Abstract
Remodeling of the acyl chains of cardiolipin (CL) is responsible for final molecular composition of mature CL after de novo CL synthesis in mitochondria. Yeast Saccharomyces cerevisiae undergoes tafazzin-mediated CL remodeling, in which tafazzin serves as a transacylase from phospholipids to monolyso-CL (MLCL). In light of the diversity of the acyl compositions of mature CL between different organisms, the mechanism underlying tafazzin-mediated transacylation remains to be elucidated. We investigated the mechanism responsible for transacylation using purified S. cerevisiae tafazzin with liposomes composed of various sets of acyl donors and acceptors. The results revealed that tafazzin efficiently catalyzes transacylation in liposomal membranes with highly ordered lipid bilayer structure. Tafazzin elicited unique acyl chain specificity against phosphatidylcholine (PC) as follows: linoleoyl (18:2) > oleoyl (18:1) = palmitoleoyl (16:1) ≫ palmitoyl (16:0). In these reactions, tafazzin selectively removed the sn-2 acyl chain of PC and transferred it into the sn-1 and sn-2 positions of MLCL isomers at equivalent rates. We demonstrated for the first time that MLCL and dilyso-CL have inherent abilities to function as an acyl donor to monolyso-PC and acyl acceptor from PC, respectively. Furthermore, a Barth syndrome-associated tafazzin mutant (H77Q) was shown to completely lack the catalytic activity in our assay. It is difficult to reconcile the present results with the so-called thermodynamic remodeling hypothesis, which premises that tafazzin reacylates MLCL by unsaturated acyl chains only in disordered non-bilayer lipid domain. The acyl specificity of tafazzin may be one of the factors that determine the acyl composition of mature CL in S. cerevisiae mitochondria.
Highlights
Cardiolipin (CL),2 bearing two phosphate headgroups and four acyl chains, is a major phospholipid in the inner mitochondrial membrane [1, 2]
They proposed the so-called thermodynamic remodeling hypothesis to explain acyl specificity in the tafazzin-mediated remodeling of CL [26]; tafazzin functions at non-bilayer-type relaxed lipid domains, which occur in curved membrane domains, because of the preferential mixing of phospholipids and MLCL species, and its acyl specificity is driven by the packing properties of these domains
Effects of the glutathione S-transferase (GST) Tag and Ca2ϩ on the Catalytic Activity of Tafazzin—We investigated transacylation from PC to MLCL catalyzed by isolated S. cerevisiae GST-tagged tafazzin with
Summary
Effects of the GST Tag and Ca2ϩ on the Catalytic Activity of Tafazzin—We investigated transacylation from PC to MLCL catalyzed by isolated S. cerevisiae GST-tagged tafazzin with. Sn-2 Acyl Chain of PC Is Selectively Transferred to Monolyso-CL—In tafazzin-mediated acyl transfer from PC to MLCL, the acyl chain of PC (sn-1, sn-2, or both positions) that is deacylated has not yet been identified This previously unresolved issue is important to obtain a more comprehensive understanding of the mechanism underlying the tafazzin-mediated transacylation. The averaged ratios of sn-2-LPC(18:1-OH):sn-1-LPC(OH-18:1) determined by HPLC and ESI-LC/MS analyses after a 40-min reaction were 98:2 (Fig. 3A, panel iii) and 93:7 (Fig. 3B, panel iii), respectively, indicating that the deacylation of PC occurred predominantly at the sn-2 position. We confirmed this regiospecificity in the deacylation of PC in the following transacylation experiments.
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