The imino amino acid, proline, has roles in both cellular nutrition and response to stress. Proline uptake in Saccharomyces cerevisiae is largely mediated by a high affinity, specific permease, Put4p, and a low affinity general amino acid permease, Gap1p. Both are subject to nitrogen catabolite repression (NCR) and nitrogen catabolite inactivation (NCI). In order for proline to be fully exploited, its transport must be derepressed, as occurs upon depletion of preferred nitrogen sources, and molecular oxygen must be present to allow the first step of catabolism via proline oxidase. This study focuses on the isolation of variants of Put4p, which are insensitive to repression by a preferred nitrogen source (ammonia) and their subsequent effect on proline transport and stress tolerance. Specific amino acid residues in the carboxy-terminal region of Put4p were targeted by site-directed mutagenesis. Substitution at Serine⁶⁰⁵, a potential phosphorylation target, led to the amelioration of ammonia-induced down-regulation of Put4p. When combined with a promoter mutation (−160), the S⁶⁰⁵A mutation resulted in increased proline uptake and accumulation. This increase in proline accumulation was associated with increased cell viability in conditions of high temperature and osmotic stress raising possible benefits in industrial fermentation applications.