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. 2019 Jul 31;24(15):2789.
doi: 10.3390/molecules24152789.

Long-Chain Polyisoprenoids Are Synthesized by AtCPT1 in Arabidopsis thaliana

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Free PMC article

Long-Chain Polyisoprenoids Are Synthesized by AtCPT1 in Arabidopsis thaliana

Przemyslaw Surowiecki et al. Molecules. .
Free PMC article

Abstract

Arabidopsis roots accumulate a complex mixture of dolichols composed of three families, (i.e., short-, medium- and long-chain dolichols), but until now none of the cis-prenyltransferases (CPTs) predicted in the Arabidopsis genome has been considered responsible for their synthesis. In this report, using homo- and heterologous (yeast and tobacco) models, we have characterized the AtCPT1 gene (At2g23410) which encodes a CPT responsible for the formation of long-chain dolichols, Dol-18 to -23, with Dol-21 dominating, in Arabidopsis. The content of these dolichols was significantly reduced in AtCPT1 T-DNA insertion mutant lines and highly increased in AtCPT1-overexpressing plants. Similar to the majority of eukaryotic CPTs, AtCPT1 is localized to the endoplasmic reticulum (ER). Functional complementation tests using yeast rer2Δ or srt1Δ mutants devoid of medium- or long-chain dolichols, respectively, confirmed that this enzyme synthesizes long-chain dolichols, although the dolichol chains thus formed are somewhat shorter than those synthesized in planta. Moreover, AtCPT1 acts as a homomeric CPT and does not need LEW1 for its activity. AtCPT1 is the first plant CPT producing long-chain polyisoprenoids that does not form a complex with the NgBR/NUS1 homologue.

Keywords: Arabidopsis thaliana; T-DNA insertion mutant; cis-prenyltransferase; dolichols; polyisoprenoids; polyprenols; protein N-glycosylation; rer2Δ and srt1Δ Saccharomyces cerevisiae mutant.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Structure of polyprenol and dolichol. α and ω represent terminal isoprene units. The internal isoprene residues in trans and cis configuration are indicated.
Figure 2
Figure 2
The effect of AtCPT1 deficiency or overexpression on long-chain dolichols accumulation—in planta analysis. (A) AtCPT1 gene structure. The start codon (ATG) and the stop codon (TGA) are indicated. Gray boxes indicate exons, and lines between boxes indicate introns. The T-DNA insertion sites in cpt1-1, cpt1-2 and cpt1-3 mutant lines are shown. (B) Relative expression of AtCPT1 in Arabidopsis roots of the wild type (Col-0), three independent T-DNA insertion mutant lines (cpt1-1, cpt1-2 and cpt1-3) and AtCPT1 overexpressing line (CPT1-OE). AtCPT1 mRNA abundance was quantified by Real-Time PCR and is presented as a percentage of the wild type (100%). (C) Representative HPLC/UV chromatograms of total polyisoprenoids extracted from the roots of Arabidopsis wild typeT-DNA insertion mutants and CPT1-OE plants. The dominating homologues of polyprenols and dolichols accumulated in Arabidopsis roots, Dol-13, Dol-16 and Pren/Dol-21, respectively are indicated. (D) Quantification of the long-chain polyprenols and dolichols extracted from roots of studied plants. Data represents three independent experiments. Asterisk (*) indicates significant difference (0.01 < P < 0.05 in Student’s t-test) between WT and mutant plants.
Figure 3
Figure 3
Growth phenotype of AtCPT1 mutants. The wild type (Col-0), T-DNA insertion mutants (cpt1-1, cpt1-2 and cpt1-3) and AtCPT1 overexpressing (CPT1-OE) Arabidopsis seedlings were grown for seven days on vertically oriented agar plates. Scale bar = 1 cm. Representative data are shown.
Figure 4
Figure 4
Subcellular localization of AtCPT1. Tobacco leaves transiently expressing AtCPT1 containing a C-terminal GFP-tag fused at the C-terminus to GFP. AtCPT1-GFP signal is co-localized mostly with the markers of the endoplasmic reticulum (A,B) while no co-localization with Golgi marker (C) is observed. PS35::AtCPT1-GFP (green) and organelle markers ER-CFP (A), ER-mCherry (B) or Golgi-mCherry (C) were transiently expressed in N. benthamiana leaves by agroinfiltration. Scale bars = 10 µm. Representative pictures are shown.
Figure 5
Figure 5
Analysis of enzymatic activity of AtCPT1 in the S. cerevisiae CPT mutants, rer2Δ or srt1Δ. (A) Expression of AtCPT1 in rer2Δ mutant complements defects in growth. rer2Δ cells were transformed with the empty plasmid pYES-DEST52 and AtCPT1 construct, respectively. Serially diluted yeast cultures were plated on solid YP-2% galactose medium and cultured for five days at 23 or 37 °C. (B) AtCPT1 expression in rer2Δ yeast strain restores glycosylation status of carboxypeptidase Y (CPY). Protein extracts from yeast transformants were separated by SDS-PAGE and analyzed by Western blot with anti-CPY antibody. The positions of mature CPY (mCPY) and hypoglycosylated glycoforms lacking between one and four N-linked glycans (−1 to −4) are indicated. (C) Polyisoprenoid profiles of rer2Δ or srt1Δ yeast mutants transformed with empty plasmid, AtCPT1, AtCPT1/Lew1 and Lew1, respectively. Presented are representative HPLC/UV chromatograms.
Figure 6
Figure 6
Phylogenetic analysis of CPTs. Neighbor-joining phylogenetic tree of CPTs and CPT related protein from plants (green or blue branches), animals (red), yeast (orange) and bacteria (yellow). The analyzed proteins sequences are divided into three groups: (I) the homomeric CPTs, (II) heteromeric CTPs that form a complex with a CPT-binding protein and (III) CPT-binding protein including the human NogoB receptor and its orthologs. Amino acid sequences were aligned using http://www.phylogeny.fr/index.cgi and the tree was constructed using https://itol.embl.de/. Species abbreviations: At, Arabidopsis thaliana; Dr, Danio rerio; Ec, Escherichia coli; Hb, Hevea brasiliensis; h, Homo sapiens; Hi, Handroanthus impetiginosus; Ir, Ixodes ricinus; Is, Ixodes scapularis; LL, Lilium longiflorum; Ls, Lactuca sativa; Mt, Mycobacterium tuberculosis; Np, Nostoc punctiforme; Os, Oryza sativa; Pa, Parthenium argentatum; Pt, Populus trichocarpa; Sb, Sorghum bicolour; Sc, Saccharomyces cerevisiae; Shz, Solanum habrochaites; Sl, Solanum lycopersicum; Sp, Streptococcus pneumoniae; Sy, Synechococcus; Syc, Synechocystis; Tb, Taraxacum brevicorniculatum; Vv, Vitis vinifera; Zm, Zea mays.

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