The Calcium-Dependent Protein Kinase 3 of Toxoplasma Influences Basal Calcium Levels and Functions beyond Egress as Revealed by Quantitative Phosphoproteome Analysis
Figure 1
Generation of phosphoproteome samples.
A) Illustration of the experimental conditions used in this study and the functional consequences of treatment with the calcium ionophore A23187. Upper panels show “intracellular” conditions: infected cells containing mutant or wild type parasites were labeled with “heavy” or “light” SILAC media, treated with A23187 ionophore or DMSO for 30 sec and subsequently lysed for phosphoproteome analysis. Whereas wildtype parasites rapidly egress upon ionophore treatment (after 2 minutes, 100% have exited the host cell), TgCDPK3 mutant parasites extrude the conoid, a sign that they have sensed the ionophore, but fail to egress in a timely matter. Lower panels show “extracellular” conditions: all as for “intracellular” except the cultures were scraped and passed through a syringe to release the parasites followed by ionophore treatment for 30 seconds. All conditions were performed in “forward” and “reverse” (where the heavy and light labeling are swapped). B) 5 dishes (“rep”) per condition (or 5 vials containing extracellular parasites) were individually treated for 30 seconds prior to lysis, mixing, reduction and alkylation. SCX chromatography and IMAC were used for phosphopeptide enrichment. Blue and green color-coding represents parasites grown in “light” or “heavy” media, respectively. In total, 96 samples were analyzed in duplicate by LC-MS/MS followed by bioinformatic filtering to reduce the site and protein FDR to <1% and 3%, respectively. C) A total of 32,147 phosphorylation sites were identified in the three datasets: intracellular treated with ionophore (IC/ION+) or DMSO (IC/ION−) and extracellular treated with ionophore (EC/ION+). Further filtering based on criteria specified in the text, including signal/noise ratio >8 (SN>8) reduced the set of quantified phosphorylation sites to 19,257 in 3064 proteins (See also Table S1). D) Histogram of median-centered log2 H/L SILAC ratios for each dataset comparing a TgCDPK3 mutant and wild type RH. MBE indicates the MBE1.1 mutant that is resistant to the ionophore and KO indicates the TgCDPK3 knock-out mutant. The conditions are as described above.