Genetic and biochemical analysis of the yeast plasma membrane Ssy1p-Ptr3p-Ssy5p sensor of extracellular amino acids

Abstract

Ssy1p and Ptr3p are known components of a yeast plasma membrane system that functions to sense the presence of amino acids in the extracellular environment. In response to amino acids, this sensing system initiates metabolic signals that ultimately regulate the functional expression of several amino acid-metabolizing enzymes and transport proteins, including multiple, genetically distinct amino acid permeases. We have found that SSY5 encodes a third component of this amino acid sensing system. Mutations in SSY5 manifest phenotypes that are indistinguishable from those resulting from either single ssy1 and ptr3 mutations or ssy5 ssy1 and ssy5 ptr3 double mutations. Although Ssy5p is predicted to be a soluble protein, it exhibits properties indicating that it is a peripherally associated plasma membrane protein. Each of the three sensor components, Ssy1p, Ptr3p, and Ssy5p, adopts conformations and modifications that are dependent upon the availability of amino acids and on the presence of the other two components. These results suggest that these components function as part of a sensor complex localized to the plasma membrane. Consistent with a sensor complex, the overexpression of SSY1 or the unique N-terminal extension of this amino acid permease homologue inactivates the amino acid sensor in a dominant-negative manner. Each of the components of the Ssy1p-Ptr3p-Ssy5p (SPS) signaling system undergoes rapid physical changes, reflected in altered electrophoretic mobility, when leucine is added to cells grown in media lacking amino acids. Furthermore, the levels of each SPS sensor component present in whole-cell extracts diminish upon leucine addition. The rapid physical alterations and reduced levels of sensor components are consistent with their being downregulated in response to amino acid availability. These results reveal the dynamic nature of the amino acid-initiated signals transduced by the SPS sensor.

FIG. 1

Growth characteristics of strains carrying single and double mutant combinations of ssy5, ssy1, and ptr3 null alleles. Strains PLY126 (wild type [WT]), HKY20 (ssy1Δ13), HKY31 (ptr3Δ15), HKY77 (ssy5Δ2), HKY33 (ssy1Δ13 ptr3Δ15), HKY84 (ssy5Δ2 ssy1Δ13), and HKY85 (ssy5Δ2 ptr3Δ15) were streaked onto the following media: SPD (plus uracil and lysine) (A), SPD (plus uracil and lysine) containing 30 mM l-histidine (B), SPD (plus uracil and lysine) containing 30 mM l-methionine (C), SD (plus uracil and lysine) (D), and SD (plus uracil and lysine) containing 100 μg of l-azetidine-2-carboxylate ml⁻¹ (E). Plates were incubated for 3 days at room temperature and photographed.

FIG. 2

Expression of AGP1 and PTR2 in wild-type (WT) and ssy5 null mutant strains. Strains PLY126 (wild type) and HKY77 (ssy5Δ2) were grown in SD to an OD₆₀₀ of 0.8, and total RNA was isolated 45 min after the addition of water (lanes 1 and 3) or 0.15 mM leucine (lanes 2 and 4). Expression levels of AGP1 (A) and PTR2 (B) were determined by Northern analysis and quantitated by phosphorimaging. The levels of actin (ACT1) transcript were used to standardize quantitations (lower panels). The relative expression levels were normalized to the expression levels in the uninduced wild-type strain.

FIG. 3

SSY5 encodes a peripherally associated PM protein. (A) The membrane association of Ssy5p was examined by using whole-cell lysates prepared from strain HKY77 expressing SSY5-c-myc. Aliquots of total protein lysate (Tot) were diluted 1:1 with H₂O, 1.6 M urea, or 2 mM EDTA; mixed; and incubated on ice for 30 min. Membrane pellet (P) and soluble (S) fractions, obtained after centrifugation at 100,000 × g for 45 min at 4°C, were resolved by SDS-PAGE and analyzed by immunoblotting. As a control, the membrane association of the PM ATPase (Pma1p) was monitored. (B) The ability of Ssy5p to associate with the PM was assessed by using the SOS membrane recruitment system. Strains cdc25H (No vector) and cdc25H transformed with plasmids pSOS, pSOS-PTR3 (pHK044), pSOS-SSY5 (pHK045), pAD-SSY5 (pHK050), pSOS-Col1, and pSOS-MAFB were grown on YPD. Culture plates were incubated at room temperature (RT [permissive]) and 37°C (nonpermissive) as indicated, and after 4 days, the plates were photographed.

FIG. 4

Functional expression of Ssy5p requires SSY1 and PTR3. (A) Strains HKY77 (wild type [WT]; lane 1), HKY84 (ssy1Δ13; lane 2), and HKY85 (ptr3Δ15; lane3) expressing SSY5-c-myc were grown in SD to an OD₆₀₀ of 0.8, and the levels of Ssy5p-c-myc were analyzed in whole-cell lysates by SDS-PAGE and immunoblotting. Prior to electrophoresis, samples were denatured for 10 min at 37°C. (B) Total RNA was prepared from strains HKY77 (wild-type; lanes 1 and 2), HKY84 (ssy1Δ13; lane 3), and HKY85 (ptr3Δ15; lane 4) expressing SSY5-c-myc grown to an OD₆₀₀ of 0.8 in SD (lanes 1, 3, and 4) or SD supplemented with 1.3 mM leucine (lane 2). The levels of SSY5 mRNA were analyzed by Northern blotting, and the levels of actin (ACT1) transcripts were used to control loading.

FIG. 5

SPS sensor component interactions. (A) The pattern of Ssy1p migration during SDS-PAGE is dependent upon Ptr3p and Ssy5p and is altered in response to amino acids. Whole-cell lysates were prepared from strains HKY20 (wild-type [WT]; lanes 1 and 4), HKY33 (ptr3Δ15; lanes 2 and 5), and HKY84 (ssy5Δ2; lanes 3 and 6) transformed with pHK010 (SSY1-HA1) grown in SD (lanes 1 to 3) and SC (lanes 4 to 6) to an OD₆₀₀ of 0.8. The levels of Ssy1p-HA1 in extracts were analyzed by SDS-PAGE and immunoblotting. (B) The electrophoretic mobility of Ptr3p is altered in response to amino acids. Whole-cell lysates were prepared from wild-type strain HKY31 transformed with pHK018 (PTR3-HA1) grown in SD (lane 1) and SC (lane 2) to an OD₆₀₀ of 0.8. The levels of Ptr3p-HA1 in extracts were analyzed by SDS-PAGE and immunoblotting. (C) The pattern of Ptr3p migration during SDS-PAGE is dependent upon Ssy1p and Ssy5p. Whole-cell lysates were prepared from strains HKY31 (wild type; lanes 1 and 4), HKY33 (ssy1Δ; lanes 2 and 5), and HKY85 (ssy5Δ; lanes 3 and 6) transformed with pHK018 (PTR3-HA1) grown in SD (lanes 1 to 3) and SC (lanes 4 to 6) to an OD₆₀₀ of 0.8. The levels of Ptr3p-HA1 in extracts were analyzed by SDS-PAGE and immunoblotting. Note that lane 2 in panel B and lane 4 in panel C are derived from the same sample. A longer exposure time was used for a more detailed analysis of the protein bands in lanes 4 to 6.

FIG. 6

Overexpression of Ssy1p or the N terminus of Ssy1p exerts a dominant-negative effect on amino acid sensor function. Dilution series of strain PLY1 transformed with pRS202 (vector; lane 1), pHK012 (2μm-SSY1; lane 2), pHK039 (2μm-SSY1_NT; lane 3), and strain HKY37 (ssy1Δ13) transformed with pRS202 (vector; lane 4) were spotted onto SPD and SPD supplemented with 5 mM histidine as indicated. Culture plates were incubated for 4 days at room temperature and photographed.

FIG. 7

Time course of l-leucine-induced transcription of PTR2 and CAR1. A liquid culture of strain HKY77 carrying plasmid pHK048 (SSY5-c-myc) was grown in SD to an OD₆₀₀ of 0.5, and the culture was split into two parts (t = 0). One-half of the cell culture received an aliquot of l-leucine (+leu; lanes 2 to 6) to a final concentration of 1.3 mM, and the other received an equal volume of water (−leu; lanes 7 to 11). Both cultures were incubated at 30°C for an additional 180 min, and at the times indicated, subsamples were withdrawn and total RNA was isolated. The levels of PTR2 (A) and CAR1 (B) expression were analyzed by Northern blotting (upper panels), and after background correction, signal strengths (arbitrary units) relative to the levels of actin mRNA (ACT1) were quantitated (lower panels).

FIG. 8

Time course analysis of the physical alterations to Ssy1p, Ptr3p, and Ssy5p after induction of sensor function by l-leucine. Strains HKY20, HKY33, and HKY77 expressing SSY1-HA1, PTR3-HA1, and SSY5-c-myc, respectively, were grown in liquid cultures of SD to an OD₆₀₀ of 0.5 (t = 0; lane 1). At t = 0, each culture received an aliquot of l-leucine to a final concentration of 1.3 mM (lanes 2 to 6). At the times indicated, subsamples were removed, whole-cell lysates were prepared, and proteins were analyzed by immunoblotting (upper panel). Lane 7 shows protein levels present in an uninduced control culture similarly incubated for 180 min. The corresponding chemiluminescent signals were quantitated (lower panel).

FIG. 9

Schematic diagram summarizing the dynamic characteristics of the SPS sensor component interactions. The state I sensor is the complex present in cells grown in the absence of amino acids. The SPS components are present in high levels, represented by the heavy outlines. In analogy to the G-protein-coupled α-factor receptor complex in MATa cells (39), the state I conformation may represent a preactivation complex. States IIa and IIb are transient complexes that rapidly form when cells grown in the absence of amino acids are induced by amino acids. The components in the transient state IIa and IIb sensor undergoing dynamic changes in expression levels are represented by the dashed outlines. The state III conformation is the downregulated complex, and the diminished levels of the components are represented by light outlines. For additional details, see text.