Mitochondrial phosphatidylserine decarboxylase from higher plants. Functional complementation in yeast, localization in plants, and overexpression in Arabidopsis

Abstract

Plants are known to synthesize ethanolamine (Etn) moieties by decarboxylation of free serine (Ser), but there is also some evidence for phosphatidyl-Ser (Ptd-Ser) decarboxylation. Database searches identified diverse plant cDNAs and an Arabidopsis gene encoding 50-kD proteins homologous to yeast (Saccharomyces cerevisiae) and mammalian mitochondrial Ptd-Ser decarboxylases (PSDs). Like the latter, the plant proteins have putative mitochondrial targeting and inner membrane sorting sequences and contain near the C terminus a Glycine-Serine-Threonine motif corresponding to the site of proteolysis and catalytic pyruvoyl residue formation. A truncated tomato (Lycopersicon esculentum) cDNA lacking the targeting sequence and a chimeric construct in which the targeting and sorting sequences were replaced by those from yeast PSD1 both complemented the Etn requirement of a yeast psd1 psd2 mutant, and PSD activity was detected in the mitochondria of the complemented cells. Immunoblot analysis of potato (Solanum tuberosum) mitochondria demonstrated that PSD is located in mitochondrial membranes, and mRNA analysis in Arabidopsis showed that the mitochondrial PSD gene is expressed at low levels throughout the plant. An Arabidopsis knockup mutant grew normally but had 6- to 13-fold more mitochondrial PSD mRNA and 9-fold more mitochondrial PSD activity. Total membrane PSD activity was, however, unchanged in the mutant, showing mitochondrial activity to be a minor part of the total. These results establish that plants can synthesize Etn moieties via a phospholipid pathway and have both mitochondrial and extramitochondrial PSDs. They also indicate that mitochondrial PSD is an important housekeeping enzyme whose expression is strongly regulated at the transcriptional level.

Figure 1.

The origins of Etn moieties in plant phospholipids. Dotted arrows show the Kennedy pathway, which proceeds from Etn to PtdEtn via phospho and CDP derivatives of Etn. CDP-DAG, CDP diacylglycerol; CMP, cytidine 5′-phosphate; P, phospho; Ptd, phosphatidyl; SDC, Ser decarboxylase; PSD, Ptd-Ser decarboxylase; PSS, Ptd-Ser synthase; BE, base exchange. Note that when PSD and base exchange act together, they constitute a cycle.

Figure 2.

Alignment of the tomato and Arabidopsis PSD homologs with yeast and Chinese hamster mitochondrial PSDs. Identical residues are shaded in black, similar residues are shaded in gray. Dashes are gaps introduced to maximize alignment. The arrowhead marks the conserved Ser residue implicated in autocatalytic cleavage into α- and β-subunits, and pyruvoyl prosthetic group formation. The bar indicates a hydrophobic inner membrane sorting sequence in the tomato and Arabidopsis proteins. LePSD1, tomato PSD1 (GenBank accession no. AY093689); AtPSD1, Arabidopsis PSD1 (AY189805); ScPSD1, yeast PSD1 (L20973); CgPSD, Chinese hamster mitochondrial PSD (M62722). The diamond shows where the LePSD1 sequence was truncated for expression in yeast, and the asterisk shows the junction between the parts of the chimeric yeast-plant enzyme.

Figure 3.

Complementation of a yeast psd1 psd2 mutant by LePSD1 constructs and PSD activities in complemented cells. A, Cells of wild-type yeast strain SEY6210 (segment 1), the psd1 psd2 mutant RYY51 (segment 2), RYY51 transformed with pVT103-U alone (segment 3) or containing full-length LePSD (segment 4), truncated LePSD (segment 5), and chimeric yeast PSD1/LePSD1 (segment 6) were plated on synthetic minimal medium plus or minus 5 mm Etn. B, PSD activities in homogenates of RYY51 cells harboring YCp50-PSD1 (ScPSD1), RYY51 cells complemented with pVT103-U alone (vector) or carrying full-length LePSD1, truncated LePSD1 (LePSD1t), and chimeric yeast PSD1/LePSD1 (chimera). Numbers in parentheses beneath the labels correspond to the segments in A. Cells were grown on minimal medium supplemented with Etn. White bars, PSD activity measured with Ptd[1′-¹⁴C]Ser; shaded bars, activity measured with NBD-Ptd-[1′-¹⁴C]Ser. Values are means of three replicates ± se.

Figure 4.

Evidence for the mitochondrial location of PSD1 in plants. Potato tuber mitochondria isolated by Percoll density gradient centrifugation were ruptured by freezing/thawing and separated into matrix and membrane fractions by ultracentrifugation. A, Specific activities (in micromoles per minute) of matrix (fumarase) and inner membrane (succinate:cytochrome c oxidoreductase) markers measured on mitochondria (Mit) and their matrix (Mat) and membrane (Mem) fractions. Data are means of three replicates ± se. B, The same fractions were analyzed by SDS-PAGE (5 μg protein lane^–1) and immunoblotting with antibodies raised against recombinant LePSD1.

Figure 5.

Analysis of the gene encoding AtPSD1 in wild-type and mutant Arabidopsis. A, Structure of the coding region of the gene. Black boxes represent exons, and solid lines represent introns. Dotted lines are outside the coding region; introns could not be identified here because the cDNA comprised the coding sequence only. The horizontal bar marked A is the position of the amplicon used in quantitative RT-PCR. The arrowhead shows the position of the codons for the GST consensus. The position of the T-DNA insertion in the probable promoter region is indicated. B, Southern-blot analysis of T-DNA mutants and their wild-type siblings. The ³²P-labeled probe was specific for the BAR gene within the T-DNA. Genomic DNA was digested with EcoRI (which cuts each side of the probe) or HindIII (which cuts only at the 5′ end of the probe).