Saccharomyces cerevisiae LEP1/SAC3 gene is associated with leucine transport

• Published in: Molecular & general genetics 1999-09, Vol.262 (2), p.332-341
• Main Authors: Stella, C.A, Korch, C, Ramos, E.H, Bauer, A, Kolling, R, Mattoon, J.R
• Format: Article
• Language: English
• Subjects: active transport; Alleles; amino acid derivatives; amino acid metabolism; amino acid sequences; Amino Acid Transport Systems; antifungal properties; Base Sequence; biological resistance; Biological Transport; citrulline; Cloning, Molecular; DNA, Fungal; Drug Resistance, Microbial; Fungal Proteins - genetics; Fungal Proteins - metabolism; genbank/u35227; genbank/z47805; genes; Genes, Fungal; Genetic Complementation Test; genetic transformation; leucine; Leucine - analogs & derivatives; Leucine - metabolism; Leucine - pharmacology; Membrane Transport Proteins; Molecular Sequence Data; mutants; Mutation; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Nucleocytoplasmic Transport Proteins; nucleotide sequences; plasmids; polypeptides; Porins; proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae - drug effects; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins; trifluoroleucine; uptake
• ISSN: 0026-8925; 1432-1874
• DOI: 10.1007/s004380051091

Leucine uptake by Saccharomyces cerevisiae is mediated by three transport systems, the general amino acid transport system (GAP), encoded by GAP1, and two group-specific systems (S1 and S2), which also transport isoleucine and valine. A new mutant defective in both group-specific transport activities was isolated by employing a gap1 leu4 strain and selecting for trifluoroleucine-resistant mutants which also showed greatly reduced ability to utilize L-leucine as sole nitrogen source and very low levels of [14C]L-leucine uptake. A multicopy plasmid containing a DNA fragment which complemented the leucine transport defect was isolated by selecting for transformants that grew normally on minimal medium containing leucine as nitrogen source and subsequently assaying [14C]L-leucine uptake. Transformation of one such mutant, lep1, restored sensitivity to trifluoroleucine. The complementing gene, designated LEP1, was subcloned and sequenced. The LEP1 ORF encodes a large protein that lacks characteristics of a transporter or permease (i.e., lacks hydrophobic domains necessary for membrane association). Instead, Lep1p is a very basic protein (pI of 9.2) that contains a putative bipartite signal sequence for targeting to the nucleus, suggesting that it might be a DNA-binding protein. A database search revealed that LEP1 encodes a polypeptide that is identical to Sac3p except for an N-terminal truncation. The original identification of SAC3 was based on the isolation of a mutant allele, sac3-1, that suppresses the temperature-sensitive growth defect of an actin 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 deletion mutant, no complementation was observed, indicating that the two mutations are functionally allelic.