Limits...
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.

Show MeSH

Related in: MedlinePlus

Identification of the yECM proteins changing expression due to the mutation on GUP1 gene. a 2D DIGE analysis of the samples 1 and 2, showing the identification of the protein spots which abundance varied ≥ 2-fold and present p < 0.05. b and c Unsupervised multivariate analysis of data from the 2D DIGE experiment. b Upper panel: Principal Component Analysis showing respectively the clustering of the eight individual Cy3- and Cy5-labeled DIGE spot maps; Lower panel: score plots showing the subset of proteins whose ratios varied 2-fold or more and in which p < 0.05 in the two principle components. c Heat map of the relative protein expression values, each vertical lane corresponding to a sample from A - upper panel, and each horizontal lane a protein spot from A - lower panel. Hierarchical clustering settings are Pearson distance measurements and average linkage. The dendrogram of eight individual spot maps clustering is shown at the top, and that of individual proteins is shown on the left
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: Identification of the yECM proteins changing expression due to the mutation on GUP1 gene. a 2D DIGE analysis of the samples 1 and 2, showing the identification of the protein spots which abundance varied ≥ 2-fold and present p < 0.05. b and c Unsupervised multivariate analysis of data from the 2D DIGE experiment. b Upper panel: Principal Component Analysis showing respectively the clustering of the eight individual Cy3- and Cy5-labeled DIGE spot maps; Lower panel: score plots showing the subset of proteins whose ratios varied 2-fold or more and in which p < 0.05 in the two principle components. c Heat map of the relative protein expression values, each vertical lane corresponding to a sample from A - upper panel, and each horizontal lane a protein spot from A - lower panel. Hierarchical clustering settings are Pearson distance measurements and average linkage. The dendrogram of eight individual spot maps clustering is shown at the top, and that of individual proteins is shown on the left

Mentions: The proteomic profile of DIGE (Fig. 5a) analysed in the DeCyder Software, revealed an average of 1400 spots per sample, including protein isoforms. The biological variation analysis (not shown) matched a total of 1200 spots that were present in every sample. This spot set was used to study the proteins that are differentially abundant in the yECM of the wt and gup1Δ yECM samples. A total of 56 protein spots presented significantly different p < 0.05, abundance variation 1.5-fold or greater between the two strains. The mutant strain presented 28 spots with increased abundance, and another 28 spots that were significantly less abundant than in the wt. Among these 56 protein spots, the 15 ones presenting an abundance variation of 2-fold or greater, orange-marked spots in Fig. 5a, were excised from the gel, subjected to tryptic digestion, and the resulting peptides analysed by MALDI-TOF/TOF (Table 2). Eight spots presented ≥2-fold increased abundance in the mutant sample (Table 2 upper half), and another 7 were more abundant in the wt sample (Table 2 lower half). These results were equally obtained in four replicates. All the proteins identified with this procedure were also identified by Nano LC-MS/MS in the yECM from both wt and mutant strains (db PXD001133).Fig. 5


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)

Identification of the yECM proteins changing expression due to the mutation on GUP1 gene. a 2D DIGE analysis of the samples 1 and 2, showing the identification of the protein spots which abundance varied ≥ 2-fold and present p < 0.05. b and c Unsupervised multivariate analysis of data from the 2D DIGE experiment. b Upper panel: Principal Component Analysis showing respectively the clustering of the eight individual Cy3- and Cy5-labeled DIGE spot maps; Lower panel: score plots showing the subset of proteins whose ratios varied 2-fold or more and in which p < 0.05 in the two principle components. c Heat map of the relative protein expression values, each vertical lane corresponding to a sample from A - upper panel, and each horizontal lane a protein spot from A - lower panel. Hierarchical clustering settings are Pearson distance measurements and average linkage. The dendrogram of eight individual spot maps clustering is shown at the top, and that of individual proteins is shown on the left
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: Identification of the yECM proteins changing expression due to the mutation on GUP1 gene. a 2D DIGE analysis of the samples 1 and 2, showing the identification of the protein spots which abundance varied ≥ 2-fold and present p < 0.05. b and c Unsupervised multivariate analysis of data from the 2D DIGE experiment. b Upper panel: Principal Component Analysis showing respectively the clustering of the eight individual Cy3- and Cy5-labeled DIGE spot maps; Lower panel: score plots showing the subset of proteins whose ratios varied 2-fold or more and in which p < 0.05 in the two principle components. c Heat map of the relative protein expression values, each vertical lane corresponding to a sample from A - upper panel, and each horizontal lane a protein spot from A - lower panel. Hierarchical clustering settings are Pearson distance measurements and average linkage. The dendrogram of eight individual spot maps clustering is shown at the top, and that of individual proteins is shown on the left
Mentions: The proteomic profile of DIGE (Fig. 5a) analysed in the DeCyder Software, revealed an average of 1400 spots per sample, including protein isoforms. The biological variation analysis (not shown) matched a total of 1200 spots that were present in every sample. This spot set was used to study the proteins that are differentially abundant in the yECM of the wt and gup1Δ yECM samples. A total of 56 protein spots presented significantly different p < 0.05, abundance variation 1.5-fold or greater between the two strains. The mutant strain presented 28 spots with increased abundance, and another 28 spots that were significantly less abundant than in the wt. Among these 56 protein spots, the 15 ones presenting an abundance variation of 2-fold or greater, orange-marked spots in Fig. 5a, were excised from the gel, subjected to tryptic digestion, and the resulting peptides analysed by MALDI-TOF/TOF (Table 2). Eight spots presented ≥2-fold increased abundance in the mutant sample (Table 2 upper half), and another 7 were more abundant in the wt sample (Table 2 lower half). These results were equally obtained in four replicates. All the proteins identified with this procedure were also identified by Nano LC-MS/MS in the yECM from both wt and mutant strains (db PXD001133).Fig. 5

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