The Gβ(KlSte4p) subunit of the heterotrimeric G protein has a positive and essential role in the induction of mating in the yeast Kluyveromyces lactis

Abstract

In the yeast Saccharomyces cerevisiae the Gβγ dimer of the heterotrimeric G protein transduces a pheromone signal from serpentine receptor to a MAP kinase cascade that activates the mating response pathway. Haploid cells lacking the Gβ subunit do not respond to sexual pheromone, leading to sterility. In this work we demonstrate that the β-subunit of Kluyveromyces lactis, encoded by the KlSTE4 gene, is a component of the G protein, and that its disruption gives rise to sterile cells. However, unlike Ste4p in S. cerevisiae, its overexpression does not induce growth arrest or promote mating. It has been shown that in K. lactis, the Gα subunit has a positive role in the mating process, hence the resulting double Gα Gβ mutant was viable and sterile. Here we show that the overproduction of Gβ subunit fails to rescue Gα mutant from sterility and that expression of a constitutive active allele of Gα enhances transcription of the KlSTE4 gene. The mating pathway triggered by the Gβ-subunit requires a functional KlSte12p transcription factor. Gβ has a 10-fold higher association rate with the Gα1 subunit involved in pheromone response than with Gα2, the protein involved in cAMP regulation in K. lactis. Additionally, the Gβ-subunit from K. lactis is able to interact with the Gα-subunit from S. cerevisiae but fails to restore the mating deficiency of Scste4 mutant. The data presented indicate that the mating pathway of K. lactis is positively and cooperatively regulated by both the Gα and the Gβ subunits.

Ume6 Is Required for the MATa/MATα Cellular Identity and Transcriptional Silencing in Kluyveromyces lactis

“…We concluded that the absence of Ume6 must affect processes distinct from the a/a repression of hsgs to suppress the mating defect. ume6 is not a bypass suppressor: STE4 encodes the b-subunit of the heterotrimeric G-protein necessary for mating pheromone signaling and is essential for mating in S. cerevisiae (Whiteway et al 1989) and K. lactis (Kawasaki et al 2005). We investigated the possibility that the lack of Ume6 activated an unusual MATa mating pathway that was independent of mating pheromone signaling.…”

The KlSTE2 and KlSTE3 genes encode MATα‐ and MATa‐specific G‐protein‐coupled receptors, respectively, which are required for mating of Kluyveromyces lactis haploid cells

“…Thus, we conducted further experiments with the KlSte3p receptor. Initially we determined whether KlSte3p is able to physically interact with the G protein involved in transducing the pheromone signal during the mating response of haploid cells (Saviñón-Tejeda et al., , Kawasaki et al, 2005. Previously, we had shown that the cytoplamic C-terminus of the ScSte2p receptor interacts with both the α-and the β-subunits of the heterotrimeric G protein (Duran-Avelar et al, 2001), and that these interactions are required for proper coupling and transmission of the pheromone stimulus (OngayLarios et al, 2000).…”

“…In this experiment we measured the β-galactosidase activity induced by the magnitude of the interaction between proteins (Figure 4). As a positive control we included the activity induced by the interaction between the Gα (KlGpa1p)-and Gβ (KlSte4p)-subunits, which have shown strong association in previous reports (Kawasaki et al, 2005). Interaction of the KlGpa1p-subunit with the KlSte3p pheromone receptor is three-fold stronger than with the KlGpa2p-subunit, which is involved in regulating cAMP levels upon glucose stimulus in K. lactis (Saviñon-Tejeda et al, 1996).…”

“…The fragment encoding the C-terminus tail of KlSte3p was digested with the restriction enzyme SspI and cloned inframe into pJG4-5. Genes encoding Gα1 (KlGpa1p), Gα2 (KlGpa2p) and Gβ (KlSte4p) subunits were subcloned into the plasmid pEG202 (Kawasaki et al, 2005). Strain W303-1A was transfected with two-hybrid plasmids and grown overnight in SGal medium at 30 • C. Protein interactions were determined by the ability of hybrid proteins to induce expression of the LACZ reporter gene, located into the pSH18-34 plasmid.…”

“…Haploid MAT a and MATα cells undergo mating when they are mixed together in the same medium. In this process it has been shown that both the Gα (Saviñón-Tejeda, et al 2001) and the Gβ (Kawasaki et al, 2005) subunits of the heterotrimeric G protein transmit the pheromone signal to downstream effectors. Moreover, a constitutively active allele of Gα1 is able to rescue ∆Gα1 cells from sterility and induces moderate growth arrest in wild-type cells (Lloret et al, 2003).…”