Cardiolipin Molecular Species with Shorter Acyl Chains Accumulate in Saccharomyces cerevisiae Mutants Lacking the Acyl Coenzyme A-binding Protein Acb1p: NEW INSIGHTS INTO ACYL CHAIN REMODELING OF CARDIOLIPIN

Pieter J Rijken,Riekelt H Houtkooper,Hana Akbari,Jos F Brouwers,Martijn C Koorengevel,Ben De Kruijff,Margrit Frentzen,Frédéric M Vaz,Anton I.P.M De Kroon

doi:10.1074/jbc.m109.016311

Pieter J Rijken, Riekelt H Houtkooper + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m109.016311

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Abstract

The function of the mitochondrial phospholipid cardiolipin (CL) is thought to depend on its acyl chain composition. The present study aims at a better understanding of the way the CL species profile is established in Saccharomyces cerevisiae by using depletion of the acyl-CoA-binding protein Acb1p as a tool to modulate the cellular acyl chain content. Despite the presence of an intact CL remodeling system, acyl chains shorter than 16 carbon atoms (C16) were found to accumulate in CL in cells lacking Acb1p. Further experiments revealed that Taz1p, a key CL remodeling enzyme, was not responsible for the shortening of CL in the absence of Acb1p. This left de novo CL synthesis as the only possible source of acyl chains shorter than C16 in CL. Experiments in which the substrate specificity of the yeast cardiolipin synthase Crd1p and the acyl chain composition of individual short CL species were investigated, indicated that both CL precursors (i.e. phosphatidylglycerol and CDP-diacylglycerol) contribute to comparable extents to the shorter acyl chains in CL in acb1 mutants. Based on the findings, we conclude that the fatty acid composition of mature CL in yeast is governed by the substrate specificity of the CL-specific lipase Cld1p and the fatty acid composition of the Taz1p substrates.

Highlights

The function of the mitochondrial phospholipid cardiolipin (CL) is thought to depend on its acyl chain composition
The present study aims at a better understanding of the way the CL species profile is established in Saccharomyces cerevisiae by using depletion of the acyl-CoA-binding protein Acb1p as a tool to modulate the cellular acyl chain content
We conclude that the fatty acid composition of mature CL in yeast is governed by the substrate specificity of the CL-specific lipase Cld1p and the fatty acid composition of the Taz1p substrates

Summary

EXPERIMENTAL PROCEDURES

Yeast Strains, Plasmids, Media, and Culture Conditions— The yeast strains listed in Table 1 were maintained on YPD agar plates (1% yeast extract, 2% bactopeptone, and 2% glucose). Analysis of Phospholipid Molecular Species by Mass Spectrometry—Total lipid extracts obtained from 10 mg of cells (dry weight) corresponding to 150 to 200 nmol of phospholipid phosphorous were dissolved in 150 ␮l of chloroform/methanol/water (50:45:5, v/v/v) containing 0.01% (w/v) NH4OH, and 5 ␮l of this solution was analyzed by HPLC-MS as described [15]. The identity of the molecular species was confirmed by recording product spectra, operating the second mass filter in linear ion-trap mode with dynamic fill time In these experiments, a collision energy of 50 V with a spread of 15 V was used, which enabled the clear identification of the [M Ϫ H]Ϫ molecular ion, the corresponding lyso-lipid fragment ions, the subsequent loss of the head group from this fragment ion, the fatty acyl-derived carboxylate ions, and the glycerophosphate ion (data not shown). For analysis of unlabeled PG products were extracted and analyzed as described before molecular species, precursor scans of m/z 227 [32] were [15]

Yeast strains and plasmids used in this study

RESULTS

Are the Acyl Chains Shorter Than

Possible acyl chain compositionsb