The plasma membrane proteome of Saccharomyces cerevisiae and its response to the antifungal calcofluor.
Calcofluor is an antifungal compound known to induce structural perturbations of the cell wall by interfering with the synthesis of chitin microfibril. Proteins from a stripped plasma membrane fraction were solubilized with the neutral and non-denaturing detergent, the n-dodecyl beta-D-maltoside. Proteins were then resolved using a recently described ion-exchange chromatography (IEC)/lithium dodecyl sulfate (LDS)-PAGE procedure. Nearly 90 proteins were identified and clustered, based on their pI, molecular weight, abundance and/or hydrophobicity. This method was then applied to profile the plasma membrane response to calcofluor. The LDS-PAGE patterns obtained from whole plasma membrane proteins were similar for the non-treated and calcofluor-treated samples. However, IEC/LDS-PAGE analysis revealed subtle changes in the expression of several proteins of low abundance, in response to calcofluor. These proteins include Pil1p and Lsp1p, two sphingolipid long-chain base-responsive inhibitors of protein kinases involved in signaling pathways for cell wall integrity and Rho1p, a small GTPase. It was recently hypothesized that Pil1p and Lsp1p could associate with, and regulate, the plasma membrane beta-1-3-glucan synthase, responsible for the synthesis of another major microfibril for yeast cell wall. Results are discussed with respect to both calcofluor effects on the plasma membrane proteins and the power of the IEC/LDS-PAGE procedure in the search for new potential therapeutics targets.
Delom
Frédéric
F
UMR 5004, Plant Biochemistry and Molecular Physiology, INRA, 2 place Viala, 34060 Montpellier cedex 1, France.
Szponarski
Wojciech
W
Sommerer
Nicolas
N
Boyer
Jean-Christophe
JC
Bruneau
Jean-Michel
JM
Rossignol
Michel
M
Gibrat
Rémy
R
D000935:Antifungal Agents / Q000494:pharmacology*; D001557:Benzenesulfonates / Q000494:pharmacology*; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008565:Membrane Proteins / Q000096:biosynthesis*; D020543:Proteome / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000935:Antifungal Agents; D001557:Benzenesulfonates; D008565:Membrane Proteins; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; C007061:C.I. Fluorescent Brightening Agent 28