Moiecuiar Microbiology (1993) 8(1). 167-178 AUA1, a gene involved in ammonia regulation of amino acid transport in Saccharomyces cerevisiae Vicky Sophlanopoulou* and George Diallinas Institute of PJIolecuiar Biology and Biotechnology, Foun- dation for Research and Technology, Hellas, P.O. Box 1527, 711 WHeraktion, Crete, Greece. Summary In Saccharomyces cerevisiae the general amino acid (GAP1) permease catalyses active transport of appar- ently all amino acids across the plasma membrane. GAP1 activity is regulated by control of synthesis and control of activity in response to the nitrogen source supplied; ammonia and giutamine Inactivate GAP1 function while proline and urea allow its maximum expression. We have isolated and characterized a gene, AUAI, involved in ammonia regulation of GAP1 activity. AUAI is not essential for growth but over- expression of the AUAI transcript in a high-copy vec- tor or due to a regulatory mutation, aua1-1, present -10 bp upstream from the start of >1(y4Mranscription, releases GAP1 activity from ammonia-inactivation without affecting GAP1 transcription. The aua1-1 mutation has no phenotype when ammonia is replaced by proline or glutamate as the nitrogen source or when it is present in a gapi background. AUAI expression is itself ammonia repressible in a wild-type strain but not in the aua1-1 mutant. The AUAI gene sequence contains a unique short open reading frame of 94 codons corresponding to a polypeptide of 11714 Da. This polypeptide is highly hydrophilic and extremely basic. The AUAI product shows no significant similarity with any previousty known protein sequence. Interestingly, a 10-amino acid segment of AUA1 is directly repeated in the most basic segment of the protein. Possible roles of AUAI are discussed. Introduction Saccharomyces cerevisiae possesses a number of uptake systems which mediate active transport of amino acids across the plasma membrane. A number of these permeases have been identified and studied genetically Received 27 July, 1992; revised and accepted 27 November. 1992. "For correspondence. Tel. (81) 210361; Fax (81) 230469. and physiologically (Wiame et ai, 1985). Most of them are specific for one or a few related i-amino acids and exhibit different properties with respect to substrate affin- ity, specificity, capacity and regulation. This is necessary in order to allow S, cerevisiae to accumulate amino acids under a wide range of growth conditions. In addition to these specific permeases. the synthesis of which is usu- ally constitutive. S. cerevisiae has a highly reguiated gen- eral amino acid transport system, the GAP1 permease (Wiame et ai, 1985; Jauniaux and Grenson, 1990). Most of L- and D- amino acids, non-proteic amino acids such as citrulline and ornithine and a number of toxic analogues of amino acids are substrates of GAP1 (Wiame e/a/.. 1985). The complex regulation of the GAP1 permease is shared by the specific proline permease (PUT4) and the ureido- succinate-allantoate permease. The activity of these per- meases is highiy dependent on the nitrogen source in the medium. The presence of ammonia, giutamine or asparagine inhibits their activities (Wiame et ai, 1985), In that way. S. cerevisiae prevents a number of nitrogenous compounds which are poor nitrogen sources from being utilized when a rich nitrogen source is present in the medium. Permease mutations have been isolated as mutations conferring resistance to amino acid toxic analogues. Depending on the analogue used, but also on the nitro- gen source present in the medium, different mutants have been isolated that allowed the charactenzation and cloning of several structural genes coding for amino acid permeases (Hoffman, 1985; Tanaka and Fink, 1985; Van- denbol et al., 1989; Jauniaux and Grenson, 1990). In addition to mutations in structural genes coding for per- meases. mutations affecting pleiotropically several amino acid uptake systems have also been selected and stud- ied. Such mutations identified different regulatory circuits which control permease functioning both at the level of transcription and at a post-transcriptional level (Grenson and Hennaut. 1971; Courchesne and Magasanik, 1983; Grenson, 1983, Vandenbol etai, 1987; 1990; Courch- esne and Magasanik, 1988). Mutations affecting transcriptiona! regulation of perme- ases include gdhCR (or ure2) mutations, which relieve ammonia repression, and gln3 mutations, which prevent increased transcription of permeases when glutamate replaces giutamine as the source of nitrogen in the medium (Coschigano and Magasanik. 1991; Minehart