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.

The Unfolded Protein Response (UPR) is an adaptive pathway that restores cellular homeostasis after endoplasmic reticulum (ER) stress. The ER-resident kinase/ribonuclease Ire1 is the only UPR sensor conserved during evolution. Autophagy, a lysosomal degradative pathway, also contributes to the recovery of cell homeostasis after ER-stress but the interplay between these two pathways is still poorly understood. We describe the ER-stress response and characterize its single Ire1 orthologue, IreA. We found that tunicamycin (TN) triggers a gene-expression reprogramming that increases the protein folding capacity of the ER and alleviates ER protein load. Further, IreA is required for cell-survival after TN-induced ER-stress and is responsible for nearly 40% of the transcriptional changes induced by TN. The response of cells to ER-stress involves the combined activation of an IreA-dependent gene expression program and the autophagy pathway. These two pathways are independently activated in response to ER-stress but, interestingly, autophagy requires IreA at a later stage for proper autophagosome formation. We propose that unresolved ER-stress in cells lacking IreA causes structural alterations of the ER, leading to a late-stage blockade of autophagy clearance. This unexpected functional link may critically affect eukaryotic cell survival under ER-stress.

BackgroundGene duplication and the subsequent divergence of paralogous pairs play a central role in the evolution of novel gene functions. S. cerevisiae possesses two paralogous genes (ALT1/ALT2) which presumably encode alanine aminotransferases. It has been previously shown that Alt1 encodes an alanine aminotransferase, involved in alanine metabolism; however the physiological role of Alt2 is not known. Here we investigate whether ALT2 encodes an active alanine aminotransferase.Principal FindingsOur results show that although ALT1 and ALT2 encode 65% identical proteins, only Alt1 displays alanine aminotransferase activity; in contrast ALT2 encodes a catalytically inert protein. ALT1 and ALT2 expression is modulated by Nrg1 and by the intracellular alanine pool. ALT1 is alanine-induced showing a regulatory profile of a gene encoding an enzyme involved in amino acid catabolism, in agreement with the fact that Alt1 is the sole pathway for alanine catabolism present in S. cerevisiae. Conversely, ALT2 expression is alanine-repressed, indicating a role in alanine biosynthesis, although the encoded-protein has no alanine aminotransferase enzymatic activity. In the ancestral-like yeast L. kluyveri, the alanine aminotransferase activity was higher in the presence of alanine than in the presence of ammonium, suggesting that as for ALT1, LkALT1 expression could be alanine-induced. ALT2 retention poses the questions of whether the encoded protein plays a particular function, and if this function was present in the ancestral gene. It could be hypotesized that ALT2 diverged after duplication, through neo-functionalization or that ALT2 function was present in the ancestral gene, with a yet undiscovered function.Conclusions ALT1 and ALT2 divergence has resulted in delegation of alanine aminotransferase activity to Alt1. These genes display opposed regulatory profiles: ALT1 is alanine-induced, while ALT2 is alanine repressed. Both genes are negatively regulated by the Nrg1 repressor. Presented results indicate that alanine could act as ALT2 Nrg1-co-repressor.

The cloning of the gene encoding the KlGpa1p subunit was achieved by standard PCR techniques and by screening a Kluyveromyces lactis genomic library using the PCR product as a probe. The full-length open reading frame spans 1,344 nucleotides including the stop codon. The deduced primary structure of the protein (447 amino acid residues) strongly resembles that of Gpa1p, the G-protein ␣ subunit from Saccharomyces cerevisiae involved in the mating pheromone response pathway. Nevertheless, unlike disruption of Gpa1 from S. cerevisiae, disruption of KlGpa1 rendered viable cells with a reduced capacity to mate. Expression of a plasmidic KlGpa1 copy in a ⌬Klgpa1 mutant restores full mating competence; hence we conclude that KlGpa1p plays a positive role in the mating pathway. Overexpression of the constitutive subunit KlGpa1p(K 364 ) (GTP bound) does not induce constitutive mating; instead it partially blocks wild-type mating and is unable to reverse the sterile phenotype of ⌬Klgpa1 mutant cells. K. lactis expresses a second G␣ subunit, KlGpa2p, which is involved in regulating cyclic AMP levels upon glucose stimulation. This subunit does not rescue ⌬Klgpa1 cells from sterility; instead, overproduction of KlGpa2p slightly reduces the mating of wild-type cells, suggesting cross talk within the pheromone response pathway mediated by KlGpa1p and glucose metabolism mediated by KlGpa2p. The ⌬Klgpa1 ⌬Klgpa2 double mutant, although viable, showed the mating deficiency observed in the single ⌬Klgpa1 mutant.

In order to identify amino acid residues of Ste4p involved in receptor recognition and/or receptor-G protein coupling, we employed random in vitro mutagenesis and a genetic screening to isolate mutant Ste4p subunits with altered pheromone response. We generated a plasmid library containing randomly mutagenized Ste4 ORFs, followed by phenotypic selection of ste4p mutants by altered K K pheromone response in yeast cells. Subsequently, we analyzed mutant ste4-10 which has a replacement of the almost universally conserved leucine 132 by phenylalanine. This residue lies in the first blade of the L L propeller structure proposed by crystallographic analysis. By overexpression experiments we found that mutant ste4p subunit triggers the mating pathway at wild type levels in both wild type and receptorless strains. When expressed in a ste4 background, however, the mutant G protein is activated inefficiently by mating pheromone in both a and K K cells. The mutant ste4-10p was tested in the two-hybrid system and found to be defective in its interaction with the Gpa1p, but has a normal association with the C-termini end of the Ste2p receptor. These observations strongly suggest that the Leu-132 of the Ste4p subunit is essential for efficient activation of the G protein by the pheromonestimulated receptor and that this domain could be an important point for physical interaction between the GL L and the GK K subunits.z 2000 Federation of European Biochemical Societies.