The yeast counterparts of human 'MELAS' mutations cause mitochondrial dysfunction that can be rescued by overexpression of the mitochondrial translation factor EF-Tu

Abstract

We have taken advantage of the similarity between human and yeast (Saccharomyces cerevisiae) mitochondrial tRNA(Leu)(UUR), and of the possibility of transforming yeast mitochondria, to construct yeast mitochondrial mutations in the gene encoding tRNA(Leu)(UUR) equivalent to the human A3243G, C3256T and T3291C mutations that have been found in patients with the neurodegenerative disease MELAS (for mitochondrial 'myopathy, encephalopathy, lactic acidosis and stroke-like episodes'). The resulting yeast cells (bearing the equivalent mutations A14G, C26T and T69C) were defective for growth on respiratory substrates, exhibited an abnormal mitochondrial morphology, and accumulated mitochondrial DNA deletions at a very high rate, a trait characteristic of severe mitochondrial defects in protein synthesis. This effect was specific at least in the pathogenic mutation T69C, because when we introduced A or G instead of C, the respiratory defect was absent or very mild. All defective phenotypes returned to normal when the mutant cells were transformed by multicopy plasmids carrying the gene encoding the mitochondrial elongation factor EF-Tu. The ability to create and analyse such mutated strains and to select correcting genes should make yeast a good model for the study of tRNAs and their interacting partners and a practical tool for the study of pathological mutations and of tRNA sequence polymorphisms.

Figure 1

Comparison of yeast and human tRNA^Leu(UUR) sequences and structures. (A) Alignment of yeast and human mitochondrial tRNA. Numbers refer to positions in the tRNA^Leu(UUR) gene for yeast and to positions in the mitochondrial DNA sequence for humans. Plus signs indicate nucleotides that are conserved between the two tRNAs. The anticodon is underlined. Bold nucleotides represent bases that were found mutated in patients (see MITOMAP, www.mitomap.org); the asterisk indicates the only pathological position not conserved in yeast. The arrows indicate the mutations studied in this work. (B) Cloverleaf structure of yeast (left) and human (right) mitochondrial tRNA^Leu(UUR). Nucleotides in grey are non-conserved bases. Arrows indicate the positions of the mutations studied and specify the substitutions studied.

Figure 2

Phenotype of yeast mutant strains harbouring mutations equivalent to A3243G and C3256T. Left panels show the growth of isogenic wild-type and mutant strains after 4 d at 28 °C on complete glucose medium (fermentative medium); right panels show the same strains and conditions on complete glycerol medium (respiratory medium).

Figure 3

Phenotypic effect of the three different mutations at position T69 of yeast (equivalent to T3291 in humans). Growth comparison of the four strains (isogenic wild-type and T→G/T→C/T→A mutants) on fermentative and respiratory media. Conditions are the same as in Fig 2. Bases at position 69 are indicated on the corresponding sectors of the plates.

Figure 4

Effect of overexpression of mt EF-Tu on the defective phenotype arising from the yeast mutation equivalent to C3256T in humans. Growth comparison on respiratory medium of the wild-type strain, the strain carrying the C3265T equivalent mutation and the same mutant strain transformed with a multicopy plasmid carrying the TUF1 gene. The corrected phenotype is correlated with the presence of the plasmid (data not shown).

Figure 5

Mitochondrial morphology observed by DASPMI staining. Cells from the wild type (A), the mutant carrying the C3256T equivalent (B) and the same mutant transformed by plasmids carrying the TUF1 gene (C) were observed by fluorescence microscopy after staining with DASPMI (the magnification is the same in all pictures, 100×). The vital dye DASPMI is supposed to reveal only functional mitochondrial membranes.

Figure 6

Northern blot analysis of mitochondrial transcripts in the wild type and in mutant strains transformed or not by plasmids containing the TUF1 gene. Total mtRNA (8 μg) purified from the wild-type cells (lane 1), from cells bearing the pathogenic C3256T equivalent mutation C26T (lane 2), from the same cells transformed with pTUF (lane 3) and from non-transformed cells bearing the non-pathogenic T3291G equivalent mutation T69G (lane 4) were loaded on partly denaturing 6% polyacrylamide–8 M urea gels. Procedure and probes were as described by Francisci et al. (1998). Hybridization was with the Leu probe (A) or with the Val probe (B).

Figure 7

Correction of mutant phenotypes by the regulated expression of TUF1. Mutant yeast strains harbouring the mutations T69C (A) and A14G (B) were transformed by a multicopy plasmid bearing the TUF1 gene under the control of the Tet promoter. This promoter is fully active in the absence of doxycycline and is inactivated in its presence. Growth of the transformed strains was compared with mutant and wild-type strains on respiratory (glycerol) and fermentative (glucose) media, with and without doxycycline.