Constitutive and hyperresponsive signaling by mutant forms of Saccharomyces cerevisiae amino acid sensor Ssy1

Abstract

Sensing of extracellular amino acids results in transcriptional induction of amino acid permease genes in yeast. Ssy1, a membrane protein resembling amino acid permeases, is required for signaling but is apparently unable to transport amino acids and is thus believed to be a sensor. By using a novel genetic screen in which potassium uptake was made dependent on amino acid signaling, we obtained gain-of-function mutations in SSY1. Some alleles confer inducer-independent signaling; others increase the apparent affinity for inducers. The results reveal that amino acid transport is not required for signaling and support the notion that sensing by Ssy1 occurs via its direct interaction with extracellular amino acids.

FIG. 1.

AGP1::KAT1 reporter system reflects SSY1-dependent signaling by amino acids with widely different potencies. (A) Yeast cell suspensions were spotted onto minimal plates with proline as the nitrogen source and supplemented with an inducer (0.2 mM leucine) or a permissive concentration of potassium (100 mM KCl). The cells were strain RGY4990 (trk1 trk2 ssy1) harboring plasmids with and without SSY1 and with and without the AGP1::KAT1 reporter. (B) Suspensions of strain RGY5004 (trk1 trk2 ssy1 [SSY1] [P_AGP1::KAT1]) were spotted onto minimal plates with the indicated concentrations of amino acids. Cit, l-citrulline. Plates were incubated for 4 days at 30°C.

FIG. 2.

The T382K substitution in Ssy1p activates the AGP1 promoter in the absence of amino acids. (A) Suspensions of strain M5025 (trk1 trk2 ssy1 AGP1::P_AGP1-KAT1) transformed with centromere-based plasmids carrying the indicated SSY1 alleles were spotted onto minimal medium (SD), minimal medium with 0.2 mM l-leucine, and minimal medium with 100 mM KCl. The plates were incubated for 5 days at 30°C. (B) β-Galactosidase activity (Miller units) in permeabilized cells of strain M5031 (ssy1 agp1::P_AGP1-lacZ-KanMX4) transformed with centromere-based plasmids carrying the indicated SSY1 alleles and grown in minimal medium (open bars) or minimal medium with 0.2 mM l-leucine (closed bars).

FIG. 3.

Constitutive signaling by SSY1-102 is dependent on PTR3 and SSY5. Centromere-based plasmids pSSY1 (with SSY1), pSSY1-102 (with SSY1-102), and pRS316 (no insert) were introduced into strains M4871 (gap1 ssy1), M4737 (gap1 ssy1 ptr3), and M4873 (gap1 ssy1 ssy5). (A) Signaling was assayed by growth as affected by an inhibitor of isoleucine-leucine-valine biosynthesis, MM, causing a need for uptake of the three amino acids, and thereby also a need for signaling. Droplets of cell suspensions were spotted onto plates of SC without uracil (growth control) and YPD with 100 mg of MM per liter, which were incubated at 30°C for 3 days. (B) Signaling was assayed by growth as affected by l-leucyl l-ethionine, a toxic dipeptide taken up through Ptr2, which is subject to SSY-dependent transcriptional induction. A filter disk containing 4 mg of the dipeptide was placed at the end of each streak of indicated cells. Plates were incubated at 30°C for 3 days.

FIG. 4.

Constitutive and supersensitive phenotypes of SSY1 mutants affected at position 382. Droplets of strain M4941 (ssy1 trk1 trk2 [P_AGP1-KAT1]) transformed with the SSY1 alleles on a centromere-based vector were spotted onto minimal media supplemented as indicated. Plates were incubated at 30°C for 4 days. Cit, l-citrulline; WT, wild type.

FIG. 5.

Dose response of AGP1 promoter activity shows that l-citrulline is 10-fold more potent than the wild type when cells contain His at position 382 of Ssy1. β-Galactosidase activity (Miller units) was assayed on permeabilized cells of strain M5031 (gap1 ssy1 agp1::P_AGP1-lacZ-kanMX4) transformed with a centromeric plasmid expressing either SSY1-106 (open circles) or SSY1 (closed circles). The curves show the best fits to the assumptions that (i) the β-galactosidase activity is linear with the amount of complex between Ssy1 and l-citrulline and (ii) this complex is in simple chemical equilibrium with its components. This leads to a β-galactosidase activity of Y = Y_max{(K_d/[l-citrulline]) + 1}⁻¹ + Y_min, where K_d is the apparent dissociation constant. Fitting yielded apparent dissociation constants (K_ds) of 800 μM for the wild type and 80 μM for SSY1-106 cells.