Prohibitins, Phb1 and Phb2, function as Atg8 receptors to support yeast mitophagy and also play a negative regulatory role in Atg32 processing.

The prohibitins Phb1 and Phb2 assemble at the mitochondrial inner membrane to form a multi-dimeric complex. These scaffold proteins are highly conserved in eukaryotic cells, from yeast to mammals, and have been implicated in a variety of mitochondrial functions including aging, proliferation, and degenerative and metabolic diseases. In mammals, PHB2 regulates PINK1-PRKN mediated mitophagy by interacting with lipidated MAP1LC3B/LC3B. Despite their high conservation, prohibitins have not been linked to mitophagy in budding yeasts. In this study, we demonstrate that both Phb1 and Phb2 are required to sustain mitophagy in Saccharomyces cerevisiae. Prohibitin-dependent mitophagy requires formation of the Phb1-Phb2 complex and a conserved AIM/LIR-like motif identified in both yeast prohibitins. Furthermore, both Phb1 and Phb2 interact and exhibit mitochondrial colocalization with Atg8. Interestingly, we detected a basal C terminus processing of the mitophagy receptor Atg32 that depends on the presence of the i-AAA Yme1. In the absence of prohibitins this processing is highly enhanced but reverted by the inactivation of the rhomboid protease Pcp1. Together our results revealed a novel role of yeast prohibitins in mitophagy through its interaction with Atg8 and regulating an Atg32 proteolytic event. Abbreviation: AIM/LIR: Atg8-family interacting motif/LC3-interacting region; ANOVA: analysis of variance; ATG/Atg: autophagy related; C terminus/C-terminal: carboxyl terminus/carboxyl-terminal; GFP: green fluorescent protein; HA: human influenza hemagglutinin; Idh1: isocitrate dehydrogenase 1; MAP1C3B/LC3B: microtubule associated protein 1 light chain 3 beta; mCh: mCherry; MIM: mitochondrial inner membrane; MOM: mitochondrial outer membrane; N starvation: nitrogen starvation; N terminus: amino terminus; PARL: presenilin associated rhomboid like; Pcp1: processing of cytochrome c peroxidase 1; PCR: polymerase chain reaction; PGAM5: PGAM family member 5 mitochondrial serine/threonine protein phosphatase; PHBs/Phb: prohibitins; PINK1: PTEN induced kinase 1; PMSF: phenylmethylsulfonyl fluoride; PRKN: parkin RBR E3 ubiquitin protein ligase; SD: synthetic defined medium; SDS: sodium dodecyl sulfate; SMD-N: synthetic defined medium lacking nitrogen; WB: western blot; WT: wild type; Yme1: yeast mitochondrial escape 1; YPD: yeast extract-peptone-dextrose medium; YPLac: yeast extract-peptone-lactate medium.

The yeast two-component SLN1 branch of the HOG pathway and the scaffolding activity of Pbs2 modulate the response to endoplasmic reticulum stress induced by tunicamycin / La rama SLN1 de dos componentes de levadura de la vía HOG y la actividad de andamiaje de Pbs2 modulan la respuesta al estrés del retículo endoplásmico inducido por tunicamicina

In addition to the UPR pathway, yeast cells require components of the HOG pathway to respond to ER stress. In this work, we found that unphosphorylated Sln1 and Ssk1 are required to mount an appropriate response to Tn. We also found that the MAPKKKs Ssk2 participates in the Tn response, but its osmo-redundant protein Ssk22 does not. We also found that the Pbs2 docking sites for Ssk2 (RDS-I and KD) are partially dispensable when mutated separately; however, the prevention of Ssk2 binding to Pbs2, by the simultaneous mutation of RDS-I and KD, caused strong sensitivity to Tn. In agreement with the lack of Hog1 phosphorylation during Tn treatment, a moderate resistance to Tn is obtained when a Pbs2 version lacking its kinase activity is expressed; however, the presence of mutual Pbs2-Hog1 docking sites is essential for the Tn response. Finally, we detected that Tn induced a transcriptional activation of some components of the SLN1 branch. These results indicate that the Tn response requires a complex formed by the MAPK module and components of the SLN1 branch but not their canonical osmoregulatory activities.(AU)

The yeast two-component SLN1 branch of the HOG pathway and the scaffolding activity of Pbs2 modulate the response to endoplasmic reticulum stress induced by tunicamycin.

In addition to the UPR pathway, yeast cells require components of the HOG pathway to respond to ER stress. In this work, we found that unphosphorylated Sln1 and Ssk1 are required to mount an appropriate response to Tn. We also found that the MAPKKKs Ssk2 participates in the Tn response, but its osmo-redundant protein Ssk22 does not. We also found that the Pbs2 docking sites for Ssk2 (RDS-I and KD) are partially dispensable when mutated separately; however, the prevention of Ssk2 binding to Pbs2, by the simultaneous mutation of RDS-I and KD, caused strong sensitivity to Tn. In agreement with the lack of Hog1 phosphorylation during Tn treatment, a moderate resistance to Tn is obtained when a Pbs2 version lacking its kinase activity is expressed; however, the presence of mutual Pbs2-Hog1 docking sites is essential for the Tn response. Finally, we detected that Tn induced a transcriptional activation of some components of the SLN1 branch. These results indicate that the Tn response requires a complex formed by the MAPK module and components of the SLN1 branch but not their canonical osmoregulatory activities.

Negative feedback-loop mechanisms regulating HOG- and pheromone-MAPK signaling in yeast.

The pheromone response and the high osmolarity glycerol (HOG) pathways are considered the prototypical MAPK signaling systems. They are the best-understood pathways in eukaryotic cells, yet they continue to provide insights in how cells relate with the environment. These systems are subjected to tight regulatory circuits to prevent hyperactivation in length and intensity. Failure to do this may be a matter of life or death specially for unicellular organisms such as Saccharomyces cerevisiae. The signaling pathways are fine-tuned by positive and negative feedback loops exerted by pivotal control elements that allow precise responses to specific stimuli, despite the fact that some elements of the systems are common to different signaling pathways. Here we describe the experimentally proven negative feedback loops that modulate the pheromone response and the HOG pathways. As described in this review, MAP kinases are central mechanistic components of these feedback loops. They have the capacity to modulate basal signaling activity, a fast extranuclear response, and a longer-lasting transcriptional process.

Tunicamycin Sensitivity-Suppression by High Gene Dosage Reveals New Functions of the Yeast Hog1 MAP Kinase.

In the yeast Saccharomyces cerevisiae, components of the High Osmolarity Glycerol (HOG) pathway are important for the response to diverse stresses including response to endoplasmic reticulum stress (ER stress), which is produced by the accumulation of unfolded proteins in the lumen of this organelle. Accumulation of unfolded proteins may be due to the inhibition of protein N-glycosylation, which can be achieved by treatment with the antibiotic tunicamycin (Tn). In this work we were interested in finding proteins involved in the ER stress response regulated by Hog1, the mitogen activated protein kinase (MAPK) of the HOG pathway. A high gene dosage suppression screening allowed us to identify genes that suppressed the sensitivity to Tn shown by a hog1Δ mutant. The suppressors participate in a limited number of cellular processes, including lipid/carbohydrate biosynthesis and protein glycosylation, vesicle-mediated transport and exocytosis, cell wall organization and biogenesis, and cell detoxification processes. The finding of suppressors Rer2 and Srt1, which participate in the dolichol biosynthesis pathway revealed that the hog1Δ strain has a defective polyprenol metabolism. This work uncovers new genetic and functional interactors of Hog1 and contributes to a better understanding of the participation of this MAPK in the ER stress response.

The Unfolded Protein Response Pathway in the Yeast Kluyveromyces lactis. A Comparative View among Yeast Species.

Eukaryotic cells have evolved signalling pathways that allow adaptation to harmful conditions that disrupt endoplasmic reticulum (ER) homeostasis. When the function of the ER is compromised in a condition known as ER stress, the cell triggers the unfolded protein response (UPR) in order to restore ER homeostasis. Accumulation of misfolded proteins due to stress conditions activates the UPR pathway. In mammalian cells, the UPR is composed of three branches, each containing an ER sensor (PERK, ATF6 and IRE1). However, in yeast species, the only sensor present is the inositol-requiring enzyme Ire1. To cope with unfolded protein accumulation, Ire1 triggers either a transcriptional response mediated by a transcriptional factor that belongs to the bZIP transcription factor family or an mRNA degradation process. In this review, we address the current knowledge of the UPR pathway in several yeast species: Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida glabrata, Cryptococcus neoformans, and Candida albicans. We also include unpublished data on the UPR pathway of the budding yeast Kluyveromyces lactis. We describe the basic components of the UPR pathway along with similarities and differences in the UPR mechanism that are present in these yeast species.

IreA Controls Endoplasmic Reticulum Stress-Induced Autophagy and Survival through Homeostasis Recovery.

The unfolded protein response (UPR) is an adaptive pathway that restores cellular homeostasis after endoplasmic reticulum (ER) stress. The ER-resident kinase/RNase 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 Dictyostelium discoideum ER stress response and characterize its single bona fide 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 Dictyostelium 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.

Activation of the Hog1 MAPK by the Ssk2/Ssk22 MAP3Ks, in the absence of the osmosensors, is not sufficient to trigger osmostress adaptation in Saccharomyces cerevisiae.

Yeast cells respond to hyperosmotic stress by activating the high-osmolarity glycerol (HOG) pathway, which consists of two branches, Hkr1/Msb2-Sho1 and Sln1, which trigger phosphorylation and nuclear internalization of the Hog1 mitogen-activated protein kinase. In the nucleus, Hog1 regulates gene transcription and cell cycle progression, which allows the cell to respond and adapt to hyperosmotic conditions. This study demonstrates that the uncoupling of the known sensors of both branches of the pathway at the level of Ssk1 and Ste11 impairs cell growth in hyperosmotic medium. However, under these conditions, Hog1 was still phosphorylated and internalized into the nucleus, suggesting the existence of an alternative Hog1 activation mechanism. In the ssk1ste11 mutant, phosphorylated Hog1 failed to associate with chromatin and to activate transcription of canonical hyperosmolarity-responsive genes. Accordingly, Hog1 also failed to induce glycerol production at the levels of a wild-type strain. Inactivation of the Ptp2 phosphatase moderately rescued growth impairment of the ssk1ste11 mutant under hyperosmotic conditions, indicating that downregulation of the HOG pathway only partially explains the phenotypes displayed by the ssk1ste11 mutant. Cell cycle defects were also observed in response to stress when Hog1 was phosphorylated in the ssk1ste11 mutant. Taken together, these observations indicate that Hog1 phosphorylation by noncanonical upstream mechanisms is not sufficient to trigger a protective response to hyperosmotic stress.

Role of the Sln1-phosphorelay pathway in the response to hyperosmotic stress in the yeast Kluyveromyces lactis.

The Kluyveromyces lactis SLN1 phosphorelay system includes the osmosensor histidine kinase Sln1, the phosphotransfer protein Ypd1 and the response regulator Ssk1. Here we show that K. lactis has a functional phosphorelay system. In vitro assays, using a heterologous histidine kinase, show that the phosphate group is accepted by KlYpd1 and transferred to KlSsk1. Upon hyperosmotic stress the phosphorelay is inactivated, KlYpd1 is dephosphorylated in a KlSln1 dependent manner, and only the version of KlSsk1 that lacks the phosphate group interacts with the MAPKKK KlSsk2. Interestingly, inactivation of the KlPtp2 phosphatase in a ΔKlsln1 mutant did not lead to KlHog1 constitutive phosphorylation. KlHog1 can replace ScHog1p and activate the hyperosmotic response in Saccharomyces cerevisiae, and when ScSln1 is inactivated, KlHog1 becomes phosphorylated and induces cell lethality. All these observations indicate that the phosphorelay negatively regulates KlHog1. Nevertheless, in the absence of KlSln1 or KlYpd1, no constitutive phosphorylation is detected and cells are viable, suggesting that a strong negative feedback that is independent of KlPtp2 operates in K. lactis. Compared with S. cerevisiae, K. lactis has only a moderate accumulation of glycerol and fails to produce trehalose under hyperosmotic stress, indicating that regulation of osmolyte production is different in K. lactis.

Ineffective Phosphorylation of Mitogen-Activated Protein Kinase Hog1p in Response to High Osmotic Stress in the Yeast Kluyveromyces lactis.

When treated with a hyperosmotic stimulus, Kluyveromyces lactis cells respond by activating the mitogen-activated protein kinase (MAPK) K. lactis Hog1 (KlHog1) protein via two conserved branches, SLN1 and SHO1. Mutants affected in only one branch can cope with external hyperosmolarity by activating KlHog1p by phosphorylation, except for single ΔKlste11 and ΔKlste50 mutants, which showed high sensitivity to osmotic stress, even though the other branch (SLN1) was intact. Inactivation of both branches by deletion of KlSHO1 and KlSSK2 also produced sensitivity to high salt. Interestingly, we have observed that in ΔKlste11 and ΔKlsho1 ΔKlssk2 mutants, which exhibit sensitivity to hyperosmotic stress, and contrary to what would be expected, KlHog1p becomes phosphorylated. Additionally, in mutants lacking both MAPK kinase kinases (MAPKKKs) present in K. lactis (KlSte11p and KlSsk2p), the hyperosmotic stress induced the phosphorylation and nuclear internalization of KlHog1p, but it failed to induce the transcriptional expression of KlSTL1 and the cell was unable to grow in high-osmolarity medium. KlHog1p phosphorylation via the canonical HOG pathway or in mutants where the SHO1 and SLN1 branches have been inactivated requires not only the presence of KlPbs2p but also its kinase activity. This indicates that when the SHO1 and SLN1 branches are inactivated, high-osmotic-stress conditions activate an independent input that yields active KlPbs2p, which, in turn, renders KlHog1p phosphorylation ineffective. Finally, we found that KlSte11p can alleviate the sensitivity to hyperosmotic stress displayed by a ΔKlsho1 ΔKlssk2 mutant when it is anchored to the plasma membrane by adding the KlSho1p transmembrane segments, indicating that this chimeric protein can substitute for KlSho1p and KlSsk2p.

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

Yeast mating signal transduction pathways require a heterotrimeric G protein composed of Gα, Gß, 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 KlGß 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.

Aspergillus nidulans transcription factor AtfA interacts with the MAPK SakA to regulate general stress responses, development and spore functions.

Fungi utilize a phosphorelay system coupled to a MAP kinase module for sensing and processing environmental signals. In Aspergillus nidulans, response regulator SskA transmits osmotic and oxidative stress signals to the stress MAPK (SAPK) SakA. Using a genetic approach together with GFP tagging and molecular bifluorescence we show that SakA and ATF/CREB transcription factor AtfA define a general stress-signalling pathway that plays differential roles in oxidative stress responses during growth and development. AtfA is permanently localized in the nucleus, while SakA accumulates in the nucleus in response to oxidative or osmotic stress signals or during normal spore development, where it physically interacts with AtfA. AtfA is required for expression of several genes, the conidial accumulation of SakA and the viability of conidia. Furthermore, SakA is active (phosphorylated) in asexual spores, remaining phosphorylated in dormant conidia and becoming dephosphorylated during germination. SakA phosphorylation in spores depends on certain (SskA) but not other (SrrA and NikA) components of the phosphorelay system. Constitutive phosphorylation of SakA induced by the fungicide fludioxonil prevents both, germ tube formation and nuclear division. Similarly, Neurospora crassa SakA orthologue OS-2 is phosphorylated in intact conidia and gets dephosphorylated during germination. We propose that SakA-AtfA interaction regulates gene expression during stress and conidiophore development and that SAPK phosphorylation is a conserved mechanism to regulate transitions between non-growing (spore) and growing (mycelia) states.

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

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.

Protein kinases involved in mating and osmotic stress in the yeast Kluyveromyces lactis.

Systematic disruption of genes encoding kinases and mitogen-activated protein kinases (MAPKs) was performed in Kluyveromyces lactis haploid cells. The mutated strains were assayed by their capacity to mate and to respond to hyperosmotic stress. The K. lactis Ste11p (KlSte11p) MAPK kinase kinase (MAPKKK) was found to act in both mating and osmoresponse pathways while the scaffold KlSte5p and the MAPK KlFus3p appeared to be specific for mating. The p21-activated kinase KlSte20p and the kinase KlSte50p participated in both pathways. Protein association experiments showed interaction of KlSte50p and KlSte20p with Galpha and Gbeta, respectively, the G protein subunits involved in the mating pathway. Both KlSte50p and KlSte20p also showed interaction with KlSte11p. Disruption mutants of the K. lactis PBS2 (KlPBS2) and KlHOG1 genes of the canonical osmotic response pathway resulted in mutations sensitive to high salt and high sorbitol but dispensable for mating. Mutations that eliminate the MAPKK KlSte7p activity had a strong effect on mating and also showed sensitivity to osmotic stress. Finally, we found evidence of physical interaction between KlSte7p and KlHog1p, in addition to diminished Hog1p phosphorylation after a hyperosmotic shock in cells lacking KlSte7p. This study reveals novel roles for components of transduction systems in yeast.

Response regulators SrrA and SskA are central components of a phosphorelay system involved in stress signal transduction and asexual sporulation in Aspergillus nidulans.

Among eukaryotes, only slime molds, fungi, and plants contain signal transduction phosphorelay systems. In filamentous fungi, multiple sensor kinases appear to use a single histidine-containing phosphotransfer (HPt) protein to relay signals to two response regulators (RR). In Aspergillus nidulans, the RR SskA mediates activation of the mitogen-activated protein kinase SakA in response to osmotic and oxidative stress, whereas the functions of the RR SrrA were unknown. We used a genetic approach to characterize the srrA gene as a new member of the skn7/prr1 family and to analyze the roles of SrrA in the phosphorelay system composed of the RR SskA, the HPt protein YpdA, and the sensor kinase NikA. While mutants lacking the HPt protein YpdA are unviable, mutants lacking SskA (DeltasskA), SrrA (DeltasrrA), or both RR (DeltasrrA DeltasskA) are viable and differentially affected in osmotic and oxidative stress responses. Both RR are involved in osmostress resistance, but DeltasskA mutants are more sensitive to this stress, and only SrrA is required for H(2)O(2) resistance and H(2)O(2)-mediated induction of catalase CatB. In contrast, both RR are individually required for fungicide sensitivity and calcofluor resistance and for normal sporulation and conidiospore viability. The DeltasrrA and DeltasskA sporulation defects appear to be related to decreased mRNA levels of the key sporulation gene brlA. In contrast, conidiospore viability defects do not correlate with the activity of the spore-specific catalase CatA. Our results support a model in which NikA acts upstream of SrrA and SskA to transmit fungicide signals and to regulate asexual sporulation and conidiospore viability. In contrast, NikA appears dispensable for osmotic and oxidative stress signaling. These results highlight important differences in stress signal transmission among fungi and define a phosphorelay system involved in oxidative and osmotic stress, cell wall maintenance, fungicide sensitivity, asexual reproduction, and spore viability.

Kluyveromyces lactis sexual pheromones. Gene structures and cellular responses to alpha-factor.

The Kluyveromyces lactis genes for sexual pheromones have been analyzed. The alpha-factor gene encodes a predicted polypeptide of 187 amino acid residues containing four tridecapeptide repeats (WSWITLRPGQPIF). A nucleotide blast search of the entire K. lactis genome sequence allowed the identification of the nonannotated putative a-pheromone gene that encodes a predicted protein of 33 residues containing one copy of the dodecapeptide a-factor (WIIPGFVWVPQC). The role of the K. lactis structural genes KlMFalpha1 and KlMFA1 in mating has been investigated by the construction of disruption mutations that totally eliminate gene functions. Mutants of both alleles showed sex-dependent sterility, indicating that these are single-copy genes and essential for mating. MATalpha, Klsst2 mutants, which, by analogy to Saccharomyces cerevisiae, are defective in Galpha-GTPase activity, showed increased sensitivity to synthetic alpha-factor and increased capacity to mate. Additionally, Klbar1 mutants (putatively defective in alpha-pheromone proteolysis) showed delay in mating but sensitivity to alpha-pheromone. From these results, it can be deduced that the K. lactis MATa cell produces the homolog of the S. cerevisiaealpha-pheromone, whereas the MATalpha cell produces the a-pheromone.

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

Mating in yeast is initiated by binding of pheromone to G-protein-coupled receptors expressed in haploid cells. We analysed the role of KlSte2p and KlSte3p receptors in the Kluyveromyces lactis mating pathway. By sequence analysis, KlSte2p and KlSte3p are the homologues of the Saccharomyces cerevisiae alpha-pheromone and a-pheromone receptors, respectively. However, by expression experiments, we determined that KlSTE2 gene is expressed in the cells typified as MATalpha and therefore is the receptor for the K. lactis a-pheromone and KlSTE3 gene is expressed in the MATa cells and binds the alpha-pheromone. The KlSTE2 gene is silent in MATa cells, while it is highly expressed in MATalpha cells, and conversely the KlSTE3 gene is expressed in MATa cells and repressed in MATalpha cells. Disruption mutants of both genes were found to be sterile, and this defect is reversed by plasmidic copies of each gene. The cytoplasmic C-terminus of KlSte3p interacts strongly with the KlGpa1p (Galpha) subunit, which is involved in the transduction of the pheromone stimulus to induce mating. Remarkably, this same domain does not interact with a constitutive active allele of the Galpha subunit, indicating that the C-terminus is able to discriminate between the active (GTP-bound) and inactive (GDP-bound) forms of the Galpha subunit.

The pheromone response pathway of Kluyveromyces lactis.

The mating pheromone response pathway in Saccharomyces cerevisiae is one of the best understood signalling pathways in eukaryotes. Comparison of this system with pathways in other fungal species has generated surprises and insights. Cloning and targetted disruption of genes encoding components of the pheromone response pathway has allowed the attribution of specific functions to these signal transduction components. In this review we describe current knowledge of the Kluyveromyces lactis mating system, and compare it with the well-understood S. cerevisiae pathway, emphasizing the similarities and differences in the heterotrimeric G protein activity. This mating pathway is controlled positively by both the Galpha and the Gbeta subunits of the heterotrimeric G protein.

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

In the yeast Saccharomyces cerevisiae the Gbetagamma 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 Gbeta subunit do not respond to sexual pheromone, leading to sterility. In this work we demonstrate that the beta-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 Galpha subunit has a positive role in the mating process, hence the resulting double GalphaDelta GbetaDelta mutant was viable and sterile. Here we show that the overproduction of Gbeta subunit fails to rescue GalphaDelta mutant from sterility and that expression of a constitutive active allele of Galpha enhances transcription of the KlSTE4 gene. The mating pathway triggered by the Gbeta-subunit requires a functional KlSte12p transcription factor. Gbeta has a 10-fold higher association rate with the Galpha1 subunit involved in pheromone response than with Galpha2, the protein involved in cAMP regulation in K. lactis. Additionally, the Gbeta-subunit from K. lactis is able to interact with the Galpha-subunit from S. cerevisiae but fails to restore the mating deficiency of Scste4Delta mutant. The data presented indicate that the mating pathway of K. lactis is positively and cooperatively regulated by both the Galpha and the Gbeta subunits.