Cells of Saccharomyces cerevisiae showed differential growth inhibition when cultured on various carbon sources in the presence of the proline analogue thiazolidine-4-carboxylic acid (TZ). On 0.5% yeast extract, 2% glucose and TZ (10 mg/ml) medium, growth lags from 8 to 10 h were observed, after which cells recovered and growth proceeded normally. Growth was totally inhibited on a medium of 0.5% yeast extract, 3% ethanol, and 5 mg of TZ per ml. This inhibition was not due to the inability of cells to undergo aerobic respiration, since similar media containing glycerol instead of ethanol allowed growth. Proline added to the culture medium reversed the lag on glucose and TZ medium but did not promote recovery on ethanol and TZ medium. TZ was found to have two probable modes of action in yeast. It was a noncompetitive inhibitor of yeast alcohol dehydrogenase, and it was also found to be incorporated into cellular protein. Uptake studies using "4C-labeled TZ showed that the recovery on glucose was correlated with the progressive exclusion of the analogue from cells.Our study of thiazolidine-4-carboxylic acid (TZ) was undertaken as part of a program to screen amino acid analogues in an attempt to find those incorporated specifically into mitochondrially synthesized protein of yeast cells. It was reasoned that amino acid analogues that inhibited growth differentially on fermentable and nonfermentable carbon sources would be useful in the study of mitochondrial biogenesis as well as cellular metabolism. Such differential growth inhibition should result from differences in specificity of the cytoplasmic and mitochondrial transfer ribonucleic acid synthetase (18).Although TZ does not specifically affect the mitochondrial system, its modes of action were investigated. In this report, we define specific factors affecting the response of yeast cells to this analogue.
MATERIALS AND METHODSOrganisms and culture conditions. A haploid p+ strain, 1493-10C, with the genotype a, his4, gal3, MAL+, SUC+, MEL+, was used in all of the following studies. This strain was chosen because of its high susceptibility to TZ on several media. Cultures were routinely grown at 30 C in a New Brunswick Psycrotherm incubator with shaking at 250 rpm. Inocula were usually grown to stationary phase in 0.5% yeast extract (Difco) and 2% glucose (glucose medium), or 1% yeast extract, 2% peptone, and 2% glucose. Cells were washed by centrifugation before use. For differential growth experiments, 0.5% yeast extract was supplemented with one of the following carbon sources: 2% glucose (glucose medium), 3% glycerol (glycerol medium), 3% ethanol (ethanol medium), 3% glycerol and 2% ethanol (glycerol and ethanol medium), or 0.5% sodium acetate (acetate medium). Media were prepared at double strength so that an equal volume of either a stock TZ solution (20 mg/ml, filter sterilized) or sterile distilled water could be added as required.Growth experiments were carried out in 6-ml cultures. Cell number was determined by measuring the optical density of the cultur...