Quantitative cell wall protein profiling of invasive and non-invasive Saccharomyces cerevisiae strains.
A new, simple approach for the isolation and quantitative analysis of the cell wall (CW) proteins from invasively growing Saccharomyces cerevisiae strains is described in this contribution. The proposed method was proved compatible with agar-invasion assays and was demonstrated to be useful as a screening tool for rapid analysis of CW protein determinants related to yeast adhesion and invasion processes. CW protein isolation was performed enzymatically on viable cells by using mild, isosmotic reaction conditions and pure, proteinase free glucanase, thus avoiding destruction of cells and protein structures, which is a drawback of the existing methods based on hot SDS, DTT or NaOH treatment. Moreover, the method requires as low as 10mg of collected cell biomass for sufficient protein yield, which makes it suitable for the study of yeast invasion at the proteomic level. The extraction protocol was optimized for fast, direct analysis of multiple protein samples by SDS-PAGE, avoiding pre-concentration or purification steps, but still preserving high resolution of protein bands. The developed method was used to compare CW protein profile of i) invasive and non-invasive strains, ii) invasive and non-invasive morphological part of the colony and iii) cells cultivated at optimal and increased growth temperature. Results of quantitative SDS-PAGE analysis of S. cerevisiae CW proteins revealed the presence of up to 20 protein bands with molecular masses in the range 60-220 kDa. In addition, comparative analysis of CW protein profiles resulted in significant changes in the protein profile expression relevant to different cultivation temperature, cell morphology (invasive vs. non-invasive growth) and yeast strain.
Zupan
Jure
J
Chair of Biotechnology, Microbiology and Food Safety, Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia.
Mavri
Jan
J
Raspor
Peter
P
D018929:Cell Culture Techniques; D002473:Cell Wall / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D042541:Intracellular Space / Q000737:chemistry; D010449:Peptide Mapping / Q000379:methods; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis* / Q000378:metabolism; D013696:Temperature
D029701:Saccharomyces cerevisiae Proteins