Hamster cell mutants unable to grow on galactose and exhibiting an overlapping complementation pattern are defective in the electron transport chain.

Abstract

O2 consumption which is sensitive to specific inhibitors of the electron transport chain is markedly decreased in whole cells of Chinese hamster lung cell mutants unable to grow on galactose in place of glucose, Activity of the respiratory chain, as measured by NADH oxidase activity which is sensitive to the specific inhibitor rotenone, is significantly reduced in mitochondria of the mutants.These mutants (Gal 50, 13, 73, 49, 17, and 3) are related by an overlapping complementation pattern where the complementing units are connected by noncomplementing units.There is a high degree of correlation between the biochemical data and the complementation map.The component of the respiratory chain most greatly modified varies with the mutant according to its position in the complementation map.For example, Gal 13 and Gal 50, which are located at the left hand end of the map, are most drastically modified in Complex I activity; rotenone-sensitive NADH-ubiquinone-1 reductase activity is 5 and 7% o f the wild type value.T h i s activity is increased 6-to 7fold in revertants of Gal 13 and Gal 50, which have regained the ability to grow on galactose, Gal 49 and Gal 73, which are located in the center of the complementation map, are most greatly affected in Complex ZLT; ubiquinol-cytochrome c reductase activity is 9 and 118, respectively, of the wild type level and 71% in a revertant of Gal 49.Gal I7 and Gal 3, which are located at the right hand end of the map, are primarily deficient in the level of ubiquinone.The content of ubiquinone is substantidy decreased in Gal 3, at 8% of the wild type value, and in Gal 17, at 33%.A revertant of Gal 3 has a %fold higher ubiquinone content than the mutant.Furthermore, most mutants are simultaneously deficient in some or all of the following: Complex I, Complex III, ubiquinone content, or uncoupling of respiration from phosphorylation of ADP.These pleiotropic alterations appear to be caused by a single mutation, considering that components diminished in the mutants are simultaneously elevated in their revertants.Using an established, near diploid Chinese hamster lung cell line (V79) in culture, Chu et al. ( 1 , Z ) isolated 67 mutants