Janus-faced enzymes yeast Tgl3p and Tgl5p catalyze lipase and acyltransferase reactions

Abstract

In the yeast, mobilization of triacylglycerols (TAGs) is facilitated by the three TAG lipases Tgl3p, Tgl4p, and Tgl5p. Motif search analysis, however, indicated that Tgl3p and Tgl5p do not only contain the TAG lipase motif GXSXG but also an H-(X)(4)-D acyltransferase motif. Interestingly, lipid analysis revealed that deletion of TGL3 resulted in a decrease and overexpression of TGL3 in an increase of glycerophospholipids. Similar results were obtained with TGL5. Therefore, we tested purified Tgl3p and Tgl5p for acyltransferase activity. Indeed, both enzymes not only exhibited lipase activity but also catalyzed acylation of lysophosphatidylethanolamine and lysophosphatidic acid, respectively. Experiments using variants of Tgl3p created by site-directed mutagenesis clearly demonstrated that the two enzymatic activities act independently of each other. We also showed that Tgl3p is important for efficient sporulation of yeast cells, but rather through its acyltransferase than lipase activity. In summary, our results demonstrate that yeast Tgl3p and Tgl5p play a dual role in lipid metabolism contributing to both anabolic and catabolic processes.

Figure 1.

Overexpression of TGL3 increases the amount of phospholipids. (A) Amount of total phospholipids per mg protein from whole cell extracts was quantified as described under Materials and Methods, and the value of wild type was set at 100%. (B) Amount of [¹⁴C]acetate incorporated into individual phospholipids is shown. Data are expressed as picomoles of [¹⁴C]acetate incorporated per A₆₀₀ units. Black bars, wild type; white bars, tgl3Δ; and gray bars, Tgl3p overexpressing strain. Mean values of three independent experiments ± SD are shown.

Figure 2.

Dual signature motifs of Tgl3p. The patatin domain (P40308) structure of TGL3 as determined by pfam analysis is indicated by the shaded region. The ²⁹⁸HXXXXD³⁰³ is a signature motif for all glycerolipid acyltransferases; and ²³⁵GXSXG²³⁹ is a typical lipase motif.

Figure 3.

Tgl3p mediates oleoyl-CoA dependent acylation of lysophosphatidylethanolamine. A Tgl3p-His₆ fusion protein was used to analyze enzyme activities. Acyltransferase assays were performed as described in Materials and Methods. (A) Purified Tgl3p was examined for the acyl acceptor specificity using LPA, LPC, LPE, LPI, and LPS as acceptors. (B) Acyl-CoA selectivity of purified Tgl3p. (C) Lineweaver-Burk plot for variable amounts of LPE used as substrate. (D) Lineweaver-Burk plot for variable amounts of oleoyl-CoA used as substrate. The mean values of three independent experiments ± SD are shown. NE, no enzyme; NS, no substrate.

Figure 4.

Effect of amino acid substitution on lipase and acyltransferase activity of Tgl3p. Amino acid residues of the acyltransferase motif HGYSQD were replaced by site-directed mutagenesis as indicated. Mutagenized variants were overexpressed using a galactose inducible promoter, lipid particles were isolated, solubilized, and assayed for LPE acyltransferase activity. (A) Western Blot analysis of lipid particles from Tgl3p variants. (B) Enzymatic activity of Tgl3p variants. White bars, lipase activity; and black bars, acyltransferase activity. The values of three independent experiments ± SD are shown.

Figure 5.

Role of Tgl3p on cell growth. Deletion mutant tgl3Δ tgl4Δ was transformed with empty pYES2 vector, pYES2-TGL3^wt, pYES2-TGL3^S237A, and pYES2-TGL3^H298. All transformants were pre-cultured in minimal glucose medium without uracil at 30°C for 48 h to reach the stationary phase. Then, cells were inoculated into fresh induction medium containing galactose as the carbon source for growth phenotype analysis. Open circles: tgl4Δ/pYES2^vector; Open squares, tgl3Δ tgl4Δ/pYES2-TGL3^wt; filled triangles, tgl3Δ tgl4Δ/pYES2-TGL3^S237A; and filled diamonds, tgl3Δ tgl4Δ/pYES2-TGL3^H298A.

Figure 6.

Functional role of Tgl3p during yeast spore formation. A tgl3Δ tgl3Δ homozygous diploid and tgl3Δ tgl3Δ transformed with plasmids bearing wild-type TGL3 and variants, respectively, were grown in sporulation medium as described in Materials and Methods. (A) DAPI staining. (B) Spores in cultures were counted and sporulation efficiency was calculated as percentages. Mean values of three independent experiments ± SD are shown.

Figure 7.

Heterologous overexpression of Tgl5p in P. pastoris enhances synthesis of phosphatidic acid. (A) Amount of total phospholipids per milligram of protein from whole cell extracts was quantified as described under Materials and Methods, and the value of wild type was set at 100%. (B) Cells overexpressing Tgl5p and the wild type were labeled in the presence of [¹⁴C]acetate. Incorporation of [¹⁴C]acetate into individual phospholipids was measured and used to calculate phospholipid synthesis. Black bars, wild type cells; and white bars, Tgl5p-overexpressing strain. (C) Incorporation of [¹⁴C]acetate into neutral lipids. Black bars, TAG; and white bars, SE. Mean values of three independent experiments ± SD are shown.

Figure 8.

Tgl5p catalyzes oleoyl-CoA–dependent acylation of lysophosphatidic acid. A purified Tgl5p-His₆ fusion protein was examined for lysophospholipid acyltransferase activity. (A) Examination of different lysophospholipid acceptors in the presence of 18:1 acyl-CoA. (B) Lineweaver-Burk plot for variable amounts of LPA used as substrate. (C) Lineweaver-Burk plot for variable amounts of oleoyl-CoA used as substrate. The mean values of three independent experiments ± SD are shown.

Figure 9.

Characterization of the purified Tgl5p. (A) Acyl chain preference of purified Tgl5p. (B) Effect of detergents on LPA acyltransferase activity. Open circles, CHAPS; and filled circles, Triton X-100.