Subcellular Localization and Light-Regulated Expression of Protoporphyrinogen IX Oxidase and Ferrochelatase in Chlamydomonas reinhardtii

Abstract

Protoporphyrinogen IX oxidase (PPO) catalyzes the last common step in chlorophyll and heme synthesis, and ferrochelatase (FeC) catalyzes the last step of the heme synthesis pathway. In plants, each of these two enzymes is encoded by two or more genes, and the enzymes have been reported to be located in the chloroplasts or in the mitochondria. We report that in the green alga Chlamydomonas reinhardtii, PPO and FeC are each encoded by a single gene. Phylogenetic analysis indicates that C. reinhardtii PPO and FeC are most closely related to plant counterparts that are located only in chloroplasts. Immunoblotting results suggest that C. reinhardtii PPO and FeC are targeted exclusively to the chloroplast, where they are associated with membranes. These results indicate that cellular needs for heme in this photosynthetic eukaryote can be met by heme that is synthesized in the chloroplast. It is proposed that the multiplicity of genes for PPO and FeC in higher plants could be related to differential expression in differently developing tissues rather than to targeting of different gene products to different organelles. The FeC content is higher in C. reinhardtii cells growing in continuous light than in cells growing in the dark, whereas the content of PPO does not significantly differ in light- and dark-grown cells. In cells synchronized to a light/dark cycle, the level of neither enzyme varied significantly with the phase of the cycle. These results indicate that heme synthesis is not directly regulated by the levels of PPO and FeC in C. reinhardtii.

Proteomic analysis of a toxic dinoflagellate Alexandrium catenella under different growth phases and conditions

“…Previous studies have shown that, in plants, cryptochromes and ferrochelatase protect cells from high light intensity and are regulated by light, at least at the mRNA level [47,48]. In C. reinhardtii, the ferrochelatase content is higher in cells grown in continuous light than those grown in the dark [41], consistent with our results. Chaperone Hsps have been found in dinoflagellates and have been shown to be upregulated in proportion to the severity of the stress encountered [18,49].…”

“…Heme is also a cofactor in some component of the mitochondrial electron transport chain and in various nonchloroplast hemoproteins (for example, the catalases and peroxidases). In the green algae Chlamydomonas reinhardtii, the level of ferrochelatase in the cells is synchronized to the light/dark cycle and shows no significant variation with the phase of the cycle [41]. In A. catenella, the highest cryptochrome and ferrochelatase expressions were detected in the exponential phase, indicating that the highly light-regulated physiological activities (photosynthesis, signal transduction and biosynthesis) occurred in the exponential growth phase of the cells.…”

Tetrapyrrole Metabolism inArabidopsis thaliana

“…These data indicate that PPO isoforms have dual localizations in both plastids and mitochondria in higher plants. However, only a single gene encoding PPO is present in a green alga (Chlamydomonas reinhardtii), the product of which is exclusively found in plastids (van Lis et al 2005). In the future, it will be important to clarify the physiological function of the PPO2 subfamily whose distribution is restricted in land plants.…”

“…More recently, Woodson et al (2011) showed that in Arabidopsis FC1 and FC2 are co-localized within plastids. In C. reinhardtii, only a single gene encoding FeCh is present, the product of which is exclusively found in plastids (van Lis et al 2005). Obornik and Green (2005) performed the phylogenetic analysis of genes involved in heme biosynthesis in eukaryotes.…”

Concerted Up-regulation of Aldehyde/Alcohol Dehydrogenase (ADHE) and Starch in Chlamydomonas reinhardtii Increases Survival under Dark Anoxia

“…Organelle Isolation and Partial Purification of Chloroplast ADHE-Isolation of chloroplasts and mitochondria from mutant strain 10-6C was essentially as described in (60). For the ADHE partial purification, the chloroplasts were resuspended in 50 mM HEPES (pH 7.0) in presence of protease inhibitors (0.5 mM benzamidine and 1 mM aminocaproic acid) to a final protein concentration of 5 mG/ml, frozen and thawed twice, and centrifuged at 11,000 ϫ g for 15 min at 4°C.…”