Proteome of the secondary plastid of Euglena gracilis reveals metabolic quirks and colourful history

Abstract

Correspondence to mfield@mac.com (MCF, proteomics) and vlada@natur.cuni.cz (VH, plastid evolution) MCF, JL, and VH conceived the original research plans; JL and MCF supervised the experiments; ELD performed the cell fractionation; MZ and SK performed the mass spectrometry analysis; AMGNV and TGE performed protein annotation and sorting, AMGNV, KZ, and ZF interpreted the annotation results, AMGNV performed the signal domain analysis, PS performed the phylogenetic analysis, AMGNV conceived the project and wrote the article with contributions of all the authors; VH, ME, MCF, and JL supervised and complemented the writing. VH agrees to serve as the author responsible for contact and ensures communication. AbstractEuglena gracilis is a well-studied biotechnologically exploitable phototrophic flagellate harbouring secondary green plastids. Here we describe its plastid proteome obtained by high-resolution proteomics. We identified 1,345 candidate plastid proteins and assigned functional annotations to 774 of them. More than 120 proteins are affiliated neither to the host lineage nor the plastid ancestor and may represent horizontal acquisitions from various algal and prokaryotic groups. Reconstruction of plastid metabolism confirms both the presence of previously studied/predicted enzymes/pathways and also provides direct evidence for unusual features of its metabolism including uncoupling of carotenoid and phytol metabolism, a limited role in amino acid metabolism and the presence of two sets of the SUF pathway for FeS cluster assembly. Most significantly, one of these was acquired by lateral gene transfer (LGT) from the chlamydiae. Plastidial paralogs of membrane traffickingassociated proteins likely mediating a poorly understood fusion of transport vesicles with the outermost plastid membrane were identified, as well as derlin-related proteins that potentially act as protein translocases of the middle membrane, supporting an extremely simplified TIC complex. The proposed innovations may be also 2 linked to specific features of the transit peptide-like regions described here. Hence the Euglena plastid is demonstrated to be a product of several genomes and to combine novel and conserved metabolism and transport processes.

The cryptic plastid of Euglena longa defines a new type of non-photosynthetic plastid organelles

“…The MEP pathway in E. gracilis provides precursors for the synthesis in the plastid of terpenoid compounds connected to photosynthesis, namely carotenoids and plastoquinone (Kim et al 2004; Novák Vanclová et al 2019). As expected, the respective enzymes are all missing from E. longa .…”

“…Photosynthetic eukaryotes generally produce three types of phytol derivatives, tocopherols (vitamin E), phylloquinone (PhQ; vitamin K 1 ) and chlorophyll (its phytyl chain), starting with a common precursor phytyl-PP, which is (directly or indirectly via salvage of phytol liberated by chlorophyll degradation) made by reduction of geranylgeranyl-PP synthesized in the plastid by the MEP pathway (Gutbrod et al 2019). E. gracilis proved to be unusual not only because of lacking the conventional geranylgeranyl-PP reductase (Novák Vanclová et al 2019), but also for making phytol from precursors provided by the MVA pathway (Disch et al 1998; Kim et al 2004). The route of phytol synthesis from the cytosolic isoprenoids is currently unknown, though it was proposed that phytyl-PP is synthesized in the E. gracilis plastid exclusively by step-wise phosphorylation of phytol by phytol kinase (VTE5) and phytyl phosphate kinase (VTE6), which are enzymes normally employed for recycling phytol from chlorophyll degradation (Novák Vanclová et al 2019).…”

“…E. gracilis proved to be unusual not only because of lacking the conventional geranylgeranyl-PP reductase (Novák Vanclová et al 2019), but also for making phytol from precursors provided by the MVA pathway (Disch et al 1998; Kim et al 2004). The route of phytol synthesis from the cytosolic isoprenoids is currently unknown, though it was proposed that phytyl-PP is synthesized in the E. gracilis plastid exclusively by step-wise phosphorylation of phytol by phytol kinase (VTE5) and phytyl phosphate kinase (VTE6), which are enzymes normally employed for recycling phytol from chlorophyll degradation (Novák Vanclová et al 2019). This scheme is supported by the fact that E. longa has retained VTE5 as well as VTE6, both proteins being highly similar to their E. gracilis orthologs and exhibiting putative plastid targeting presequences (Fig.…”

“…In addition of its role in chlorophyll synthesis, phytyl-PP is used by E. gracilis to make tocopherols and a PhQ derivative, 5’-monohydroxyphylloquinone (OH-PhQ; Ziegler et al 1989; Watanabe et al 2017; Novák Vanclová et al 2019). All four enzymes mediating synthesis of α-tocopherol from phytyl-PP and homogentisate were identified in E. gracilis and are localized to its plastid (Novák Vanclová et al 2019).…”

“…To chart the main paths of the metabolic map of the E. longa plastids, we searched for homologs of enzymes underpinning pathways known from plastids of other species. The reconstruction was greatly facilitated by the recent characterization of the E. gracilis plastid metabolic network based on a proteomic analysis of the organelle (Novák Vanclová et al 2019). N-terminal regions of the candidates were evaluated for characteristics of presequences predicting a specific subcellular localization to distinguish those likely representing plastid-targeted proteins from enzymes located to other parts of the cell.…”