Publicação
Characterization of ScGup1 partners in the yeast putative Hedgehog-like morphogenic pathway
| Resumo: | Saccharomyces cerevisiae Gup1 is a membrane bound O-acyltransferase firstly associated with glycerol uptake, and then involved in a wide range of cellular processes, including: (i) plasma membrane and cell wall composition, (ii) rafts assembly and integrity, (iii) lipid metabolism and GPI anchor remodeling, (iv) trafficking, (v) cytoskeleton polarization and budding pattern, (vi) telomere length, (vii) cell death, and (viii) ECM composition among others. Candida albicans Gup1 was also associated with morphogenesis and differentiation. The disruption of GUP1 in this pathogenic yeast reduces virulence, affecting its capacity to adhere/invade, to differentiate into hyphae and to form biofilms. Yeast Gup1 and Gup2 proteins in higher Eukaryotes, respectively HHATL and HHAT, are regulators of the morphogenic cell-cell signalling Hedgehog pathway. HHAT is responsible for the palmitoylation of the Hedgehog secreted morphogen, and HHATL for its negative regulation. The existence of a paracrine signaling pathway similar to Hedgehog was never described in microbial cells. However, unicellular organism can form large aggregates of cells like colonies or biofilms that have a tissue-like behavior, where cells differentiate, specialize, and spatially organize, supported by a complex saccharide and proteinatious ECM. Therefore, cell-cell communication must underlie these numerous communities. It remains unclear, however, whether this occurs through a diffusible chemical, like ammonia or quorum-sensing chemicals, or through a peptide signal like the Hh morphogen from higher Eukaryotes. The presence of a Gup/HHAT(L) protein in all Eukaryotes suggests a conserved mechanism in which these proteins might be involved. The main goal of this work was to identify and characterize the proteins interacting physically with Gup1 in S. cerevisiae, as a first step to disclose the function(s) of Gup proteins in yeast. Several proteins were previously suggested to putatively interact with Gup1, though only one did not arise from HTP surveys, the ammonium transceptor Mep2. In this work, two novel Gup1 physical interactions were found: the yeast outer mitochondrial membrane VDAC (Por1), and the eisosome core component Pil1. The interaction between Gup1 and the newly identified Por1 and Pil1 partners, as well as the previously identified Mep2, was studied: (i) the expression and localization of these partners was assessed by RT-PCR and GFP fluorescence respectively, and (ii) several processes commonly associated to Gup1 were evaluated phenotypically, for which purpose new single and double deleted strains were built. |
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| Autores principais: | Moreira, Joana Isabel da Silva Tulha |
| Assunto: | Ciências Naturais::Ciências Biológicas |
| Ano: | 2017 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Saccharomyces cerevisiae Gup1 is a membrane bound O-acyltransferase firstly associated with glycerol uptake, and then involved in a wide range of cellular processes, including: (i) plasma membrane and cell wall composition, (ii) rafts assembly and integrity, (iii) lipid metabolism and GPI anchor remodeling, (iv) trafficking, (v) cytoskeleton polarization and budding pattern, (vi) telomere length, (vii) cell death, and (viii) ECM composition among others. Candida albicans Gup1 was also associated with morphogenesis and differentiation. The disruption of GUP1 in this pathogenic yeast reduces virulence, affecting its capacity to adhere/invade, to differentiate into hyphae and to form biofilms. Yeast Gup1 and Gup2 proteins in higher Eukaryotes, respectively HHATL and HHAT, are regulators of the morphogenic cell-cell signalling Hedgehog pathway. HHAT is responsible for the palmitoylation of the Hedgehog secreted morphogen, and HHATL for its negative regulation. The existence of a paracrine signaling pathway similar to Hedgehog was never described in microbial cells. However, unicellular organism can form large aggregates of cells like colonies or biofilms that have a tissue-like behavior, where cells differentiate, specialize, and spatially organize, supported by a complex saccharide and proteinatious ECM. Therefore, cell-cell communication must underlie these numerous communities. It remains unclear, however, whether this occurs through a diffusible chemical, like ammonia or quorum-sensing chemicals, or through a peptide signal like the Hh morphogen from higher Eukaryotes. The presence of a Gup/HHAT(L) protein in all Eukaryotes suggests a conserved mechanism in which these proteins might be involved. The main goal of this work was to identify and characterize the proteins interacting physically with Gup1 in S. cerevisiae, as a first step to disclose the function(s) of Gup proteins in yeast. Several proteins were previously suggested to putatively interact with Gup1, though only one did not arise from HTP surveys, the ammonium transceptor Mep2. In this work, two novel Gup1 physical interactions were found: the yeast outer mitochondrial membrane VDAC (Por1), and the eisosome core component Pil1. The interaction between Gup1 and the newly identified Por1 and Pil1 partners, as well as the previously identified Mep2, was studied: (i) the expression and localization of these partners was assessed by RT-PCR and GFP fluorescence respectively, and (ii) several processes commonly associated to Gup1 were evaluated phenotypically, for which purpose new single and double deleted strains were built. |
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