolation and genetic analysis of spontaneous BFA-resistant mutants.
- Gene sequencing and overexpression studies.
- Plasmid-based complementation to identify resistance genes.
Main Results:
- BFA treatment inhibited secretion, caused Golgi disappearance, and endoplasmic reticulum accumulation in S. pombe, mirroring effects in mammalian cells.
- Five dominant BFA-resistant mutations were identified, mapping to two linkage groups.
- One mutation was allelic to crm1. Overexpression of obr1 was observed but insufficient for resistance.
- Six genes, including the transcription factor pap1, were identified from resistant mutants that confer resistance in high copy.
Conclusions:
- BFA affects Golgi morphology in fungi similarly to mammalian cells.
- Multiple genes and potentially diverse mechanisms contribute to BFA resistance in yeast.
- Pap1 may confer resistance indirectly by regulating other gene expressions.