Introduction of cytochrome b mutations in Saccharomyces cerevisiae by a method that allows selection for both functional and non-functional cytochrome b proteins

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.

Fig. 1. Creating the recipient strain containing ARG8^m instead of cytochrome b in its mitochondria

Panel A depicts the creation of a plasmid for biolistic transformation containing an ARG8^m cassette flanked by cytochrome b 5’- and 3’- UTRs. The template for the ARG8 gene using mitochondrial codons was plasmid pDS24 (9). The primers COB-ARG8-F and COB-ARG8-R were used for the gene amplification, resulting in an ARG8^m gene flanked by approximately 100 bps of COB 5’-UTR and 3’-UTR, respectively, encoded by the 3’- ends in both primers. Both primers also contained the restriction sites shown, via which the gene was cloned into the pBluescript vector KS to create the vector pSCSI. The plasmid also contains the β-lactamase gene (“bla“). Panel B depicts how ARG8^m was introduced into the mitochondrial DNA of the recipient strain. The strain used for bombardment was DFS160, which is auxotrophic for leucine and arginine and does not contain mitochondrial DNA. The synthetic ρ^- DFS160 containing the plasmid pSCSI was named CAB50. Replacement of cytochrome b by ARG8^m can be confirmed by arginine prototrophy.

Fig. 2. Introduction of cytochrome b mutations into recipient strain YTE31

Panel A depicts the creation of a plasmid containing the cytochrome b gene (COB) cassette flanked by cytochrome b 5’- and 3’-UTRs and a functional COX2 gene, as a template for primer mutagenesis. The template for the COB gene was an intron-less version of the cytochrome b gene, which was amplified with primers which resulted in a COB gene flanked by approximately 340 bps of UTRs on each side, and HincII and ApaI sites for cloning into the Bluescript vector, named pCB6. The COX2 gene, which allows selection of cytochrome b mutants that are impaired or respiratory deficient, was amplified with primers that resulted in a PCR product containing the ORF of COX2 flanked by approximately 250 bps of the 5’- and 3’- UTRs. This was cloned via a PstI site into vector pCB6, resulting in vector pMD2. Panel B depicts a flowchart with the sequence of events needed to introduce plasmid pMDx, containing a cytochrome b mutation, into recipient strain YTE31.

Fig. 3. Screening for cytochrome b mutations

Panel A shows non-fermentable medium plates (YPEG) carrying the crosses of the synthetic ρ^- with recipient strain YTE31 (left) or the COX2 rescue strain NB97 (right). The upper half shows the Y16A mutant strain, which is respiratory competent and grows in both crosses. The lower panel shows the R79E mutant strain, the growth of which is only possible with the rescue strain. Panel B shows schematically how the COX2 rescue works. After mating on fermentable medium, the cells are replica plated on non-fermentable medium. The tester strain, NB97, is ρ⁺ but contains a deletion in COX2 and thus is respiratory deficient unless corrected with a functional COX2 gene. Hence, the rescue is dependent on receiving the COX2 gene on the plasmid that was introduced by the biolistic transformation of the recipient strain and thus confirms the existence of the plasmid in that strain. One has to go back to the original plate to localize the corresponding colony, and perform this procedure at least three more times before finally mating the synthetic ρ^- with YTE31.

Fig. 4. Testing the strains with cytochrome b mutations for their growth on non-fermentable medium

Shown in panel A are the strains with the cytochrome b mutations created in this study, grown on YPEG plates. The plates on the left depict the growth of serial dilutions of the strains at 30°C. The plates on the right are the same strains grown at 34°C. WT is the strain that was created by bombarding DFS160 with non-mutated, intron-less cytochrome b, followed by cytoduction and crossing as with the cytochrome b mutant strains. For these experiments, the cytochrome b mutations were in a MR6 nuclear background (see Material and Methods). Panel B shows the growth of the strains in the liquid non-fermentable medium N3. The left panel shows the center N mutants and the right panel shows the center P mutants. The growth curve for the wild-type strain is shown in both panels to permit comparison of the mutants.