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Quantitative differential proteomics of yeast extracellular matrix: there is more to it than meets the eye.

Faria-Oliveira F, Carvalho J, Ferreira C, Hernáez ML, Gil C, Lucas C - BMC Microbiol. (2015)

Bottom Line: In yeast, the deletion of GUP1 was associated with a vast number of diverse phenotypes including the cellular differentiation that accompanies biofilm formation.Two strains were compared, wild type and the mutant defective in GUP1.Accordingly, the correspondent differences in proteome unveiled acetic and citric acid producing enzymes as putative players in structural integrity maintenance.

View Article: PubMed Central - PubMed

Affiliation: CBMA - Centro de Biologia Molecular e Ambiental, Departamento de Biologia, Universidade do Minho, Campus de Gualtar, 4710-057, Braga, Portugal.

ABSTRACT

Background: Saccharomyces cerevisiae multicellular communities are sustained by a scaffolding extracellular matrix, which provides spatial organization, and nutrient and water availability, and ensures group survival. According to this tissue-like biology, the yeast extracellular matrix (yECM) is analogous to the higher Eukaryotes counterpart for its polysaccharide and proteinaceous nature. Few works focused on yeast biofilms, identifying the flocculin Flo11 and several members of the HSP70 in the extracellular space. Molecular composition of the yECM, is therefore mostly unknown. The homologue of yeast Gup1 protein in high Eukaryotes (HHATL) acts as a regulator of Hedgehog signal secretion, therefore interfering in morphogenesis and cell-cell communication through the ECM, which mediates but is also regulated by this signalling pathway. In yeast, the deletion of GUP1 was associated with a vast number of diverse phenotypes including the cellular differentiation that accompanies biofilm formation.

Methods: S. cerevisiae W303-1A wt strain and gup1∆ mutant were used as previously described to generate biofilm-like mats in YPDa from which the yECM proteome was extracted. The proteome from extracellular medium from batch liquid growing cultures was used as control for yECM-only secreted proteins. Proteins were separated by SDS-PAGE and 2DE. Identification was performed by HPLC, LC-MS/MS and MALDI-TOF/TOF. The protein expression comparison between the two strains was done by DIGE, and analysed by DeCyder Extended Data Analysis that included Principal Component Analysis and Hierarchical Cluster Analysis.

Results: The proteome of S. cerevisiae yECM from biofilm-like mats was purified and analysed by Nano LC-MS/MS, 2D Difference Gel Electrophoresis (DIGE), and MALDI-TOF/TOF. Two strains were compared, wild type and the mutant defective in GUP1. As controls for the identification of the yECM-only proteins, the proteome from liquid batch cultures was also identified. Proteins were grouped into distinct functional classes, mostly Metabolism, Protein Fate/Remodelling and Cell Rescue and Defence mechanisms, standing out the presence of heat shock chaperones, metalloproteinases, broad signalling cross-talkers and other putative signalling proteins. The data has been deposited to the ProteomeXchange with identifier PXD001133.

Conclusions: yECM, as the mammalian counterpart, emerges as highly proteinaceous. As in higher Eukaryotes ECM, numerous proteins that could allow dynamic remodelling, and signalling events to occur in/and via yECM were identified. Importantly, large sets of enzymes encompassing full antagonistic metabolic pathways, suggest that mats develop into two metabolically distinct populations, suggesting that either extensive moonlighting or actual metabolism occurs extracellularly. The gup1∆ showed abnormally loose ECM texture. Accordingly, the correspondent differences in proteome unveiled acetic and citric acid producing enzymes as putative players in structural integrity maintenance.

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Related in: MedlinePlus

Compared functional distribution of the yECM-only proteins in wt and gup1∆ mutant strains. S. cerevisiae wt (dark grey bars), gup1∆ mutant (dashed bars), unknown/uncharacterised ORFs (white bars). Arrows indicate the highest percentage of reduction in numbers of proteins in mutant ECM compared to wt’s
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Fig4: Compared functional distribution of the yECM-only proteins in wt and gup1∆ mutant strains. S. cerevisiae wt (dark grey bars), gup1∆ mutant (dashed bars), unknown/uncharacterised ORFs (white bars). Arrows indicate the highest percentage of reduction in numbers of proteins in mutant ECM compared to wt’s

Mentions: From all the proteins found in S. cerevisiae ECM (db PXD001133), 150 are involved in the synthesis, folding and degradation of other proteins (Fig. 4). These include the proteins from the HSP70 family, Ssa1/2/3/4, Ssb1, Ssc1, Sse1/2 and Kar2, and the proteases, Lap4, Dug1, Ecm14, Ape2, Prd1 and Zps1. The HSP70 are chaperones responsible for the folding and membrane translocation of other proteins [47]. In particular, Ssa1 and Ssa2 are implicated in the biosynthesis and assembly of the cell wall [48]. These two proteins, as well as Ssb2 and Sse1 were previously reported to be present in the cell surface in both S. cerevisiae and C. albicans [39, 45, 46], and were found in this work in the top 12 most scored proteins in yECM above mentioned (db PXD001133). Moreover, also a number of proteins performing roles of cellular organization were identified. The wt yECM samples present several proteins involved in the cytoskeleton organization, namely the tubulin Tub2, the Arc19, Arc35, Arp2, Ent2 and Ent3 proteins have a role in the assembly of actin cortical patches, and the Rvs161 and Vps1 modulate the cytoskeleton assembly.Fig. 4


Quantitative differential proteomics of yeast extracellular matrix: there is more to it than meets the eye.

Faria-Oliveira F, Carvalho J, Ferreira C, Hernáez ML, Gil C, Lucas C - BMC Microbiol. (2015)

Compared functional distribution of the yECM-only proteins in wt and gup1∆ mutant strains. S. cerevisiae wt (dark grey bars), gup1∆ mutant (dashed bars), unknown/uncharacterised ORFs (white bars). Arrows indicate the highest percentage of reduction in numbers of proteins in mutant ECM compared to wt’s
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4660637&req=5

Fig4: Compared functional distribution of the yECM-only proteins in wt and gup1∆ mutant strains. S. cerevisiae wt (dark grey bars), gup1∆ mutant (dashed bars), unknown/uncharacterised ORFs (white bars). Arrows indicate the highest percentage of reduction in numbers of proteins in mutant ECM compared to wt’s
Mentions: From all the proteins found in S. cerevisiae ECM (db PXD001133), 150 are involved in the synthesis, folding and degradation of other proteins (Fig. 4). These include the proteins from the HSP70 family, Ssa1/2/3/4, Ssb1, Ssc1, Sse1/2 and Kar2, and the proteases, Lap4, Dug1, Ecm14, Ape2, Prd1 and Zps1. The HSP70 are chaperones responsible for the folding and membrane translocation of other proteins [47]. In particular, Ssa1 and Ssa2 are implicated in the biosynthesis and assembly of the cell wall [48]. These two proteins, as well as Ssb2 and Sse1 were previously reported to be present in the cell surface in both S. cerevisiae and C. albicans [39, 45, 46], and were found in this work in the top 12 most scored proteins in yECM above mentioned (db PXD001133). Moreover, also a number of proteins performing roles of cellular organization were identified. The wt yECM samples present several proteins involved in the cytoskeleton organization, namely the tubulin Tub2, the Arc19, Arc35, Arp2, Ent2 and Ent3 proteins have a role in the assembly of actin cortical patches, and the Rvs161 and Vps1 modulate the cytoskeleton assembly.Fig. 4

Bottom Line: In yeast, the deletion of GUP1 was associated with a vast number of diverse phenotypes including the cellular differentiation that accompanies biofilm formation.Two strains were compared, wild type and the mutant defective in GUP1.Accordingly, the correspondent differences in proteome unveiled acetic and citric acid producing enzymes as putative players in structural integrity maintenance.

View Article: PubMed Central - PubMed

Affiliation: CBMA - Centro de Biologia Molecular e Ambiental, Departamento de Biologia, Universidade do Minho, Campus de Gualtar, 4710-057, Braga, Portugal.

ABSTRACT

Background: Saccharomyces cerevisiae multicellular communities are sustained by a scaffolding extracellular matrix, which provides spatial organization, and nutrient and water availability, and ensures group survival. According to this tissue-like biology, the yeast extracellular matrix (yECM) is analogous to the higher Eukaryotes counterpart for its polysaccharide and proteinaceous nature. Few works focused on yeast biofilms, identifying the flocculin Flo11 and several members of the HSP70 in the extracellular space. Molecular composition of the yECM, is therefore mostly unknown. The homologue of yeast Gup1 protein in high Eukaryotes (HHATL) acts as a regulator of Hedgehog signal secretion, therefore interfering in morphogenesis and cell-cell communication through the ECM, which mediates but is also regulated by this signalling pathway. In yeast, the deletion of GUP1 was associated with a vast number of diverse phenotypes including the cellular differentiation that accompanies biofilm formation.

Methods: S. cerevisiae W303-1A wt strain and gup1∆ mutant were used as previously described to generate biofilm-like mats in YPDa from which the yECM proteome was extracted. The proteome from extracellular medium from batch liquid growing cultures was used as control for yECM-only secreted proteins. Proteins were separated by SDS-PAGE and 2DE. Identification was performed by HPLC, LC-MS/MS and MALDI-TOF/TOF. The protein expression comparison between the two strains was done by DIGE, and analysed by DeCyder Extended Data Analysis that included Principal Component Analysis and Hierarchical Cluster Analysis.

Results: The proteome of S. cerevisiae yECM from biofilm-like mats was purified and analysed by Nano LC-MS/MS, 2D Difference Gel Electrophoresis (DIGE), and MALDI-TOF/TOF. Two strains were compared, wild type and the mutant defective in GUP1. As controls for the identification of the yECM-only proteins, the proteome from liquid batch cultures was also identified. Proteins were grouped into distinct functional classes, mostly Metabolism, Protein Fate/Remodelling and Cell Rescue and Defence mechanisms, standing out the presence of heat shock chaperones, metalloproteinases, broad signalling cross-talkers and other putative signalling proteins. The data has been deposited to the ProteomeXchange with identifier PXD001133.

Conclusions: yECM, as the mammalian counterpart, emerges as highly proteinaceous. As in higher Eukaryotes ECM, numerous proteins that could allow dynamic remodelling, and signalling events to occur in/and via yECM were identified. Importantly, large sets of enzymes encompassing full antagonistic metabolic pathways, suggest that mats develop into two metabolically distinct populations, suggesting that either extensive moonlighting or actual metabolism occurs extracellularly. The gup1∆ showed abnormally loose ECM texture. Accordingly, the correspondent differences in proteome unveiled acetic and citric acid producing enzymes as putative players in structural integrity maintenance.

Show MeSH
Related in: MedlinePlus