CLD1 overexpression alters whole-cell monolysocardiolipin content of <i>coq7</i> hypomorphs.

<p><b>(A, B)</b> Whole-cell lipid extracts were prepared from the indicated yeast strains following growth at 25°C in liquid YEPE<sub>3%</sub> to the indicated optical density (OD<sub>600</sub>). Lipids were analyzed by thin layer chromatography (TLC). In (A), average MLCL/CL ratios (+/- range) are plotted for duplicate independent experiments, with triplicate technical replicates assayed at each OD<sub>600,</sub> in each experiment. Multiple regression analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162165#pone.0162165.s002" target="_blank">S2 Table</a>) revealed a significant (<i>p<0</i>.<i>007</i>) increase in the MLCL/CL ratio in <i>coq7-11</i><sub><i>i</i></sub><i>(W120R)</i> yeast following overexpression of Cld1p (1D and sup+). In (B) representative, raw TLC data is shown. CL, cardiolipin; MLCL, monolysocardiolipin; PA, phosphatidic acid; PE, phosphatidylethanolamine; <i>PG</i>, phosphatidylglycerol; <i>PS</i>, phosphatidylserine; <i>PI</i>, phosphatidylinositol; <i>PC</i>, phosphatidylcholine. <b>(C)</b> MLCL derivatives were extracted from yeast cultured at 25°C to mid-log phase (OD<sub>600</sub> ~7), then analyzed by HPLC-ESI-MS/MS. <i>m/z</i> for [M-H]<sup>-</sup> of CL(16:1/16:1/16:1), CL(16:1/16:1/18:1), CL(18:1/18:1/16:1) and CL(18:1/18:1/18:1) used for quantification are m/z 1107.6884, 1135.7196, 1163.7510 and 1191.7823, respectively. <i>Arrows</i> indicate C16:1 and C18:1 peaks used for MS1 identification. <b>(D)</b> Absolute quantitation of MLCL species in mid-log phase of the indicated yeast strains (pmole per 35ml culture volume, OD<sub>600</sub> 7.0). Data is presented as mean (+/- SEM), n = 4–5 independent replicates per strain. Significance testing was undertaken using Student’s <i>t-test p<0</i>.<i>05)</i>. Overexpression of wild type CLD1 (amplicon 1D) in the <i>coq7-11i(W120R)</i> background leads to a shift in C18:1 enriched MLCL species. This shift was not observed when the non-suppressing <i>cld1(W170R)</i> point mutant (encoded by amplicon 2C) was overexpressed, suggesting this allele either directly or indirectly results in an enzymatic loss-of-function protein.</p>