A polyamine metabolon involving aminopropyl transferase complexes in Arabidopsis

Abstract

The conversion of putrescine to spermidine in the biosynthetic pathway of plant polyamines is catalyzed by two closely related spermidine synthases, SPDS1 and SPDS2, in Arabidopsis. In the yeast two-hybrid system, SPDS2 was found to interact with SPDS1 and a novel protein, SPMS (spermine synthase), which is homologous with SPDS2 and SPDS1. SPMS interacts with both SPDS1 and SPDS2 in yeast and in vitro. Unlike SPDS1 and SPDS2, SPMS failed to suppress the speDelta3 deficiency of spermidine synthase in yeast. However, SPMS was able to complement the speDelta4 spermine deficiency in yeast, indicating that SPMS is a novel spermine synthase. The SPDS and SPMS proteins showed no homodimerization but formed heterodimers in vitro. Pairwise coexpression of hemagglutinin- and c-Myc epitope-labeled proteins in Arabidopsis cells confirmed the existence of coimmunoprecipitating SPDS1-SPDS2 and SDPS2-SPMS heterodimers in vivo. The epitope-labeled SPDS and SPMS proteins copurified with protein complexes ranging in size from 650 to 750 kD. Our data demonstrate the existence of a metabolon involving at least the last two steps of polyamine biosynthesis in Arabidopsis.

Figure 1.

Amino Acid Sequence Comparison and Genomic Organization of Arabidopsis Aminopropyl Transferase Proteins. (A) Comparison of amino acid sequences of Arabidopsis and T. maritima SPDS proteins. The SPDS protein sequences were aligned and compared by Baylor College of Medicine Search Launcher (http://searchlauncher.bcm.tmc.edu). Black shading indicates identical amino acid residues, whereas similarities are highlighted in gray. Asterisks indicate the positions of residues that interact with the artificial inhibitor 5-adenosyl-1,8-diamino-3-thiooctane. The arrowhead indicates a residue of putrescine N-methyltransferase that deviates from the conserved residues of SPDS. (B) Scheme of the intron/exon structure of the genomic regions of the SPDS1, SPDS2, SPMS, and ACL5 genes. Black boxes and solid lines represent exons and introns, respectively. Numbers refer to the nucleotides in each exon.

Figure 2.

Distance Relationship among Spermidine and Spermine Synthases of Diverse Origins. Protein sequences were aligned using ClustalX, and the tree was generated using the neighbor-joining method. Origins of the proteins are as follows: AtACL5 (Arabidopsis), AtSPDS1 (Arabidopsis), AtSPDS2 (Arabidopsis), AtSPMS (Arabidopsis), ScSPE3 (Saccharomyces cerevisiae), ScSPE4 (S. cerevisiae), MmSPDS (Mus musculus), MmSPMS (M. musculus), HsSPDS (Homo sapiens), HsSPMS (H. sapiens), DrSPMS (Danio rerio), FrSPMS (Fugu rubripes), TfSPMS (Tetraodon fluviatilis), PsSPDS1 (Pisum sativum), PsSPDS2 (P. sativum), LeSPDS (Lycopersicon esculentum), TmSPDS (T. maritima), BsSPDS (Bacillus subtilis), BhSPDS (Bacillus holodurans), AaSPDS (Aquifex aeolicus), CeSPDS (Caenorhabditis elegans), and NcSPDS (Neurospora crassa).

Figure 3.

Yeast Two-Hybrid Interactions between SPDS and SPMS. Yeast transformants containing either GAL4 binding domain (GBD) or GAL4 activation domain (GAD) fusion proteins were grown on nylon filters, placed on synthetic medium, and tested for β-galactosidase activity with the LacZ filter-lift assay. Photographs were taken after 12 h of β-galactosidase enzymatic reaction.

Figure 4.

Interactions of SPDS and SPMS Proteins in Vitro. (A) The SPDS and SPMS proteins were fused to N-terminal GST tags using pGEX vectors and purified from E. coli using glutathione-Sepharose affinity chromatography. Quality tests of total protein extracts from E. coli before (lanes 1, 4, and 7) and after (lanes 2, 5, and 8) induction of GST-SPDS/SPMS expression with isopropylthiogalactoside, and that of purified GST-SPDS/SPMS proteins (lanes 3, 6, and 9), were performed by SDS-PAGE. Lanes 1 to 3, GST-SPDS1; lanes 4 to 6, GST-SPMS; and lanes 7 to 9, GST-SPDS2. (B) SDPS1 and SPMS labeled with ³⁵S-Met were subjected to pulldown assay with GST-SPDS2. Beads carrying immobilized GST-SPDS2 and control GST proteins, as well as the control glutathione-Sepharose matrix (GS), were incubated with equal amounts of labeled SPDS proteins. The eluted matrix-bound proteins and aliquots from the supernatants were separated by SDS-PAGE and detected by autoradiography. A similar pulldown assay was performed using ³⁵S-SPDS1 and GST-SPMS.

Figure 5.

Yeast Complementation Studies. (A) Complementation test with the yeast speΔ3 mutant. The growth of the speΔ3 yeast strain carrying the Arabidopsis SPDS expression vectors YEpACT-SPDS1, YEpACT-SPDS2, and YEpACT-SPMS, and the empty control vector YEpACT, was monitored after diluting the cultures 1:500 every 24 h and measuring OD₆₀₀ as described (Alabadí and Carbonell, 1999). SPE3 strain 2602 was used as a control. After 4 days in culture, 100 μM spermidine was added to the medium of YEpACT and YEpACT-SPMS transformants. The OD₆₀₀ values correspond to the mean of three independent assays. (B) Complementation test with the yeast speΔ4 mutant. Putrescine, spermidine, and spermine contents were determined, as described in Methods, in the extracts of the yeast wild type (strain 2602), the spe4 mutant (Y504), and the speΔ4 mutant transformed with the Arabidopsis SPMS cDNA under the control of the yeast ADH1 promoter. (C) Conversion of ¹⁴C-spermidine into ¹⁴C-spermine in the yeast speΔ4 mutant transformed with the Arabidopsis SPMS cDNA under the control of the yeast ADH1 promoter. Yeast cells were incubated with ¹⁴C-spermidine, and radioactivity was quantified in fractions after HPLC separation of dansylated polyamines. The inset shows the radioactivity of the HPLC fractions collected every 1 min during the interval 11 to 18 min. HD, 1,6-diamine hexane; spd, spermidine; spm, spermine.

Figure 6.

Immunological Detection of Epitope-Labeled SPDS and SPMS. The formation of heterodimers and protein complexes in Arabidopsis cell extracts is shown. (A) Individual expression of epitope-tagged SPDS and SPMS in plant cells. Total protein extracts from Arabidopsis cells transformed with the indicated constructs were immunoblotted with anti-HA and anti-c-Myc antibodies. (B) Effect of double transformations on protein expression levels. Arabidopsis cells were transformed simultaneously as indicated, and total protein extracts were used for immunoblot analysis with anti-HA and anti-c-Myc antibodies. Remarkably, all pairwise combinations of SPDS1 and SPMS yielded very low protein expression levels. (C) Detection of SPDS heterodimers formed in vivo. Epitope-labeled SPDS1-myc and SPDS2-HA, as well as SPDS2-myc and SPMS-HA, proteins were coexpressed in Arabidopsis cells. The cell extracts were subjected to immunoaffinity purification on anti-c-Myc IgG beads. Aliquots from the total protein extracts (T) and eluted matrix-bound proteins (IP) were immunoblotted with anti-HA and anti-c-Myc antibodies. (D) Superose 6 size fractionation of protein extracts prepared from Arabidopsis cells expressing SPDS2-HA, SPDS1-myc, and SPMS-myc proteins. Protein complexes carrying the epitope-labeled proteins were assayed by immunoblotting with anti-HA and anti-c-Myc antibodies. The arrow indicates the position of the molecular mass marker thyroglobulin (669 kD).