Construction of Global Acyl Lipid Metabolic Map by Comparative Genomics and Subcellular Localization Analysis in the Red Alga Cyanidioschyzon merolae

Pathways of lipid metabolism have been established in land plants, such as Arabidopsis thaliana, but the information on exact pathways is still under study in microalgae. In contrast with Chlamydomonas reinhardtii, which is currently studied extensively, the pathway information in red algae is still in the state in which enzymes and pathways are estimated by analogy with the knowledge in plants. Here we attempt to construct the entire acyl lipid metabolic pathways in a model red alga, Cyanidioschyzon merolae, as an initial basis for future genetic and biochemical studies, by exploiting comparative genomics and localization analysis. First, the data of whole genome clustering by Gclust were used to identify 121 acyl lipid-related enzymes. Then, the localization of 113 of these enzymes was analyzed by GFP-based techniques. We found that most of the predictions on the subcellular localization by existing tools gave erroneous results, probably because these tools had been tuned for plants or green algae. The experimental data in the present study as well as the data reported before in our laboratory will constitute a good training set for tuning these tools. The lipid metabolic map thus constructed show that the lipid metabolic pathways in the red alga are essentially similar to those in A. thaliana, except that the number of enzymes catalyzing individual reactions is quite limited. The absence of fatty acid desaturation to produce oleic and linoleic acids within the plastid, however, highlights the central importance of desaturation and acyl editing in the endoplasmic reticulum, for the synthesis of plastid lipids as well as other cellular lipids. Additionally, some notable characteristics of lipid metabolism in C. merolae were found. For example, phosphatidylcholine is synthesized by the methylation of phosphatidylethanolamine as in yeasts. It is possible that a single 3-ketoacyl-acyl carrier protein synthase is involved in the condensation reactions of fatty acid synthesis in the plastid. We will also discuss on the redundant β-oxidation enzymes, which are characteristic to red algae.

localized in the plastid (Supplementary Figure 5B). It is likely that CMR388C is the TGD3 ortholog in C. merolae.
Flippase catalyzes the movement of polar lipids between the two membrane leaflets that does not happen spontaneously, which finally results in asymmetric distribution of lipids between the two leaflets of a membrane. In A. thaliana, the P4 subfamily of ATPases, ALA1-12, are believed to act as flippases (Gomès et al., 2000). Among them, ALA1-3 already have been characterized (López-Marqués et al., 2010Poulsen et al., 2008). Additionally, ALA-interacting subunit (ALIS) proteins are involved in the determination of subcellular localization of ALA enzymes (López-Marqués et al., 2010Poulsen et al., 2008). In C. merolae, two flippases (ALA1; CMR306C and ALA2; CMS375C) and an ALIS protein (CMT246C) were detected by the Gclust analysis (Supplementary Table 3). Both ALA1 and ALIS proteins were dually localized to the ER and cytoplasmic membrane ( Figure 1D and Supplementary Figure 5B). ALA2 was targeted to the nucleus and cytosol (Supplementary Figure 5B).
Biotin is attached to biotin-dependent enzymes, such as carboxylases or decarboxylases, by posttranslational modification catalyzed by holocarboxylase synthetase (HCS). In C. merolae, HCS (CMC080C) was dually localized in the plastid and cytosol, as in plants (Supplementary Figure 6C). Additionally, we analyzed subcellular localization of typical biotin-dependent enzymes, namely methylcrotonyl-CoA carboxylase (MCC), propionyl-CoA carboxylase (PCC) and carbamoylphosphate synthase (CAR). Individual subunits of MCC (MCCA; CMT073C and MCCB; CMT071C) and PCC (PCCA; CMN243C and PCCB; CMM132C) were localized in the mitochondrion (Supplementary Figure 6C). C. merolae has two types of CARs involved in pyrimidine synthesis and arginine synthesis. They are multifunctional type and multisubunit type, respectively. Subcellular localization of a multifunctional type CAR (CAR1; CMQ255C) was not examined yet. In multisubunit type CAR, the small subunit (CarA) is encoded in the plastid genome, and was not analyzed. A product of nuclear CarB gene (CML055C) encoding the large subunit of CAR was targeted in the plastid as expected (Supplementary Figure 6C).

Supplementary Figure 3. Subcellular localization of enzymes related to synthesis of phospholipids and TAG in C. merolae.
These fluorescence micrographs show C. merolae cells transiently expressing GFP-fused protein related to synthesis of phospholipids and TAG. Abbreviation of enzyme names is indicated to Table 1. Asterisked enzymes of subcellular localization were detected by immunostained with anti-GFP antibody. DIC; Nomarski differential interference contrast, Chlorophyll; phycobilin and chlorophyll autofluorescences, GFP; GFP fluorescence or immunofluorescence using anti-GFP antibody, Merge; merged images of phycobilin and chlorophyll autofluorescences as well as green fluorescence. Bar = 2 µm.

Supplementary Figure 4. Subcellular localization of enzymes related to β-oxidation.
Fluorescence micrographs show C. merolae cells transiently expressing GFP-fused protein related to β-oxidation (A). Three β-oxidation enzymes were examined subcellular localization using constructs of GFP-fused C-terminal peptide (B). Abbreviation of enzyme names is indicated to Table 1. DIC; Nomarski differential interference contrast, Chlorophyll; phycobilin and chlorophyll autofluorescences, GFP; GFP fluorescence, Merge; merged images of phycobilin and chlorophyll autofluorescences as well as green fluorescence. Bar = 2 µm.  Table 3. Asterisked enzymes of subcellular localization were detected by immunostained with anti-GFP antibody. DIC; Nomarski differential interference contrast, Chlorophyll; phycobilin and chlorophyll autofluorescences, GFP; GFP fluorescence or immunofluorescence using anti-GFP antibody, Merge; merged images of phycobilin and chlorophyll autofluorescences as well as green fluorescence. Bar = 2 µm.

Supplementary Tables
Supplementary Table 1

. List of primers used for making of EGFP or HA tag constructs.
A part of uppercase letters of sequence of primers indicates common sequences of pCEG1 or pBSHAb-T3´ vector required for the cloning using the In-Fusion Cloning Kit (Clontech laboratories, Mountain View, CA, USA). Because a peptide sequence of CMS056C and CMM311C is identical, the same primer is used for subcellular localization analysis of these proteins.