SummaryAmino acid transporters of the yeast plasma membrane (permeases) belong to a family of integral membrane proteins with pronounced structural similarity. We present evidence that a member of this family, encoded by the open reading frame (ORF) YDR160w (SSY1), is required for the expression of a set of transporter genes. Thus, deletion of the SSY1 gene causes loss of leucine-inducible transcription of the amino acid permease genes BAP2, TAT1 and BAP3 (ORF YDR046c) and the peptide transporter, PTR2. D-leucine can generate the signal without entering the cell. We propose that Ssy1p is situated in the plasma membrane and is involved in sensing leucine in the medium.

In order to identify genes involved in uptake of isoleucine, leucine and valine in Saccharomyces cerevisiae we isolated mutants that, on a complex medium, were sensitive to an inhibitor of the biosynthesis of the branched-chain amino acids. Mutants that in a secondary screen showed reduced uptake of isoleucine, leucine and valine when growing in synthetic complete medium were further characterized. Genetic analysis identified five loci, named ssy1 through ssy5. ssy2 corresponds to the previously characterized bap1 mutation, which we recently have found to be allelic to stp1. ssy1, ssy3 and ssy5 exhibit a reduced uptake of phenylalanine, methionine and threonine, as well. Furthermore, they are resistant to several neutral amino acid analogs. ssy4 only affects uptake of few neutral amino acids and is as sensitive as the wild type to the amino acid analogs tested. It was previously found that a C-terminal truncation of 29 codons of BAP2, which encodes a branched-chain amino acid permease, results in increased uptake of the branched-chain amino acids. We find epistasis of the C-terminally truncated BAP2 gene over the ssy4 mutation, while the other ssy mutations are epistatic over the truncated BAP2 gene. SSY1, SSY3 and SSY5 were cloned from a low-copy genomic library by complementation of the mutants. The SSY3 gene and the SSY5 gene show no significant homology to any sequence in the databases. SSY1 is a member of the major family of genes encoding amino acid permeases in yeast. We discuss possible roles of Ssy1p in amino acid uptake.

Uptake of branched-chain amino acids by Saccharomyces cerevisiae from media containing a preferred nitrogen source is mediated by the permeases encoded by BAP2, BAP3, and VAP1/TAT1. The transcriptional activity of the BAP2 promoter is affected by a number of genes, including SSY1, which encodes an amino acid permease homologue that is necessary for transcription of BAP2. Other genes that control BAP2 encode known (Leu3p, Tup1p) and putative (Stp1p, Stp2p) transcription factors. We present evidence that the zinc-finger proteins Stp1p and Stp2p bind directly to the BAP2 promoter. Binding of Stplp to the BAP2 promoter in vivo and in vitro indicates that the STP gene family indeed encodes transcription factors. The presence of a Leu3p binding site in the BAP2 promoter is required for full promoter activity on synthetic complete medium. The capacity of Leu3p to activate BAP2 transcription correlates with conditions that affect the level of alpha-isopropyl malate. The effect of a tup1 deletion on BAP2 transcription depends on SSY1. In an ssy1 strain, the phenotype of tup1 conforms to the well-established role of Tup1p as part of a repressor complex, but in the SSY1 strain deletion of TUP1 causes a decrease in transcription, indicating that Tup1p may also have an activating role at the BAP2 promoter. Our results thus suggest a complex interplay between several transcription factors in the expression of BAP2.

Vps10p is a receptor that sorts several different vacuolar proteins by cycling between a late Golgi compartment and the endosome. The cytoplasmic tail of Vps10p is necessary for the recycling, whereas the lumenal domain is predicted to interact with the soluble ligands. We have studied ligand binding to Vps10p by introducing deletions in the lumenal region. This region contains two domains with homology to each other. Domain 2 binds carboxypeptidase Y (CPY), proteinase A (PrA) and hybrids of these proteases with invertase. Moreover, we show that aminopeptidase Y (APY) is a ligand of Vps10p. The native proteases compete for binding to domain 2. Binding of CPY(156)-invertase or PrA(137)-invertase, on the other hand, do not interfere with binding of CPY to Vps10p. Furthermore, the Q24RPL27 sequence known to be important for vacuolar sorting of CPY, is of little importance in the Vps10p-dependent sorting of CPY-invertase. Apparently, domain 2 contains two different binding sites; one for APY, CPY and PrA, and one for CPY-invertase and PrA-invertase. The latter interaction seems not to be sequence specific, and we suggest that an unfolded structure in these ligands is recognized by Vps10p.

The bap1 mutant of Saccharomyces cerevisiae was previously isolated by its reduced uptake of branched-chain amino acids. In the present study, the corresponding wild-type gene was cloned and partial sequencing and subsequent genetic analysis revealed identity to STP1, a gene involved in tRNA maturation. The decrease in amino-acid uptake caused by stp1 mutations is independent of GCN4. It was previously found that the BAP2 promoter can be activated by the presence of amino acids, notably leucine, in the medium. We found that this activation depends on STP1. As a simple hypothesis we propose that Stp1p is a transcription factor which activates BAP2, and probably other amino-acid permease genes.