Please use this identifier to cite or link to this item: http://hdl.handle.net/1822/50821

TitleQuantitative differential proteomics of yeast extracellular matrix: there is more to it than meets the eye
Author(s)Oliveira, Fábio Luís Silva Faria
Carvalho, Joana
Ferreira, Célia
Hernáez, Maria Luisa
Gil, Concha
Lucas, Cândida
KeywordsSaccharomyces cerevisiae
Extracellular matrix
ECM
Proteome
Proteome
Nano LC-MS/MS
DIGE
Metalloproteinases
Chaperones
Extracellular signals
Issue date2015
PublisherBioMed Central
JournalBMC Microbiology
Abstract(s)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 gup1Δ mutant were used as previously described to generate biofilmlike 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 gup1Δ 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.
TypeArticle
URIhttp://hdl.handle.net/1822/50821
DOI10.1186/s12866-015-0550-1
ISSN1471-2180
Publisher versionhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4660637/pdf/12866_2015_Article_550.pdf
Peer-Reviewedyes
AccessOpen access
Appears in Collections:DBio - Artigos/Papers

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