In the yeast S. cerevisiae
, PA, a key intermediate in lipid metabolism, can be formed in vitro from the precursors G-3-P and DHAP. Contradictory evidence has been presented about the number of enzymes involved in the first step of acylation forming LPA or 1-acyl-DHAP and the substrate specificity of the enzyme(s). Tillman and Bell (26
) concluded from their experiments that one enzyme can acylate both G-3-P and DHAP. Racenis et al. (22
), on the other hand, argued that although temperature optima and kinetics of inactivation by heat were the same for GAT and DHAPAT activities some differences exist, such as the inhibitory effect of N
-ethylmaleimide and pH optima. Tillman and Bell (26
) and Racenis et al. (22
) used crude yeast membrane preparations as enzyme sources for their in vitro assays. Thus, their results reflected an “overall” situation of acylation activities in yeast. More recent studies of G-3-P acylation with highly purified subcellular fractions and the use of the two acyltransferase mutant strains TTA1 and YMN5 revealed that more than one acyltransferase system contributes to PA formation in yeast (1
). One acylation machinery is dually localized to lipid particles and the endoplasmic reticulum; at least one other acylation system was detected only in the endoplasmic reticulum.
Results presented in this study demonstrate that the major GAT of the yeast, the hypothetical Gat1p, which is located in lipid particles and the endoplasmic reticulum, can acylate both G-3-P and DHAP. Lipid particles of the wild-type strain can acylate both precursors, whereas those of the gat1
mutant strain TTA1 showed neither GAT nor DHAPAT activity. Since lipid particles do not contain a second GAT, both enzymatic activities can be attributed to the same protein. These results are in line with the finding of Tillman and Bell (26
) that one acylating enzyme, namely Gat1p, has GAT activity as well as DHAPAT activity.
The results are more complicated for GAT and DHAPAT activities of other subcellular compartments. It has been shown during previous work in our laboratory (1
) that Gat1p is not exclusively localized to lipid particles but indeed contributes most prominently to GAT activity of the endoplasmic reticulum (30,000 × g
microsomes). Using the gat1
mutant strain TTA1 it was shown, however, that at least another GAT must be present in the microsomal fraction. Although the GAT:DHAPAT activity ratios are similar in 30,000 × g
microsomes of wild-type and TTA1 strains (Table ), the possibility that microsomal acyltransferase activity is derived from more than one enzyme cannot be excluded. In contrast to other organelles tested, mitochondria exhibit a low ratio of GAT activity to DHAPAT activity that suggests the presence of a distinct mitochondrial DHAPAT. This activity cannot be attributed to contamination with other subcellular fractions.
Cell biological and biochemical experiments presented in this paper led us to propose a model of the subcellular distribution of acyltransferase reactions involved in the formation of PA in yeast (Fig. ). One GAT, namely Gat1p, which can use G-3-P as well as DHAP as a substrate, is located in lipid particles. Gat1p is also present in the endoplasmic reticulum, which in addition contains at least another GAT, a hypothetical Gat2p. This enzyme may also have DHAPAT activity. Alternatively, besides Gat1p additional distinct enzymes for G-3-P and DHAP acylation may be present in the endoplasmic reticulum. The third site of acyltransferase activity is mitochondria, but this compartment appears to harbor a distinct DHAPAT.
Scheme of subcellular distribution of enzymes involved in PA biosynthesis in yeast. ER, endoplasmic reticulum; MITO, mitochondria; LP, lipid particles.
Double labeling experiments with [2-3H, U-14C]glycerol demonstrated that both GAT and DHAPAT reactions are relevant in vivo. The fact that the 3H/14C ratios in all glycerolipid species of the gat1 mutant strain TTA1 were lower than in the corresponding wild-type strain indicates that in the absence of Gat1p the overall contribution of the DHAP pathway to glycerolipid biosynthesis is increased. This observation is in good agreement with the cell biological model of PA formation shown in Fig. : when Gat1p, which is the only GAT and DHAPAT in lipid particles and the major GAT and DHAPAT in the endoplasmic reticulum, is inactivated, the ratio of cellular GAT activity to DHAPAT activity is shifted towards the DHAP pathway due to the contribution of the mitochondrial DHAPAT.
Similar to the result for TTA1, which lacks Gat1p, deletion of SLC1
results in a shift of the acylation pathway towards utilization of DHAP. This result may serve as another indication that Slc1p can at least in part replace Gat1p, as was suggested earlier (1
). A gat1slc1
double mutant exhibited intermediate 3
C ratios in all glycerolipids as compared to those for TTA1 and YMN5. Thus, residual acyltransferases present in the gat1slc1
mutant compensate for the lack of Gat1p and Slc1p by catalyzing formation of PA at a level sufficient for balanced cellular growth. This result also indicates that both the G-3-P pathway and the DHAP pathway are active in the double mutant and that the latter is preferred over the former, as contrasted with the situation for the wild-type.
Another important finding revealed during this investigation was the subcellular localization of ADR activity. ADR catalyzes the NADPH-dependent reduction of 1-acyl-DHAP to LPA. Among the subcellular fractions which harbor DHAPAT activity only lipid particles and 30,000 × g microsomes showed ADR activity; mitochondria lack this reductase activity.
An interesting result obtained through in vivo labeling experiments (Results section and Table ) was the relatively low 3
C ratio in cardiolipin of all strains tested compared to the other glycerolipid species. The decreased 3
C ratio indicates a greater contribution of the DHAP pathway than the G-3-P pathway to cardiolipin formation. Cardiolipin synthesis occurs in mitochondria, the organelles which have a preference for DHAP acylation over G-3-P acylation. A greater contribution of the DHAP pathway to the synthesis of cardiolipin could be due to the utilization of PA mainly formed from mitochondrion-derived 1-acyl-DHAP. For further metabolic conversion of 1-acyl-DHAP formed by DHAPAT of mitochondria, however, the acylation intermediate has to be transferred to a site of ADR activity where it is reduced to LPA prior to the second step of acylation. 1-Acyl-DHAP is reasonably soluble in an aqueous environment and might thus migrate rather easily from one compartment to another. In respect to the greater contribution of the DHAP pathway to cardiolipin synthesis, however, an alternative possibility of 1-acyl-DHAP migration may be envisaged. 1-Acyl-DHAP formed in mitochondria could be translocated to the “closest” site of ADR activity, which is the mitochondrion-associated endoplasmic reticulum, the so-called MAM fraction (7
). At this site, reduction of 1-acyl-DHAP and further acylation could occur. CDP-diacylglycerol formed in the subsequent metabolic step in the MAM subfraction of the endoplasmic reticulum or in mitochondria (14
) might serve as a substrate for cardiolipin synthesis in mitochondria. As a result of this (hypothetical) sequence of translocation and conversion steps DHAP might be preferentially incorporated into cardiolipin. Since PA formed in the MAM fraction of the endoplasmic reticulum from G-3-P can also be used for CDP-diacylglycerol formation and incorporation into cardiolipin, part of the glycerol moieties of cardiolipin is also derived from G-3-P.
The complexity of PA formation in yeast requires further characterization of enzymes involved in the first step of acylation of G-3-P and/or DHAP and in the subsequent steps involved in this process. Thus, future efforts will be focused on the identification of the enzymes involved in PA biosynthesis, their characterization at the molecular level, and the interplay of organelles during initial steps of glycerolipid biosynthesis.