Yeast sulfite reductase mutants produce wine with reduced H2S
Focus:
This module was chosen to complement a 1-semester, 3-credit laboratory class, Investigations in Molecular Cell Biology. The only prerequisite for this class is the first semester of Introductory Biology, so students still have many misconceptions about genes, enzyme function, protein structure, metabolism and evolution. In this class, students work in teams of 3 on various mutants in yeast methionine biosynthesis. The course was designed to incorporate an original research question (although the actual quantity of original research that beginners can do in a semester is small). The class research question concerns the evolutionary conservation of the various genes involved in methionine synthesis. After learning the ropes, students use plasmid complementation to test whether orthologs from other species are able to complement the MET gene deficiency in their own strain. They then design an original experiment based on the results of their complementation experiment.
Overview:
The module revolves the yeast MET5 and MET10 genes. The products of these genes form heterotetrameric protein, sulfite reductase. Sulfite reductase catalyzes a key regulatory step in the pathway and has an interesting siroheme cofactor that is itself the end-product of a pathway that includes the MET1 and MET8 genes. Students are studying a variety of other MET genes, but none are working with MET5 and MET10. These papers were chosen because they reinforce the major concepts of the course and because they employ many of the methods that the students will be using in the lab. The module also brings students through a series of articles that identified and characterized the MET5 and MET10 genes and their gene products. We would like them to go back in time and vicariously participate in some of the discoveries that led to our current understanding of these genes. The final article in the series describes a real life application of MET5/MET10 research, the development of a yeast strain with mutations in the MET5 and MET10 genes that can be used to produce wine with reduced H2S content. Because of time limitations, we will not be able to study every figure in these papers.
Applicable for Courses:
Molecular Cell BiologyEducational Level:
Lower LevelRoadmap Objectives:
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- Article: Masselot, M., and de Robichon-Szulmajster, H. 1975. Methionine biosynthesis in Saccharomyces cerevisiae. Mol. Gen. Genet. 139, 121-132.
- Content area/major concepts: In this seminal paper, the authors collected all known met strains available at the time and identified complementation groups for most of the MET genes known today.
- Methods or technology used to obtain data: complementation, tetrad analysis, auxotrophic analysis
- How the CREATE strategy was used:
- Biggest teaching challenge: Meiosis is always challenging! Students may experience difficulties with linkage analysis, and tetrads add some additional complexity.
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- Article: Hansen, J., Cherest, H., and Kieliand-Brandt, M.C. 1994. Two divergent MET10 genes, one from Saccharomyces cerevisiae and one from Saccharomyces carlsbergensis, encode the alpha subunit of sulfite reductase and specify potential binding sites for FAD and NADPH. J. Bacteriol. 176, 6050-6058.
- Content area/major concepts: The MET10 gene was identified by complementation with plasmids from a S. cerevisiae library. The MET10 gene had been previously mapped to chromosome 6 by classical genetics. Restriction enzyme mapping was used to localize the gene on the chromosome. Northern blots were used to study regulation of MET10 expression.
- Methods or technology used to obtain data: plasmids, DNA cloning, gene sequencing, chromosome mapping, northern blots
- How the CREATE strategy was used:
- Biggest teaching challenge: Students have trouble understanding library construction and the quantitative issues associated with library screening.
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- Article: Crane, B.R., Siegel, L.M., and Getzoff, E.D. 1997. Probing the catalytic mechanism of sulfite reductase by X-ray crystallography: Structures of the Escherichia coli hemoprotein in complex with substrates, inhibitors, intermediates, and products. Biochemistry 36, 12120-12137.
- Content area/major concepts: Yeast sulfite reductase has not been crystallized. This paper presents multiple crystal structures of the orthologous E. coli enzyme. The papers show how substrates bind to the active site and how substrate binding brings about conformational changes in an enzyme.
- Methods or technology used to obtain data: Protein overexpression, X-ray crystallography
- How the CREATE strategy was used:
- Biggest teaching challenge: We will do just a portion of this paper, which is beyond the students. I will use this paper together with some Protein Data Bank searches and exercises. We also will spend some time using molecular visualization software to manipulate the structures from this paper.
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- Article: Cordente, A.G., Heinrich, A., Pretorius, I.S., and Swiegers, J.H. 2009. Isolation of sulfite reductase variants of a commercial wine yeast with significantly reduced hydrogen sulfide production. FEMS Yeast Research 9, 446-459.
- Content area/major concepts: The authors are interested in developing a wine yeast strain that produces lower amounts of hydrogen sulfide. They focus on sulfite reductase as a target for mutagenesis, hypothesizing that reductions in sulfite reductase will reduce hydrogen sulfide production. Following a genetic screen for sulfide production, the MET5 and MET10 genes are sequenced in mutant strains and mutant alleles are identified. Plasmid complementation is used to evaluate whether the mutations have reduced sulfide production. The paper provides a good example of structure/function relationships.
- Methods or technology used to obtain data: genetic screen, gene cloning, PCR, site-directed mutagenesis, DNA sequencing, plasmid complementation
- How the CREATE strategy was used:
- Biggest teaching challenge: Students will need some help with the genetic screens and this will be the first paper that deals with a diploid strain, raising lots of issues about genetic background.