CLD1 overexpression alters whole-cell monolysocardiolipin content of coq7 hypomorphs.
(A, B) Whole-cell lipid extracts were prepared from the indicated yeast strains following growth at 25°C in liquid YEPE3% to the indicated optical density (OD600). 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 OD600, in each experiment. Multiple regression analysis (S2 Table) revealed a significant (p<0.007) increase in the MLCL/CL ratio in coq7-11i(W120R) 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; PG, phosphatidylglycerol; PS, phosphatidylserine; PI, phosphatidylinositol; PC, phosphatidylcholine. (C) MLCL derivatives were extracted from yeast cultured at 25°C to mid-log phase (OD600 ~7), then analyzed by HPLC-ESI-MS/MS. m/z for [M-H]- 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. Arrows indicate C16:1 and C18:1 peaks used for MS1 identification. (D) Absolute quantitation of MLCL species in mid-log phase of the indicated yeast strains (pmole per 35ml culture volume, OD600 7.0). Data is presented as mean (+/- SEM), n = 4–5 independent replicates per strain. Significance testing was undertaken using Student’s t-test p<0.05). Overexpression of wild type CLD1 (amplicon 1D) in the coq7-11i(W120R) background leads to a shift in C18:1 enriched MLCL species. This shift was not observed when the non-suppressing cld1(W170R) point mutant (encoded by amplicon 2C) was overexpressed, suggesting this allele either directly or indirectly results in an enzymatic loss-of-function protein.