Francisco Torres-Quiroz

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.

Systematic identification of signal integration by protein kinase A.

Significance Protein kinase A (PKA) complexes are versatile signaling enzymes controlling homeostasis in eukaryotes. This enzyme is involved in multiple functions under physiological and pathological conditions in humans and governs the virulence of many pathogenic fungi. Here we systematically identify PKA regulators in yeast. Notably, we describe signaling to PKA that involves feedback from the cellular recycling process, autophagy. We also uncover a posttranslational modification, acetylation, that regulates PKA activity in both yeast and mammals, and we show that this mechanism impacts aging. Thus, we identify what regulates PKA as a first step toward the ability to cure diseases and infections, for instance, by providing new candidate genes for drug targeting in health research and antifungals for agricultural and medical purposes. Cellular processes and homeostasis control in eukaryotic cells is achieved by the action of regulatory proteins such as protein kinase A (PKA). Although the outbound signals from PKA directed to processes such as metabolism, growth, and aging have been well charted, what regulates this conserved regulator remains to be systematically identified to understand how it coordinates biological processes. Using a yeast PKA reporter assay, we identified genes that influence PKA activity by measuring protein–protein interactions between the regulatory and the two catalytic subunits of the PKA complex in 3,726 yeast genetic-deletion backgrounds grown on two carbon sources. Overall, nearly 500 genes were found to be connected directly or indirectly to PKA regulation, including 80 core regulators, denoting a wide diversity of signals regulating PKA, within and beyond the described upstream linear pathways. PKA regulators span multiple processes, including the antagonistic autophagy and methionine biosynthesis pathways. Our results converge toward mechanisms of PKA posttranslational regulation by lysine acetylation, which is conserved between yeast and humans and that, we show, regulates protein complex formation in mammals and carbohydrate storage and aging in yeast. Taken together, these results show that the extent of PKA input matches with its output, because this kinase receives information from upstream and downstream processes, and highlight how biological processes are interconnected and coordinated by PKA.

Integrative avenues for exploring the dynamics and evolution of protein interaction networks.

Over the past decade, the study of protein interaction networks (PINs) has shed light on the organizing principles of living cells. However, PINs have been mostly mapped in one single condition. We outline three of the most promising avenues of investigation in this field, namely the study of first, how PINs are rewired by mutations and environmental perturbations; secondly, how inter-species interactions affect PIN achitectures; thirdly, what mechanisms and forces drive PIN evolution. These investigations will unravel the dynamics and condition dependence of PINs and will thus lead to a better functional annotation of network architecture. One major challenge to reach these goals is the integration of PINs with other cellular regulatory networks in the context of complex cellular phenotypes.

Protein Kinases Involved in Mating and Osmotic Stress in the Yeast Kluyveromyces lactis

ABSTRACT 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 Gα and Gβ, 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.

Feedback regulation between autophagy and PKA.

Protein kinase A (PKA) controls diverse cellular processes and homeostasis in eukaryotic cells. Many processes and substrates of PKA have been described and among them are direct regulators of autophagy. The mechanisms of PKA regulation and how they relate to autophagy remain to be fully understood. We constructed a reporter of PKA activity in yeast to identify genes affecting PKA regulation. The assay systematically measures relative protein-protein interactions between the regulatory and catalytic subunits of the PKA complex in a systematic set of genetic backgrounds. The candidate PKA regulators we identified span multiple processes and molecular functions (autophagy, methionine biosynthesis, TORC signaling, protein acetylation, and DNA repair), which themselves include processes regulated by PKA. These observations suggest the presence of many feedback loops acting through this key regulator. Many of the candidate regulators include genes involved in autophagy, suggesting that not only does PKA regulate autophagy but that autophagy also sends signals back to PKA.

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

ABSTRACT 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 KlSTE2 and KlSTE3 genes encode MATα- 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 α‐pheromone and a‐pheromone receptors, respectively. However, by expression experiments, we determined that KlSTE2 gene is expressed in the cells typified as MATα and therefore is the receptor for the K. lactis a‐pheromone and KlSTE3 gene is expressed in the MATa cells and binds the α‐pheromone. The KlSTE2 gene is silent in MATa cells, while it is highly expressed in MATα cells, and conversely the KlSTE3 gene is expressed in MATa cells and repressed in MATα 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 (Gα) 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 Gα subunit, indicating that the C‐terminus is able to discriminate between the active (GTP‐bound) and inactive (GDP‐bound) forms of the Gα subunit. Copyright

αβ’‐NAC cooperates with Sam37 to mediate early stages of mitochondrial protein import

The mitochondrial proteome is mostly composed of nuclear‐encoded proteins. Such polypeptides are synthesized with signals that guide their intracellular transport to the surface of the organelle and later within the different mitochondrial subcompartments until they reach their functional destination. It has been suggested that the nascent‐polypeptide associated complex (NAC) – a cytosolic chaperone that recognizes nascent chains on translationally active ribosomes – has a role in the import of nuclear‐encoded mitochondrial proteins. However, the molecular mechanisms that regulate the NAC‐mediated cotranslational import are still not clear. Here, we show that a particular NAC heterodimer formed by subunits α and β′ in Saccharomyces cerevisiae is specifically involved in the process of mitochondrial import and functionally cooperates with Sam37, an outer membrane protein subunit of the sorting and assembly machinery complex. Mutants in both components display growth defects, incorrectly accumulate precursor forms of mitochondrial proteins in the cytosol, and have an altered mitochondrial protein content. We propose that αβ′‐NAC and Sam37 are members of the system that recognizes mitochondrial proteins at early stages of their synthesis, escorting them to the import machinery of mitochondria.

YAAM: Yeast Amino Acid Modifications Database

Abstract Proteins are dynamic molecules that regulate a myriad of cellular functions; these functions may be regulated by protein post-translational modifications (PTMs) that mediate the activity, localization and interaction partners of proteins. Thus, understanding the meaning of a single PTM or the combination of several of them is essential to unravel the mechanisms of protein regulation. Yeast Amino Acid Modification (YAAM) (http://yaam.ifc.unam.mx) is a comprehensive database that contains information from 121 921 residues of proteins, which are post-translationally modified in the yeast model Saccharomyces cerevisiae. All the PTMs contained in YAAM have been confirmed experimentally. YAAM database maps PTM residues in a 3D canvas for 680 proteins with a known 3D structure. The structure can be visualized and manipulated using the most common web browsers without the need for any additional plugin. The aim of our database is to retrieve and organize data about the location of modified amino acids providing information in a concise but comprehensive and user-friendly way, enabling users to find relevant information on PTMs. Given that PTMs influence almost all aspects of the biology of both healthy and diseased cells, identifying and understanding PTMs is critical in the study of molecular and cell biology. YAAM allows users to perform multiple searches, up to three modifications at the same residue, giving the possibility to explore possible regulatory mechanism for some proteins. Using YAAM search engine, we found three different PTMs of lysine residues involved in protein translation. This suggests an important regulatory mechanism for protein translation that needs to be further studied. Database URL: http://yaam.ifc.unam.mx/

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.