Fig 4.TIF (4.59 MB)

CLD1 overexpression alters whole-cell monolysocardiolipin content of coq7 hypomorphs.

Download (4.59 MB)
figure
posted on 07.09.2016 by Adwitiya Kar, Haley Beam, Megan B. Borror, Michael Luckow, Xiaoli Gao, Shane L. Rea

(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.

History

Licence

Exports