Abstract
The plasma membrane separates the cell from the external environment and plays an important role in the stress response of the cell. In this study, we compared plasma membrane proteome modifications of yeast cells exposed to mild (0.4 m NaCl) or high (1 m NaCl) salt stress for 10, 30, or 90 min. Plasma membrane-enriched fractions were isolated, purified, and subjected to iTRAQ labeling for quantitative analysis. In total, 88-109 plasma membrane proteins were identified and quantified. The quantitative analysis revealed significant changes in the abundance of several plasma membrane proteins. Mild salt stress caused an increase in abundance of 12 plasma membrane proteins, including known salt-responsive proteins, as well as new targets. Interestingly, 20 plasma membrane proteins, including the P-type H(+)-ATPase Pma1, ABC transporters, glucose and amino acid transporters, t-SNAREs, and proteins involved in cell wall biogenesis showed a significant and rapid decrease in abundance in response to both 0.4 m and 1 m NaCl. We propose that rapid protein internalization occurs as a response to hyper-osmotic and/or ionic shock, which might affect plasma membrane morphology and ionic homeostasis. This rapid response might help the cell to survive until the transcriptional response takes place.
Highlights
Exposure of yeast cells to saline stress implies exposure to both specific cation toxicity and osmotic stress [1]
The response after 10 min of salt stress involved transcripts coding for proteins involved in nucleotide and amino acid metabolism, intracellular transport, and protein synthesis, after 30 min of stress the response involved transcripts related to respiration and energy production, and after 90 min of stress the response involved transcripts the response involved cellular detoxification, major facilitator superfamily transporters, and enzymes involved in nitrogen or sulfur metabolism and lipid or fatty acid biosynthesis
Yeast Plasma Membrane Quantitative Proteomics After Salt Stress response of yeast cells to salt stress has been restricted to soluble proteins and has used a two-dimensional electrophoresis gel-based approach (16 –18); using this method, most proteins showing changes in abundance were found to be involved in various aspects of carbohydrate metabolism, such as the synthesis of the osmo-protectant glycerol, in protein folding, and in protein degradation
Summary
Yeast Cultures and Salt Stress—S. cerevisiae strain W303 (MAT ␣ ade, leu112 his trp ura can1–100) was used. The supernatant was centrifuged for 45 min at 14,000 rpm (JA-30.50Ti, Beckman) to pellet crude membranes, which were resuspended in 5 ml of suspension medium (10 mM imidazole, 2 mM MgCl2, pH 7.5, containing the protease inhibitor mix) per 10 g of cells. Control and salt stress cultures were harvested, washed in ice-cold water, and the cell pellets resuspended in lysis buffer (25 mM Tris, 5 mM EDTA, pH 7.5) containing the. Quantification of Relative Change—To determine differences in expression of proteins between the salt-treated cells and the control, the average ratio of the identified protein was calculated by ProteinPilot based on the weighted average log ratios of the peptides. Bias correction for unequal mixing during the combination of the different labeled samples was performed This correction is based on the assumption that most proteins do not change in expression. The signal of Cdc48p was used for quantification normalization of Gas1p signal
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