The beta subunit of the heterotrimeric G protein triggers the Kluyveromyces lactis pheromone response pathway in the absence of the gamma subunit

Abstract

The Kluyveromyces lactis heterotrimeric G protein is a canonical Galphabetagamma complex; however, in contrast to Saccharomyces cerevisiae, where the Ggamma subunit is essential for mating, disruption of the KlGgamma gene yielded cells with almost intact mating capacity. Expression of a nonfarnesylated Ggamma, which behaves as a dominant-negative in S. cerevisiae, did not affect mating in wild-type and DeltaGgamma cells of K. lactis. In contrast to the moderate sterility shown by the single DeltaKlGalpha, the double DeltaKlGalpha DeltaKlGgamma mutant displayed full sterility. A partial sterile phenotype of the DeltaKlGgamma mutant was obtained in conditions where the KlGbeta subunit interacted defectively with the Galpha subunit. The addition of a CCAAX motif to the C-end of KlGbeta, partially suppressed the lack of both KlGalpha and KlGgamma subunits. In cells lacking KlGgamma, the KlGbeta subunit cofractionated with KlGalpha in the plasma membrane, but in the DeltaKlGalpha DeltaKlGgamma strain was located in the cytosol. When the KlGbeta-KlGalpha interaction was affected in the DeltaKlGgamma mutant, most KlGbeta fractionated to the cytosol. In contrast to the generic model of G-protein function, the Gbeta subunit of K. lactis has the capacity to attach to the membrane and to activate mating effectors in absence of the Ggamma subunit.

Figure 1.

(A) Ribbon representation of the Gβγ dimers from K. lactis and S. cerevisiae, showing Gβ in green and Gγ in red. Regions not modeled due to disorder are not shown. (B) Alignment of Gγs with identical (|), conserved (:), and nonconserved (.) residues. The CAAX motif in the C-termini of Gγs is boxed. (C) Residues forming the coiled-coil domain in Gβ and Gγ from both species. Amino acid residues are colored following the same pattern as the ribbon model, except for amino acids that make contact between the coiled-coil domains of Gβ and Gγ, which are shown in blue. Numbers in parentheses indicate the position of the last residue shown.

Figure 2.

(A) Disruption of the KlGγ gene. Cells were grown overnight in YPD medium, and genomic DNA from MATa (155) and MATα (12/8) wild-type strains and their ΔKlGγ mutants was obtained by a standard phenol-extraction protocol. DNA was digested with HindIII, subjected to Southern blot analysis and probed with the full radiolabeled Gγ gene. (B) Expression of the KlGγ gene. Cells were grown to midlog phase in YPD medium, harvested, and resuspended in water. Total RNA from wild-type and disrupted strains was extracted by the standard acidic-phenol protocol and was subjected to Northern blot analysis using a KlGγ gene probe. Large and small rRNA are indicated.

Figure 3.

Effect of inactivation of G protein subunits on mating. Mating was done by replica-plating strain 155 (WT) or its mutants onto YPD plates containing a lawn of MATα cells (strain 12/8), followed by incubation overnight at 30°C. Diploid selection was done by replica-plating onto SD. Pictures were taken after 48-h incubation at 30°C. For strains carrying plasmids YEpKD-HA-STE4 (+HAGβ) and YEpKD-HA-STE4R¹³⁰ (+HAGβR¹³⁰), mating was carried on YPGal plates.

Figure 4.

Physical association of G protein subunits determined by the two-hybrid system. The binding domain corresponds to LexA-fused proteins cloned into pEG202 and activation domain corresponds to B42-fused proteins cloned into pJG4-5. Gβ and Gγ from S. cerevisiae are named as Sc. Two-hybrid plasmids were introduced into strain EGY48, and two independent clones were plated on SGal plates containing 1 mg/ml X-Gal. Pictures were taken 24 h after incubation at 30°C. Quantitation of β-galactosidase activity was determined as described (Ongay-Larios et al., 2000). The β-galactosidase activity corresponds to the average value of three independent clones. The S. cerevisiae endochitinase (Cts1p) was used as negative interaction control.

Figure 5.

Immunodetection of the KlGβ subunit. Cells expressing the wild type (HAGβ) and mutant (HAGβR¹³⁰) HA epitope-tagged versions of KlGβ under the control of the GAL1 promoter (YEpKD plasmid) were fractioned as indicated in Materials and Methods. Fifteen micrograms of protein from the membrane (M) or cytosolic (C) fractions was resolved in SDS-PAGE and analyzed by immunoblotting with either anti-HA, anti-Gpa1 (Gα subunit), or anti-Hog1.

Figure 6.

Ribbon representation of the Gαβγ complexes from K. lactis and S. Cerevisiae. Gα is shown in blue, Gβ in green, and Gγ in red. Regions not modeled due to disorder are not shown. The K. lactis Trp130 (136 in S. cerevisiae) in Gβ is shown in yellow, relative to Ile278 (magenta) and Glu280 (orange) of the Gα subunit (positions 303 and 305, respectively, in S. cerevisiae).

Figure 7.

(A) Effect of the expression of Gβ-CCAAX on mating of ΔGαΔGγ mutant. Mating was done by replica-plating mutant strain ΔGαΔGγ (carrying YEpKD alone or YEpKD-HASTE4-CCAAX plasmid) onto YPGal plate containing a lawn of MATα cells (strain 12/8), followed by incubation overnight at 30°C. Diploid selection was done by replica-plating onto SD. Pictures were taken after 48-h incubation at 30°C. (B) Immunodetection of the KlGβ-CCAAX subunit. ΔGαΔGγ cells expressing the chimeric HAGβ-CCAAX protein under the control of the GAL1 promoter (YEpKD plasmid) were fractioned as indicated in Materials and Methods. Fifteen micrograms of protein from the membrane (M) or cytosolic (C) fractions was resolved in SDS-PAGE and analyzed by immunoblotting with either anti-HA or anti-Hog1.

Figure 8.

Role of G protein subunits in the K. lactis mating pathway. The three subunits of the trimeric protein play positive roles in the transmission of the pheromone stimulus. Although the Gβ subunit is the main transducer, Gα is required at some degree, and Gγ is dispensable. To be active, Gβ has to be tethered to the membrane by action of Gα and/or Gγ. Soluble Gβ is unable to activate the mating system. Gβ fused to a CCAAX motif (curved arrow) partially bypasses lack of Gα and Gγ. Gβ subunit defective in its interaction with Gα (indicated by the black star) can function only in presence of Gγ.