Roberto Coria

Heterologously expressed fungal transient receptor potential channels retain mechanosensitivity in vitro and osmotic response in vivo

The budding yeast Saccharomyces cerevisiae has a mechanosensitive channel, TrpY1, a member of the Trp superfamily of channels associated with various sensations. Upon a hyperosmotic shift, a yeast cell releases Ca2+ from the vacuole to the cytoplasm through this channel. The TRPY1 gene has orthologs in other fungal genomes, including TRPY2 of Kluyveromyces lactis and TRPY3 of Candida albicans. We subcloned TRPY2 and TRPY3 and expressed them in the vacuole of S. cerevisiae deleted of TRPY1. The osmotically induced Ca2+ transient was restored in vivo as reported by transgenic aequorin. Patch-clamp examination showed that the TrpY2 or the TrpY3 channel was similar to TrpY1 in unitary conductance, rectification properties, Ca2+ sensitivity, and mechanosensitivity. The retention of mechanosensitivity of transient receptor potential channels in a foreign setting, shown here both in vitro and in vivo, implies that these mechanosensitive channels, like voltage-gated or ligand-gated channels, do not discriminate their settings. We discuss various mechanisms, including the possibility that stress from the lipid bilayer by osmotic force transmits forces to the transmembrane domains of these channels.

The Activity of Yeast Hog1 MAPK Is Required during Endoplasmic Reticulum Stress Induced by Tunicamycin Exposure

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers the so-called unfolded protein response (UPR), a conserved signaling pathway that drives the transcription of genes such as chaperones and folding enzymes. Nevertheless, the activity of the UPR accounts only for a part of the gene expression program activated upon ER stress. Moreover, the mechanism(s) for how cells adapt and survive to this stress are largely unknown. Here, we show that the yeast high osmolarity glycerol (HOG) pathway plays a role in ER stress resistance. Strains lacking the MAPK Hog1p displayed sensitivity to tunicamycin or β-mercaptoethanol, whereas hyperactivation of the pathway enhanced their resistance. However, these effects were not due to Hog1p-mediated regulation of the UPR. Northern blot analysis demonstrated that Hog1p controls the tunicamycin-induced transcriptional change of GPD1 and that wild-type cells exposed to the drug accumulated glycerol in a Hog1p-dependent manner. Consistent with this, deletion of genes involved in glycerol synthesis caused increased sensitivity to tunicamycin, whereas overexpression of GPD1 provided higher tolerance to both wild-type and hog1Δ mutant cells. Quite remarkably, these effects were mediated by the basal activity of the MAPK because tunicamycin exposure does not trigger the phosphorylation of Hog1p or its nuclear import. Hence, our results describe new aspects of the yeast response to ER stress and identify additional functions of glycerol and the Hog1p MAPK to provide stress resistance.

Isolation of a gene encoding a G protein α subunit involved in the regulation of cAMP levels in the yeast Kluyveromyces lactis

Using chromosomal DNA from Kluyveromyces lactis as template and oligodeoxynucleotides designed from conserved regions of various G protein alpha subunits we were able to amplify by the polymerase chain reaction two products of approximately 0·5 kb (P‐1) and 0·8 kb (P‐2). Sequencing showed that these two fragments share high homology with genes coding for the Gα subunits from different sources. Using the P‐1 fragment as a probe we screened a genomic library from K. lactis and we cloned a gene (KlGPA2) whose deduced amino acid sequence showed, depending on the exact alignment, 62% similarity and 38% identity with Gpa1p and 76% similarity and 63% identity with Gpa2p, the G protein α subunits from Saccharomyces cerevisiae. KlGPA2 is a single‐copy gene and its disruption rendered viable cells with significantly reduced cAMP level, indicating that this Gα subunit may be involved in regulating the adenylyl cyclase activity, rather than participating in the mating pheromone response pathway. KlGpa2p shares some structural similarities with members of the mammalian Gαs family (stimulatory of adenylyl cyclase) including the absence in its N‐terminus of a myristoyl‐modification sequence. The sequence reported in this paper has been deposited in the GenBank data base (Accession No. L45105).

Yeast communities associated with artisanal mezcal fermentations from Agave salmiana

The aims of this work were to characterize the fermentation process of mezcal from San Luis Potosi, México and identify the yeasts present in the fermentation using molecular culture-dependent methods (RFLP of the 5.8S-ITS and sequencing of the D1/D2 domain) and also by using a culture-independent method (DGGE). The alcoholic fermentations of two separate musts obtained from Agave salmiana were analyzed. Sugar, ethanol and major volatile compounds concentrations were higher in the first fermentation, which shows the importance of having a quality standard for raw materials, particularly in the concentration of fructans, in order to produce fermented Agave salmiana must with similar characteristics. One hundred ninety-two (192) different yeast colonies were identified, from those present on WL agar plates, by RFLP analysis of the ITS1-5.8S- ITS2 from the rRNA gene, with restriction endonucleases, HhaI, HaeIII and HinfI. The identified yeasts were: Saccharomyces cerevisiae, Kluyveromyces marxianus, Pichia kluyveri, Zygosaccharomyces bailii, Clavispora lusitaniae, Torulaspora delbrueckii, Candida ethanolica and Saccharomyces exiguus. These identifications were confirmed by sequencing the D1-D2 region of the 26S rRNA gene. With the PCR-DGGE method, bands corresponding to S. cerevisiae, K. marxianus and T. delbrueckii were clearly detected, confirming the results obtained with classic techniques.

The Leu-132 of the Ste4(Gβ) subunit is essential for proper coupling of the G protein with the Ste2 α factor receptor during the mating pheromone response in yeast

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 α 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 β 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 α 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 pheromone‐stimulated receptor and that this domain could be an important point for physical interaction between the Gβ and the Gα subunits.

The deduced primary structure of subunit I from cytochrome c oxidase suggests that the genus Polytomella shares a common mitochondrial origin with Chlamydomonas

We cloned and sequenced the mitochondrial gene encoding subunit I of cytochrome c oxidase (coxI) of Polytomella spp., a colorless alga related to Chlamydomonas. The purpose was to explore whether homology between the two species also exists at the level of a mitochondrial enzyme. The gene is 1512 bp long and contains no introns. The translated protein sequence exhibits 73.8% identity with its Chlamydomonas reinhardtii counterpart. The data obtained support the hypothesis that the separation of the colorless alga from the Chlamydomonas lineage was a late event in evolution, that occurred after the endosymbiotic process that gave rise to mitochondria.

The KlGpa1 Gene Encodes a G-Protein α Subunit That Is a Positive Control Element in the Mating Pathway of the Budding Yeast Kluyveromyces lactis

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 alpha 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 DeltaKlgpa1 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 DeltaKlgpa1 mutant cells. K. lactis expresses a second Galpha subunit, KlGpa2p, which is involved in regulating cyclic AMP levels upon glucose stimulation. This subunit does not rescue DeltaKlgpa1 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 DeltaKlgpa1 DeltaKlgpa2 double mutant, although viable, showed the mating deficiency observed in the single DeltaKlgpa1 mutant.

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

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 GαΔ GβΔ mutant was viable and sterile. Here we show that the overproduction of Gβ subunit fails to rescue GαΔ 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 Scste4Δ 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. Copyright

The yeast potassium transporter TRK2 is able to substitute for TRK1 in its biological function under low K and low pH conditions.

In S. cerevisiae, K+ transport relies principally on two structurally related membrane proteins, known as Trk1p and Trk2p. Direct involvement in cation movements has been demonstrated for Trk1p, which is a high‐affinity K+ transporter. Initially described as a low‐affinity K+ transporter, Trk2p seems to play a minor role in K+ transport, since its activity is only apparent under very specific conditions, such as in a Δsin3 background. Here we show that growth of a Δtrk1Δsin3 double mutant, under K+‐limiting conditions or at low pH, is Trk2p‐dependent, and by Northern blot analysis we demonstrate that deletion of SIN3 results in transcriptional derepression of TRK2. In addition, we show that heterologous overexpression of TRK2 with the inducible GAL1 promoter bypasses Sin3p repression in a Δtrk1Δtrk2 double mutant and fully restores growth under non‐permissive conditions. Furthermore, kinetic experiments in a Δtrk1Δsin3 double mutant revealed a K+ transporter with an apparent high affinity and a moderate capacity. Taken together, these results indicate that TRK2 encodes a functional K+ transporter that, under our experimental conditions, displays distinctive kinetic characteristics. Copyright

Autophagy in Dictyostelium: Mechanisms, regulation and disease in a simple biomedical model.

ABSTRACT Autophagy is a fast-moving field with an enormous impact on human health and disease. Understanding the complexity of the mechanism and regulation of this process often benefits from the use of simple experimental models such as the social amoeba Dictyostelium discoideum. Since the publication of the first review describing the potential of D. discoideum in autophagy, significant advances have been made that demonstrate both the experimental advantages and interest in using this model. Since our previous review, research in D. discoideum has shed light on the mechanisms that regulate autophagosome formation and contributed significantly to the study of autophagy-related pathologies. Here, we review these advances, as well as the current techniques to monitor autophagy in D. discoideum. The comprehensive bioinformatics search of autophagic proteins that was a substantial part of the previous review has not been revisited here except for those aspects that challenged previous predictions such as the composition of the Atg1 complex. In recent years our understanding of, and ability to investigate, autophagy in D. discoideum has evolved significantly and will surely enable and accelerate future research using this model.