Biogenesis of Mitochondria, XVIII. A New Class of Cytoplasmically Determined Antibiotic Resistant Mutants in Saccharomyces cerevisiae

Abstract

Abstract. New mutant yeasts resistant to the antibiotics chloramphenicol and mikamycin were isolated. They are mitochondrial mutants, characterized by several criteria as cytoplasmically determined. Biochemical studies show that amino acid incorporation into protein in vitro by mitochondria isolated from cells resistant or sensitive to mnikamycin or chloramphenicol is inhibited by these antibiotics. Although aerobically-grown resistant strains of Saccharomyces cerevisiae are not affected by mikamycin or chloramphenicol, it is found that the mitochondrial protein-synthesizing system of anaerobically grown cells is inhibited in vivo. Cross resistance among the antibiotics chloramphenicol, mikamycin, erythromycin, lincomycin, carbomycin, and spiramycin is reported. All erythromycin resistant mutants, unlike the others, are resistant to erythromycin in vivo and in vitro. The results indicate that some of the cytoplasmic mutations (mikamycin and chloramphenicol resistance) are expressed at the mitochondrial membrane, whereas others (erythromycin resistance) possibly reflect changes in mitochondrial ribosomal proteins. We further suggest that conformational changes, either in the membranes or ribosomes, are likely to account for the observed antibiotic cross resistances.The mitochondrial protein synthesizing system of Saccharomyces cerevisiae is specifically inhibited, in vivo and in vitro, by a number of antibacterial antibiotics: these include chloramphenicol, lincomycin, erythromycin, carbomycin, and spiramycin. '2 Yeast grown in the presence of these antibiotics lack the particulate mitochondrial cytochromes a, a3, b, and cl, thus are unable to grow on nonfermentable substrates. An erythromycin-resistant strain of S. cerevisiae showing cytoplasmic inheritance has been isolated and partially characterized in this laboratory.3 4 In vivo and in vitro, its mitochondrial protein synthesizing system is unaffected by high levels of erythromycin. We have suggested, by analogy with bacterial mutants, that a change in the response of the mitochondrial protein-synthesizing system to erythromycin is a result of an alteration in a mitochondrial ribosomal protein. 3'4 In this communication we report the isolation and some biochemical properties of two new cytoplasmically-determined mutant types of S. cerevisiae resistant to mikamycin and chloramphenicol. In contrast to the cytoplasmically-deter-1233

Mitochondrial ribosomes

“…Moreover, when the mutant is grown anaerobically on a limited supply of unsaturated fatty acids and ergosterol, a procedure that strongly modifies the permeability properties of the mitochondrial membranes, induction of mitochondrial differentiation by O2 has become sensitive to inhibition by mikamycin etc. Bunn et al [94] suggest that resistance to antibiotics in the mikamycin-type mutants is due to a permeability barrier in the mitochondrial membrane which is lost when the cells lack unsaturated fatty acids and ergosterol, or when the membrane is damaged during isolation of the mitochondria, whereas mutation to erythromycin resistance is due to a change in a ribosomal protein. The authors further suggest that changes in the mitochondrial inner membrane and in mitochondrial ribosomal proteins could co-exist and "that interaction between them may occur" [94] , the link apparently being that the ribosome is part of the membrane.…”

“…Linnane and coworkers [46,94,95] have recently raised the possibility that mitochondrial ribosomes are attached to the mitochondrial membrane and might even be an integral part of the membrane. Three arguments were brought forward to support this idea:…”

“…1) The ribosomes of yeast mitochondria are intimately associated with the organelle membranes; the difficulty of their separation by the use of detergents suggests a strong interaction [94].…”

Altered mitochondrial ribosomes in a cytoplasmic mutant of yeast

“…Bunn et al [9] have shown that two types of cytoplasmic mutants can be distinguished. The first type is isolated as resistant to mikamycin, but is crossresistant to several other unrelated antibiotics.…”

The hunt for mitochondrially synthesized proteins

“…In 1979, Buse and his colleagues at the University of Aachen in Germany finally managed to sequence the first large subunits of mammalian cytochrome oxidase, establishing them as hydrophobic, but otherwise "normal" proteins (Steffens & Buse, 1979). Perhaps the most decisive breakthrough was the discovery of specific mitochondrial DNA mutations that altered only a single mitochondrially made protein (Bunn et al, 1970;Wilkie 1970;Tzagoloff et al, 1975;Douglas & Butow, 1976). Some of these mutations made the FIFo-ATPase or cytochrome b resistant to antibiotics; others selectively inactivated one of the enzyme complexes of oxidative phosphorylation; and still others were naturally occurring mutations that had no physiological effect, but altered the electrophoretic mobility of a mitochondrially made protein.…”