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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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Global analysis of the proteins identified by LC-MS/MS in the yECM and LGM of wt and gup1∆ mutant strains. a Numbers of proteins unequivocally identified by GC-MS/MS in yECM and liquid growth medium LGM, of S. cerevisiae wt and gup1∆ mutant: 401 proteins are exclusive of wt yECM, 213 of which are common to gup1∆ mutant, while 159 are exclusive of this mutant yECM. In opposition, and as previously reported [26], the gup1∆ mutant secretes 3.5x more proteins into liquid growth medium than wt strain. b Compared functional distribution of the proteins found by LC-MS/MS in S. cerevisiae wt yECM (upper panel) and in liquid medium LGM (lower panel). Functional classes absent from LGM are mentioned in grey insert. c Identical compared functional distribution from the proteins found in gup1∆ yECM (upper panel) and in LGM (lower panel)
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Fig3: Global analysis of the proteins identified by LC-MS/MS in the yECM and LGM of wt and gup1∆ mutant strains. a Numbers of proteins unequivocally identified by GC-MS/MS in yECM and liquid growth medium LGM, of S. cerevisiae wt and gup1∆ mutant: 401 proteins are exclusive of wt yECM, 213 of which are common to gup1∆ mutant, while 159 are exclusive of this mutant yECM. In opposition, and as previously reported [26], the gup1∆ mutant secretes 3.5x more proteins into liquid growth medium than wt strain. b Compared functional distribution of the proteins found by LC-MS/MS in S. cerevisiae wt yECM (upper panel) and in liquid medium LGM (lower panel). Functional classes absent from LGM are mentioned in grey insert. c Identical compared functional distribution from the proteins found in gup1∆ yECM (upper panel) and in LGM (lower panel)

Mentions: From the S. cerevisiae wt ECM 694 proteins (db PXD001133), only 62 are presently uncharacterized ORFs or have no attributed function. The remaining 630 have known or predicted roles spread by a wide range of functional groups, from which Metabolism, broadly taken, and Protein Fate/Remodelling account for 51 % of the yECM proteins, while ≈ 10 % are proteins involved in Cell Rescue and Defence mechanisms (Fig. 3b, c), which includes signalling proteins.Fig. 3


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)

Global analysis of the proteins identified by LC-MS/MS in the yECM and LGM of wt and gup1∆ mutant strains. a Numbers of proteins unequivocally identified by GC-MS/MS in yECM and liquid growth medium LGM, of S. cerevisiae wt and gup1∆ mutant: 401 proteins are exclusive of wt yECM, 213 of which are common to gup1∆ mutant, while 159 are exclusive of this mutant yECM. In opposition, and as previously reported [26], the gup1∆ mutant secretes 3.5x more proteins into liquid growth medium than wt strain. b Compared functional distribution of the proteins found by LC-MS/MS in S. cerevisiae wt yECM (upper panel) and in liquid medium LGM (lower panel). Functional classes absent from LGM are mentioned in grey insert. c Identical compared functional distribution from the proteins found in gup1∆ yECM (upper panel) and in LGM (lower panel)
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig3: Global analysis of the proteins identified by LC-MS/MS in the yECM and LGM of wt and gup1∆ mutant strains. a Numbers of proteins unequivocally identified by GC-MS/MS in yECM and liquid growth medium LGM, of S. cerevisiae wt and gup1∆ mutant: 401 proteins are exclusive of wt yECM, 213 of which are common to gup1∆ mutant, while 159 are exclusive of this mutant yECM. In opposition, and as previously reported [26], the gup1∆ mutant secretes 3.5x more proteins into liquid growth medium than wt strain. b Compared functional distribution of the proteins found by LC-MS/MS in S. cerevisiae wt yECM (upper panel) and in liquid medium LGM (lower panel). Functional classes absent from LGM are mentioned in grey insert. c Identical compared functional distribution from the proteins found in gup1∆ yECM (upper panel) and in LGM (lower panel)
Mentions: From the S. cerevisiae wt ECM 694 proteins (db PXD001133), only 62 are presently uncharacterized ORFs or have no attributed function. The remaining 630 have known or predicted roles spread by a wide range of functional groups, from which Metabolism, broadly taken, and Protein Fate/Remodelling account for 51 % of the yECM proteins, while ≈ 10 % are proteins involved in Cell Rescue and Defence mechanisms (Fig. 3b, c), which includes signalling proteins.Fig. 3

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