The Gα subunit signals through the Ste50 protein during the mating pheromone response in the yeast Kluyveromyces lactis

Abstract

Yeast mating signal transduction pathways require a heterotrimeric G protein composed of Gα, Gβ, and Gγ subunits connected to a mitogen-activated protein kinase (MAPK) module. While in Saccharomyces cerevisiae elimination of Gα induces constitutive activation of the mating pathway, in Kluyveromyces lactis it produces partial sterility, which indicates that K. lactis Gα (KlGα) is required to positively activate mating. We use physical interaction experiments to determine that KlGα interacts with the adaptor protein KlSte50p. The Ras association (RA) domain of KlSte50p favored interaction with the GDP-bound KlGα subunit, and when the KlGα protein is constitutively activated, the interaction drops significantly. Additionally, KlSte50p strongly associates with the MAPK kinase kinase (MAPKKK) KlSte11p through its sterile alpha motif (SAM) domain. Genetic experiments placed KlSte50p downstream of the G protein α subunit, indicating that KlGα may stimulate the mating pathway via KlSte50p. Fusion of KlSte50p to the KlGβ subunit partially eliminated the requirement of KlGα for mating, indicating that one contribution of KlGα to the mating pathway is to facilitate plasma membrane anchoring of KlSte50p.

Fig. 1.

Mating capacity of mutant strains carrying YEpKD plasmids. The wild-type (WT) strain (155) or its mutant derivatives were streaked on SD medium selective for YEpKDGal plasmids and replicated onto YPGal plates containing a lawn of the tester strain (strain 12/8). Plates were incubated for 12 h at 30°C to allow cells to mate, and cells were replicated onto SD plates for diploid selection. Plates were photographed 48 h later. All mutant strains were treated with 5-fluoroorotic acid (FOA) for negative selection of the URA3 cassette used for gene disruption. WT or mutant strains were transformed with empty YEpKDGal (not shown), YEpKD-STE50, or YEpKD-STE4 plasmid prior to mating. Mating efficiency was determined by mixing 1 × 10⁶ cells of each parent in YPGal medium and incubating the cells for 12 h at 30°C. Cells were collected by centrifugation, diluted, and plated on SD medium until colonies appeared. Numbers are the average of three independent experiments and are relative to the mating efficiency of the wild-type cross (155 × 12/8).

Fig. 2.

(A) Schematic representation of K. lactis Ste50p showing the SAM and RA domains and the Ser/Thr-rich region. Numbers represent KlSte50p coordinates according to the alignment with ScSte50p (26). Fragments used for two-hybrid assays are shown. (B) Protein interactions determined by the two-hybrid system. Full-length KlSTE4, KlSTE50, and the indicated fragments were cloned into the activation plasmid pJG4-5. The wild-type KlGPA1 and KlGPA1^Q298L alleles, which encode GDP- and GTP-bound Gα subunits, respectively, were cloned into the binding plasmid pEG202. Plasmids were introduced into strain EGY48. The β-galactosidase activity (24) of the KlSTE50 constructs is relative to the KlGα-KlGβ (pEG202KlGPA1-pJG4-5KlSTE4) interaction, and data represent the average of three independent clones. The S. cerevisiae endochitinase gene (CTS1) cloned into pEG202 was used as negative-interaction control for each KlSTE50 construct. (C) KlSte50p-KlSte11p interaction. Full-length KlSTE50 and the indicated fragments, cloned into pJG4-5, were assayed for the interaction with KlSTE11, which was cloned into pEG202. β-Galactosidase activity is relative to the interaction of full-length KlSTE50 and KlSTE11 and is the average value of three independent assays.

Fig. 3.

Immunodetection of KlSte50 adaptor protein. Wild-type and ΔKlgpa1 cells expressing the wild-type KlSte50p tagged with His₆ and expressed under the control of the GAL1 promoter (YEpKDGal plasmid) were fractionated as indicated in Materials and Methods. A total of 150 μg of protein from the cytosolic fractions (C1 and C2) and the membrane fraction (M) was resolved by SDS-PAGE and analyzed by immunoblotting with either monoclonal anti-His₆-peroxidase (Roche) or rabbit anti-Hog antibodies. α, anti.

Fig. 4.

Schematic representation of KlGβ-KlSte50p fusion protein and its effect on mating. The hybrid protein KlSte4p with the hemagglutinin (HA) epitope fused at its N terminus and KlSte50p fused at its C terminus is shown along with the last five codons of KlSTE4 and the first five codons of KlSTE50 linked by the Leu codon (gray). The ΔKlgpa1 ΔKlste50 and ΔKlgpa1 ΔKlste4 double mutants were transformed with empty YEpKDGal (−) or YEpKDGal-STE4/STE50 plasmids. Cells were streaked on SD medium and replicated onto YPGal plates containing a lawn of the tester strain (12/8). Plates were incubated for 12 h at 30°C to allow cells to mate, and cells were replicated onto SD plates for diploid selection. Plates were photographed 48 h later. Mating efficiency was determined by mixing 1 × 10⁶ cells of each parent in YPGal medium and incubating the cells for 12 h at 30°C. Cells were collected by centrifugation, diluted, and plated on SD medium until colonies appeared. Numbers are the average of three independent experiments and are relative to the mating efficiency of the wild-type cross (155 × 12/8 [data not shown]).