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Updated: Jul 5, 2026
Mitochondrial Transformation in Baker's Yeast to Study Translation and Respiratory Complex Assembly
Published on: June 7, 2024
Introduction of cytochrome b mutations in Saccharomyces cerevisiae by a method that allows selection for both
Martina G Ding1, Christine A Butler, Scott A Saracco
1Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755, USA.
Abstract:
We have previously used inhibitors interacting with the Qn site of the yeast cytochrome bc(1) complex to obtain yeast strains with resistance-conferring mutations in cytochrome b as a means to investigate the effects of amino acid substitutions on Qn site enzymatic activity [M.G. Ding, J.-P. di Rago, B.L. Trumpower, Investigating the Qn site of the cytochrome bc1 complex in Saccharomyces cerevisiae with mutants resistant to ilicicolin H, a novel Qn site inhibitor, J. Biol. Chem. 281 (2006) 36036-36043.]. Although the screening produced various interesting cytochrome b mutations, it depends on the availability of inhibitors and can only reveal a very limited number of mutations. Furthermore, mutations leading to a respiratory deficient phenotype remain undetected. We therefore devised an approach where any type of mutation can be efficiently introduced in the cytochrome b gene. In this method ARG8, a gene that is normally encoded by nuclear DNA, replaces the naturally occurring mitochondrial cytochrome b gene, resulting in ARG8 expressed from the mitochondrial genome (ARG8(m)). Subsequently replacing ARG8(m) with mutated versions of cytochrome b results in arginine auxotrophy. Respiratory competent cytochrome b mutants can be selected directly by virtue of their ability to restore growth on non-fermentable substrates. If the mutated cytochrome b is non-functional, the presence of the COX2 respiratory gene marker on the mitochondrial transforming plasmid enables screening for cytochrome b mutants with a stringent respiratory deficiency (mit(-)). With this system, we created eight different yeast strains containing point mutations at three different codons in cytochrome b affecting center N. In addition, we created three point mutations affecting arginine 79 in center P. This is the first time mutations have been created for three of the loci presented here, and nine of the resulting mutants have never been described before.
Insights
Researchers developed a new method to create yeast cytochrome b mutations, enabling the study of enzyme activity and respiratory function. This approach overcomes limitations of previous inhibitor-based screening methods.
Area of Science:
- Mitochondrial genetics
- Molecular biology
- Biochemistry
Background:
- Previous methods for studying yeast cytochrome b mutations relied on specific inhibitors, limiting the scope of discoverable mutations.
- These inhibitor-based approaches could not identify mutations causing respiratory deficiency.
Purpose of the Study:
- To develop a versatile method for introducing any type of mutation into the yeast cytochrome b gene.
- To enable the investigation of amino acid substitutions on Qn site enzymatic activity and respiratory function.
Main Methods:
- Replaced the mitochondrial cytochrome b gene with ARG8, creating ARG8(m).
- Introduced mutated cytochrome b versions, inducing arginine auxotrophy.
- Utilized non-fermentable growth for selecting respiratory-competent mutants.
- Employed the COX2 respiratory gene marker for screening respiratory-deficient (mit(-)) mutants.
Main Results:
- Successfully created eight yeast strains with point mutations in cytochrome b affecting center N.
- Generated three point mutations affecting arginine 79 in center P.
- Nine of the generated mutants are novel, and three loci were mutated for the first time.
Conclusions:
- The developed method efficiently introduces diverse mutations in yeast cytochrome b.
- This system facilitates the study of structure-function relationships in the cytochrome bc1 complex.
- The approach allows for the identification of both functional and non-functional cytochrome b mutants.
