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GAS1, GAS2, GAS3 and GAS4 : four developmentally regulated genes with specialized roles at different stages of the yeast life cycle
No full textThe formation of Saccharomyces cerevisiae cell wall requires the coordinated activity of enzymes involved in the biosynthesis and modification of its components, such as glucans.
The β-(1,3)-glucan synthase complexes, that have Fks proteins as putative catalytic subunits, use UDP-glucose as a substrate and catalyse the synthesis and vectorial extrusion of glucan chains into the outer space. Then, β-(1,3)-glucan chains are branched, elongated and remodelled in order to create a robust texture capable of counteracting the high internal pressure and determining cell morphology. β-(1,3)-glucan is the main component of the vegetative cell wall and one of the most abundant polymers of the spore wall. Several enzymes belonging to the family GH72 of glycosyl hydrolases have been identified in fungi. These enzymes are responsible of the lateral elongation of β-(1,3)-glucan, thus contributing to the assembly and organization of the glucan layer.
The multigene GAS family of S. cerevisiae is composed of five members, GAS1-5, involved in cell wall maintenance. They share significant similarity with Aspergillus fumigatus GEL1 and GEL2, and with Candida albicans PHR1 and PHR2. Similar to the most extensively characterized member, Gas1p, the remaining Gas proteins are β-(1,3)-glucanosyltransferases involved in cell wall assembly and maintenance. Based on their expression patterns, they appear to play partially overlapping roles throughout the yeast life cycle: GAS1 and GAS5 are expressed during vegetative growth, whereas GAS2 and GAS4 are expressed exclusively during sporulation and required for normal spore wall formation, finally GAS3 is a weakly expressed gene. Thus these enzymes could satisfy the cellular needs to remodel β-(1,3)-glucan in different physiological conditions and in different conformations along the yeast life cell cycle. Moreover, considering its role in yeast cell biology, the GAS enzyme family represents a very promising molecular target for new antifungal drugs.
During my PhD thesis I focused my interest on the functional characterization of GAS1, 2, 3 and 4 in various stages of the yeast life cycle: vegetative growth, meiosis, sporulation and spore germination. This study is aimed to understand the biological significance of the developmentally regulated requirement of the specific members of the GAS redundant family in the morphological stages of yeast life cycle.
GAS2 and GAS4 genes are specifically induced during sporulation and encode for glycoproteins. The effects of the loss of Gas2 and Gas4 proteins on spore wall morphogenesis are dramatic. Synthesis of all the layers of the spore cell wall occurs, but the accumulation and organization of wall material is abnormal. The lack of the elongase activity of Gas2 and Gas4 proteins in the double mutant might cause the formation of shorter or less branched β(1,3)-glucan chains in the inner layer of the spore wall. Thus, the connection of the outermost layers to a less compact glucan network could make the spore wall more fragile and easily stripped under harsh conditions. These defects cause an increase in spore permeability to exogenous substances, a decrease in refractivity, and a marked decrease in spore viability. The possible execution point for GAS2 and GAS4 could be between the synthesis and organization of β(1,3)-glucan and, more specifically, in the elongation of the β(1,3)-glucan chains. Consistently with their role, during sporulation Gas2 and Gas4 proteins localize at the newly assembling prospore membrane during the meiotic divisions and in mature ascospore the proteins decorate the spore periphery. A slight difference in the protein patterns of fluorescence on the spore suggests that Gas2p and Gas4p final localization could be respectively the spore wall and the prospore membrane.
In this work, an extensive study of the localization of the Gas1 protein during the yeast life cycle was performed, taking advantage of a GFP-tagged version of the protein. During vegetative growth Gas1p has a dual localization: in the plasma membrane and at the site of bud emergence, particularly in the neck, in the chitin ring that surrounds the neck region and in the bud scars where Gas1p remains after cytokinesis. At the neck region Gas1p appears to absolve important functions in yeast as a part of the mechanisms that ensure the resistance of the neck region and the morphogenesis of the septum. The size and morphology of the neck region is severely affected both in the gas1Δ and gas1Δ chs3Δ mutant, suggesting an involvement of the protein in the maintenance of the integrity of the mother-bud neck region. The presence of Gas1p in the chitin ring could be part of the mechanism necessary to prevent new incorporation of glucan chains into the neck region or alternatively the protein could be required for a particular type of remodelling necessary for the septum region in preparation to cell division. Additionally, Gas1p could act as landmark protein for the choice of the site of bud emergencee. As to Gas1p localization at the plasma membrane, our study supports the validity of Gas1p-GFP as a marker to follow the dynamics of lipid raft.
At the induction of sporulation, GAS1 mRNA levels steadily decrease and by 10h it is completely declined. Surprisingly, Gas1p levels are roughly constant during the entire sporulation processs and the protein is very stable, being detectable also at 43h after the induction of sporulation. During spore development, a translocation event occurs through which at the completion of meiosis II, Gas1p, synthesized during vegetative growth, is removed from the plasma membrane and internalized. Later, Gas1p is detected associated to the nascent prospore membrane surrounding the nuclear lobes and finally in mature spores it localizes at the spore periphery. This translocation event suggests that Gas1p delivery to the spore surface is not part of the developmentally reprogramming of the secretory pathway from the trans Golgi to the prospore membrane, whereas it involves at least in part the endocytic pathway. We demonstrated that END3-mediated endocytosis is one of the mechanisms required for the removal of the Gas1p from the plasma membrane and its efficient re-localization at the prospore membrane. Moreover in a sps1Δ mutant, Gas1p remains localized at the plasma membrane and fails to reach the spore surface. Sps1p is a member of the Ste20 protein kinase family and regulates the trafficking to the prospore membrane of enzymes involved in spore wall synthesis, such as the glucan synthase Fks2p and chitin synthase Chs3p. Thus Sps1p could regulate the traffic of Gas1p most likely in an indirect way by interacting and modifying the components of the intracellular trafficking machinery.
Gas1p translocation during sporulation
To test a possible involvement of Gas1p in spore wall formation, in this study we tried to characterize the sporulation phenotype of a gas1Δ mutant. Unfortunately our analysis was complicated by the mutant reduced cell viability when grown in presence of a poor carbon source such as acetate. gas1Δ sporulation defect could rely in a unsatisfied energetic request as the cell wall perturbations, typical of a gas1Δ mutant, enhance carbon and energy mobilization to efficiently combat cell wall weakening and the metabolism of acetate as the sole carbon source could be not sufficient to satisfy this energetic request. Moreover the addition of sorbitol to the sporulation medium only partially rescues gas1Δ defective phenotype during spore development. Even though sorbitol can mitigate the gas1Δ cell wall damages, it has no buffering effect on the gas1Δ energetic request, thus the mutant cells remained substantially unable to sporulate. Consequently, gas1Δ sporulation defective phenotype appears to be reminiscent of the mutant defects during vegetative growth, even worsened in a poor carbon source. Even though we cannot exclude a role for Gas1p during spore morphogenesis, it is our consumption that the protein translocation to the spore represents a “storage”mechanisms to ensure the presence of the Gas1p during spore germination. At 3h after the shift to a rich medium, Gas1p exhibits a highly polarized distribution, decorating exclusively half of the germinating spore in its growing pole. The protein localization is consistent with its role in glucan layer remodelling of the cell wall at the growing portion of the germinating cell. Besides gas1Δ germinating spore inability to support the elongation during the polarized growth of the cell suggests that Gas1p is required for a very early step in germination. Besides the protein is involved in a post-germination stage to support the polarized growth of the newly emerging bud.
Finally, in this study we reported the preliminary results about the functional characterization of GAS3. The gene is expressed at a very low level during the vegetative growth in glucose and acetate. Consistently with the GAS3 expression pattern, Gas3p appears as a highly polydispersed glycoprotein of high molecular weight that is present in vegetative growing cells and along the sporulation process. EndoH treatment reduces the size and the aspect of the protein to a sharp band, suggesting that Gas3p is a heavily N-glycosylated protein. The experiments indicated that neither the overexpression nor the deletion of the GAS3 gene, alone or in combination with GAS2 and GAS4, lead to relevant differences in sporulation with respect toh the wild type or with the defective phenotype of the gas2 gas4 null mutant strain . The construction of a tagged version of the Gas3 protein to determine its localization will be a useful tool to understand the function ofl Gas3p during yeast life cycle
Study on the localization of Gas1, Gas2 and Gas4 proteins during vegetative grwth and during sporulation,
No full textObblighi degli amministratori. Art. 2485
No full textIl commento analizza gli obblighi degli amministratori in conseguenza del verificarsi di una causa di sciogliment
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Biosensing and rhizosphere – endosphere geochemical microprofiling of polychlorinated byphenils degradation by soil microbiota upon stimulation of root exudates
Full text linkIntroduction: Phyto-rhyzo-remediation is a promising technology for pollutant clean-up provided by the plant holobiont, composed by the host plant and its microbiota. Plant root exudation is modulated by the pollution stress and has a key role in the activation of the microbial degrading metabolism. Despite the well documented role of the plant holobiont in ecosystem services, the complex interactions between host and microbiome are poorly understood, in particular in contaminated environments.
Materials and Methods: The project will span metabolomics, bioengineering of microbial strains together with an original application of microsensor/sensor devices to profile the chemistry of the root microenvironments. The study will be applied to the site of Brescia-Caffaro, one of the largest sites in Europe contaminated by polychlorinated biphenyls (PCBs).
Results: The project aims to sort out the time-spatial synergistic interplay within the plant holobiont components and the geochemistry of rhizosphere micro-niches supporting microbial degradation. The research will combine the: i) set up and application of bacterial biosensors to examine topology and dynamics of activation of the PCB degradation pathways upon stimulation by identified plant root exudates; and ii) sensing the plant modulated chemical micro-habitats through microsensor/sensor devices during plant-microbe interaction under PCBs stress.
Conclusions: The project outcomes will provide a comprehensive understanding of the plant holobiont applied to environmental biotechnology, focusing on the the role of root exudates as boost of soil microbiome degradative potential.
Acknowledgments: This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement N° 841317
Polychlorinated byphenils degradation by soil microbiota upon stimulation of root exudates.
Full text linkPhyto-rhizo-remediation is a promising technology for pollutant clean-up provided by the plant holobiont, composed by the host plant and its associated microbiome. Through root exudation, the plant nurtures and shape the structure and functionality of the microbial communities inhabiting the root system. The complex interactions between the plant host and the microbiome are poorly understood, in particular in contaminated environments where the pollution stress induces specific root exudation profiles that could have a role in the activation of the microbial degrading metabolism.
The study will be applied to the site of Brescia-Caffaro, one of the largest sites in Europe contaminated by polychlorinated biphenyls (PCBs).
The project aims to sort out the time-spatial synergistic interplay within the plant holobiont components and the geochemistry of rhizosphere micro-niches supporting microbial degradation. The research will combine the: i) set up and application of bacterial biosensors to examine topology and dynamics of activation of the PCB degradation pathways upon stimulation by identified plant root exudates; and ii) sensing the plant modulated chemical micro-habitats through microsensor/sensor devices during plant-microbe interaction under PCBs stress.
The project outcomes will provide a comprehensive understanding of the plant holobiont applied to environmental biotechnology, focusing on the the role of root exudates as boost of soil microbiome degradative potential.
Acknowledgments: This project has received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement N° 841317
A cysteine-rich domain related to the plant CBM43 is essential for the beta(1,3)-glucanosyltransferase activity of Gas family of proteins of Saccharomyces cerevisiae
No full textThe GAS multigene family is constituted by 5 genes (GAS1 to GAS5). GAS1 is the best characterized gene to date. It encodes the major GPI-anchored plasma membrane protein in the yeast Saccharomyces cerevisiae. Gas1p is endowed of a β(1,3)-glucanosyltransferase activity that is essential for the proper assembly of the glucan network of the cell wall during vegetative growth. The absence of this activity causes a weakening of the cell wall that activates a salvage pathway. GAS2 and GAS4 are expressed during sporulation and are essential for the assembly of the spore wall. Gas proteins belong to a broader family of extracellular enzymes from fungal and yeast species that includes also Phr1 and Phr2 from Candida albicans and Gel proteins from Aspergillus fumigatus. At the moment 70 protein sequences similar to Gas1p were identified and constitute Family 72 of the Glycoside Hydrolase database. In this work we focused on the Gas family of proteins as representative of the GH72 family. Gas proteins share an N-terminal domain of about 330-350 amino acids, where two catalytic residues are located, whereas they are dissimilar in the C-terminal portion. Out of the five Gas proteins, only Gas1 and Gas2 proteins share a cysteine-enriched domain of about 100 amino acids in their C-terminal region. This module, named Cys-box is similar to a novel Carbohydrate Binding Module (CBM), namely CBM43, an independent module that tightly binds laminarin in some plant β(1,3)-glucanases. Family GH72 appears to be divided into two subfamilies: one comprehends proteins with the Cys-box (GH72+) and the other one includes proteins without the Cys-box (GH72-). First we tested the activity of all the Gas proteins. Recombinant forms were produced in soluble form and purified from P. pastoris medium. Gas2 protein exhibited in vitro a β(1,3)-glucan-transferase activity identical to that of Gas1p whereas Gas4 and Gas5 proteins an activity similar to Gas1p. In order to study the role of the Cys-box we carried out a truncation analysis from the C-terminal end of Gas1 and Gas2 proteins. The removal of the Cys-box did not affect the folding of the proteins, as assessed by different spectroscopic analysis, but totally abolished the activity and also slightly reduced the thermal stability of the proteins. More extensive truncations greatly affected folding of the recombinant proteins and the putative catalytic N-terminal domain could not be produced in a proper conformation indicating that it does not constitute a structurally and functionally independent module. The results obtained suggest a possible interdependent relation of the N-terminal and C-terminal region in the GH72+ enzymes indicating a different role of the Cys-box in the fungal glucan transferases with respect to the CBM43 of plant glucanases. The analysis of a phylogenetic tree of the N-terminal domains of Family GH72 revealed a distinct molecular evolution of the GH72- and GH72+ subfamilies providing support to the hypothesis that the type of C-terminal region imposed constraints to the evolution of the N-terminal portion
Dynamics of the localization of the glycosylphosphatidylinositol-containing protein Gas1 during the yeast life cycle
No full textGas proteins are beta-(1,3)-glucan elongases which play a crucial role in the assembly and remodeling of the cell wall at different stages of the yeast life cycle. Gas1 is the best characterized member out of a family of five (Gas1 to Gas5). GAS1 gene is actively expressed during vegetative growth and its loss causes a round cell shape, incomplete bud growth, defects in cell separation and modifications in the structure of the cell wall. In this work we have used two different fusions of Gas1p to fluorescent proteins, RFP-Gas1p and Gas1p-GFP, to investigate Gas1p localization during the entire yeast life cycle. In vegetative growth Gas1p was detected at the cell periphery, in the bud neck and in the bud scars. The fluorescence in the plasma membrane was more intense at discrete sites indicating that Gas1p is sequestered in lipid rafts. This was confirmed by a floatation assays indicating that Gas1p-GFP is recovered in the DRMs fraction as wild type Gas1p. The Gas1p associated to lipid rafts was subjected to protein turnover and represented a mobile pool of molecules. Another pool of Gas1p was immobile and consisted in the molecules which were stably bound to the chitin ring and bud scars. At the bud scar Gas1p-GFP remained fluorescent for several generations. In a chs3 null mutant the lack of the chitin ring and of the Gas1p cross-linked to it unveiled a further localization of Gas1p along the septum line in cells at cytokinesis. Thus Gas1p is also localized to the primary septum. In the chs1, chs2 and chs3 single null mutant Gas1p was released into the growth medium indicating that Gas1p is linked to all type of chitin synthesized in the cell. Finally we studied the destiny of Gas1p during the process of meiosis and sporulation and during germination. GAS1 gene expression is repressed during sporulation but the protein persists for many days. Interestingly Gas1p is translocated from the plasma membrane to the prospore membrane by an endocytotic pathway which partially relies on the END3 gene product. Moreover, our results indicate that Gas1p is required during the polarized growth of the germinating spore.
Acknowledgement: RTN project Cantrain N. 51248 to L
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