Amino acid signaling in Saccharomyces cerevisiae: a permease-like sensor of external amino acids and F-Box protein Grr1p are required for transcriptional induction of the AGP1 gene, which encodes a broad-specificity amino acid permease

Abstract

The SSY1 gene of Saccharomyces cerevisiae encodes a member of a large family of amino acid permeases. Compared to the 17 other proteins of this family, however, Ssy1p displays unusual structural features reminiscent of those distinguishing the Snf3p and Rgt2p glucose sensors from the other proteins of the sugar transporter family. We show here that SSY1 is required for transcriptional induction, in response to multiple amino acids, of the AGP1 gene encoding a low-affinity, broad-specificity amino acid permease. Total noninduction of the AGP1 gene in the ssy1Delta mutant is not due to impaired incorporation of inducing amino acids. Conversely, AGP1 is strongly induced by tryptophan in a mutant strain largely deficient in tryptophan uptake, but it remains unexpressed in a mutant that accumulates high levels of tryptophan endogenously. Induction of AGP1 requires Uga35p(Dal81p/DurLp), a transcription factor of the Cys6-Zn2 family previously shown to participate in several nitrogen induction pathways. Induction of AGP1 by amino acids also requires Grr1p, the F-box protein of the SCFGrr1 ubiquitin-protein ligase complex also required for transduction of the glucose signal generated by the Snf3p and Rgt2p glucose sensors. Systematic analysis of amino acid permease genes showed that Ssy1p is involved in transcriptional induction of at least five genes in addition to AGP1. Our results show that the amino acid permease homologue Ssy1p is a sensor of external amino acids, coupling availability of amino acids to transcriptional events. The essential role of Grr1p in this amino acid signaling pathway lends further support to the hypothesis that this protein participates in integrating nutrient availability with the cell cycle.

FIG. 1

The yeast Ydr160p protein of the amino acid permease family displays unusual features resembling those of the Snf3p and Rgt2p glucose sensors. The CBI values (13) of the genes coding for the 17 Hxt proteins of the hexose transporter family, the glucose sensors Snf3p and Rgt2p (left panel), and the 18 proteins of the amino acid permease family (right panel) are plotted against the number of amino acid residues present in the proteins.

FIG. 2

The Ydr160-Ssy1p protein displays unusual structural features compared to other members of the amino acid permease family. The amino acid sequences of the amino acid permeases Gap1p (43), Hip1p (79), Agp1p-Wap1p (65), and Ydr160p-Sys1p (42) were aligned by using the PILEUP program (23). Identical and conserved residues are indicated in black boxes. The transmembrane segments predicted by using the TMAP algorithm (72) are underlined.

FIG. 3

Deletion of the SSY1 or the AGP1 gene affects the utilization of several amino acids in cells lacking the general amino acid permease (Gap1p). Cells were spread on minimal medium with the indicated amino acid at the final concentration of 1 mM (A) or 10 mM (B) as the sole nitrogen source. The strains were 23344c (ura3), 32501b (gap1Δ ura3), 32501d (gap1Δ ssy1Δ ura3), 30633c (gap1Δ agp1Δ ura3), and 32502b (gap1Δ ssy1Δ agp1Δ ura3).

FIG. 4

Deletion of the SSY1 or AGP1 gene alters incorporation of amino acids. The time course of ¹⁴C-labeled leucine, isoleucine, phenylalanine, and tyrosine (initial concentration, 0.1 mM) accumulation measured in cells growing on minimal medium with urea as the sole nitrogen source is shown. The strains were 30629c (gap1Δ ura3) (■), 30633c (gap1Δ agp1Δ ura3) (□) and 32501d (gap1Δ ssy1Δ ura3) (○).

FIG. 5

Noninduction of the AGP1 gene in the ssy1Δ mutant is not due to inducer exclusion. The time course of accumulation of ¹⁴C-labeled amino acids (initial concentration, 0.1 mM) (solid symbols) and of the increase in β-galactosidase activity (open symbols) in strains 32501a (ura3) (squares) and 32501c (ssy1Δ ura3) (circles) transformed with the YCpAGP1-lacZ plasmid and initially growing on minimal medium with urea as the sole nitrogen source is shown.

FIG. 6

The gap1-92 agp1-1 strain is largely defective in tryptophan transport. The time course of ¹⁴C-labeled tryptophan accumulation (initial external concentration, 5 mM) in strains 23344c (ura3) (•) and 30622a (gap1-92 agp1-1 ura3) (○) initially growing on minimal medium with urea (5 mM) as the sole nitrogen source is shown.

FIG. 7

The Ssy1p amino acid sensor affects expression of multiple amino acid permease genes. The results of RT-PCR analysis of the total RNA from strains 23344c (ura3) and 32501c (ssy1Δ ura3), with oligonucleotide primers specific to the actin gene (ACT1) and to several genes encoding amino acid permeases (see Materials and Methods), are shown. The cells were grown on minimal urea medium. At time zero, phenylalanine (5 mM) (Phe) or leucine (5 mM) (Leu) was added to part of the cultures, and the cells were collected after 90 min.

FIG. 8

Schematic presentation of the role of the permease-like amino acid sensor Ssy1p in the transcriptional regulation of amino acid permease genes in S. cerevisiae.