The Saccharomyces cerevisiae LEP1/SAC3 gene is associated with leucine transport

Leucine uptake by Saccharomyces cerevisiae is mediated by three transport s ystems, the general amino acid transport system (GAP), encoded by GAP1, and two group-specific systems (S1 and S2), which also transport isoleucine an d valine. A new mutant defective in both group-specific transport activitie s was isolated by employing a gap1 leu4 strain and selecting for trifluorol eucine-resistant mutants which also showed greatly reduced ability to utili ze L-leucine as sole nitrogen source and very low levels of [C-14]L-leucine uptake. A multicopy plasmid containing a DNA fragment which complemented t he leucine transport defect was isolated by selecting for transformants tha t grew normally on minimal medium containing leucine as nitrogen source and subsequently assaying [C-14]L-leucine uptake. Transformation of one such m utant, lep1, restored sensitivity to trifluoroleucine. The complementing ge ne, designated LEP1, was subcloned and sequenced. The LEP1 ORF encodes a la rge protein that lacks characteristics of a transporter or permease (i.e., lacks hydrophobic domains necessary for membrane association). Instead, Lep 1p is a very basic protein (pI of 9.2) that contains a putative bipartite s ignal sequence for targeting to the nucleus, suggesting that it might be a DNA-binding protein. A database search revealed that LEP1 encodes a polypep tide that is identical to Sac3p except for an N-terminal truncation. The or iginal identification of SAC3 was based on the isolation of a mutant allele , sac3-1, that suppresses the temperature-sensitive growth defect of an act in mutant containing the allele act1-1. Sac3p has been previously shown to be localized in the nucleus. When a lep1 mutant was crossed with a sac3 del etion mutant, no complementation was observed, indicating that the two muta tions are functionally allelic.