Subcellular evidence for the involvement of peroxisomes in plant isoprenoid biosynthesis

Abstract

The role of peroxisomes in isoprenoid metabolism, especially in plants, has been questioned in several reports. A recent study of Sapir-Mir et al.¹ revealed that the two isoforms of isopentenyl diphosphate (IPP) isomerase, catalyzing the isomerisation of IPP to dimethylallyl diphosphate (DMAPP) are found in the peroxisome. In this addendum, we provide additional data describing the peroxisomal localization of 5-phosphomevalonate kinase and mevalonate 5-diphosphate decarboxylase, the last two enzymes of the mevalonic acid pathway leading to IPP.² This finding was reinforced in our latest report showing that a short isoform of farnesyl diphosphate, using IPP and DMAPP as substrates, is also targeted to the organelle.³ Therefore, the classical sequestration of isoprenoid biosynthesis between plastids and cytosol/ER can be revisited by including the peroxisome as an additional isoprenoid biosynthetic compartment within plant cells.

In plants, the classical view of the isoprenoid metabolism compartmentalisation is based on a physical separation of biosynthetic enzymes between plastid, mitochondria and cytosol/RE.⁴ Such compartments house the two biosynthetic pathways that provide the C₅ universal precursor of isoprenoid compounds, isopentenyl diphosphate (IPP), including the methyl-D-erythritol 5-phosphate (MEP) pathway localized in plastids and the mevalonic acid (MVA) pathway, generally regarded as cytosolic. IPP undergoes a reversible isomerisation step catalyzed by the IPP isomerase (IDI), which leads to the generation of dimethylallyl diphosphate (DMAPP). Sequential condensations of IPP with DMAPP lead to the formation of polyprenyl diphosphates, which are the precursors for the biosynthesis of the different families of isoprenoid end products. These polyprenyl diphosphates are synthesized by prenyltransferases including GPP synthase (GPS), FPP synthase (FPS) and GGPP synthase (GGPS), belonging to the short-chain prenyltransferase subfamily.⁴ Short-chain prenyltransferases have been localized in the subcellular compartments mentioned above, suggesting that the C₅ precursors originating from the MEP and MVA pathways lead to plastidial and cytosolic/ER isoprenoid end products, respectively, but are also transported to the mitochondria for ubiquinone side-chain production. While the MEP pathway enzymes have been entirely localized within plastids,⁵ the compartmentalisation of the MVA pathway has been less characterized. For a long time, it has been considered cytosolic although an additional peroxisomal localization has been suggested.⁶ In mammals, the group of Krisans issued a clarification by confirming previous data showing peroxisomal localization of the early steps in the isoprenoid/cholesterol pathway.⁷ As a consequence, the involvement of peroxisomes in isoprenoid biosynthesis becomes gradually clearer. Besides mammals, trypanosomatid parasites contain the MVA pathway, for which it was proposed that the last steps are localized within peroxisome-related microbodies called glycosomes.⁸ In addition, the GGPS PaxG of the filamentous fungus Penicillium paxilli, which is required for the synthesis of the secondary metabolite paxilline, is also targeted to the peroxisome.⁹

In plants, recent studies have identified the peroxisome as an additional site for isoprenoid biosynthesis. The involvement of this organelle in IPP biosynthesis has been highlighted by transient expression of IDI-GFP constructs in tobacco protoplasts. Arabidopsis thaliana contains two IDI genes, which are transcribed each as long and short isoforms. The long versions of protein products are targeted to chloroplasts/mitochondria whereas the short versions, lacking the N-targeting signal, are localized to peroxisomes.¹ Following this work, we extended this type of study to other isoprenoid enzymes leading us to re-evaluate the classical scheme of their cytosolic localization. Thus, transient transformations of Madagascar periwinkle (Catharanthus roseus) cells with Yellow Fluorescent Protein-fused constructs revealed that the last two enzymes of the MVA pathway from Arabidopsis and C. roseus, 5-phosphomevalonate kinase (PMK) and mevalonate 5-diphosphate decarboxylase (MVD), are localized to peroxisomes.² Furthermore, two other C. roseus enzymes related to the MVA pathway have been shown to be targeted to this organelle: a short isoform of IDI (Courdavault, personal communication), thus confirming the results obtained by Sapir-Mir et al.¹ and a short isoform of trans-type FPS.³

Two types of peroxisomal targeting signals (PTS), PTS1 and PTS2 mediate the peroxisome import of proteins by the PTS1 receptor PEX5 and the PTS2 receptor PEX7.⁶ PTS1 is commonly a C-terminal tripeptide displaying the consensus sequence (S/C/A)(K/R/H)(L/M) and PTS2 is a nonapeptide located near the N-terminus, and having the usual consensus sequence (R/K)(L/V/I/Q)X₅(H/Q)(L/A/F).¹⁰ It could be mentioned that there are peroxisomal-targeted proteins that contain alternative targeting information, differing from the canonical PTS.¹¹ PTS2-like motifs were found in the N-terminal region of Arabidopsis and C. roseus PMK and MVD. The short IDI isoforms of Arabidopsis and C. roseus contained a C-terminal PTS1 while no canonical PTS was found in the periwinkle FPS. Plants contain at least two FPS genes.¹² FPS1 encodes a long isoform FPS1L targeted to mitochondria¹² and a short isoform FPS1S, for which no localization has been reported. The FPS2 gene encodes a short isoform FPS2 that is more closely related to the periwinkle FPS, allowing in turn to suggest a potential peroxisomal localization for this type of isoform.

On the basis of these results, we proposed a new model of the subcellular distribution of the early steps leading to the biosynthesis of FPP and then to isoprenoids in plants, providing evidence for a complex compartmentalisation of IDI, FPS and the MVA pathway enzymes (Fig. 1). Such model highlights the existence of potential exchanges of intermediate metabolites between cytosol, ER and peroxisome but also raises the question of the specific role of each individual short FPS isoform in the biosynthesis of cytosolic isoprenoid end products (sesquiterpenes and farnesylated proteins) and ER-located sterols and polyisoprenoid alcohols. Finally, in light of these new data, one may wonders whether the peroxisome could be an autonomous organelle capable of synthesizing isoprenoid compounds itself from its own FPP pool.

Figure 1.

New view scheme of the subcellular distribution of the early steps leading to isoprenoid biosynthesis in plants. The subcellular localization of the mitochondrial and plastidial isoprenoid pathways are not represented. The scheme is a compilation of published results.^1-3 Enzymes from the MVA pathway are indicated in italics. Enzymes highlighted by black background have been experimentally validated for their subcellular localization in plants. No experimental data have validated the putative peroxisomal or cytosolic localization of the short isoform FPS1. Plain arrows indicate translocation of intermediates across the membranes. AACT: acetoacetyl-CoA thiolase; DMAPP: dimethylallyl diphosphate; FPP: farnesyl diphosphate; FPS1 and FPS2: short isoforms of farnesyl diphosphate synthases 1 and 2; HMG-CoA: hydroxymethylglutaryl-CoA; HMGS: hydroxymethylglutaryl-CoA synthase; HMGR: hydroxymethylglutaryl-CoA reductase; IDI: isopentenyl diphosphate isomerase; IPP: isopentenyl diphosphate; MVD: mevalonate diphosphate decarboxylase; MVK: mevalonate kinase; PMK: phosphomevalonate kinase.

Simkin A J, Guirimand G, Papon N, Courdavault V, Thabet I, Ginis O, Bouzid S, Giglioli-Guivarc'h N, Clastre M. Peroxisomal localisation of the final steps of the mevalonic acid pathway in planta. Planta. 2011;234 doi: 10.1007/s00425-011-1444-6.

Thabet I, Guirimand G, Courdavault V, Papon N, Godet S, Dutilleul C, Bouzid S, Giglioli-Guivarc'h N, Clastre M, Simkin A J. The subcellular localization of periwinkle farnesyl diphosphate synthase provides insight into the role of peroxisome in isoprenoid biosynthesis. J Plant Physiol. 2011;168 doi: 10.1016/j.jplph.2011.06.017.