Phyllosinol

Chloroplasts of plants contain an intricate membrane system, the thylakoids, which harbor the complexes of the photosynthetic machinery. Chloroplasts are confined by two membranes, the inner and outer envelope. The major glycerolipids of chloroplasts are the glycolipids monogalactosyl diacylglycerol (MGD), digalactosyl diacylglycerol (DGD), and sulfoquinovosyl diacylglycerol (SQD). Furthermore, two phospholipids, phosphatidyl glycerol (PG) and phosphatidyl choline (PC), are found in chloroplast membranes. The photosystems and light-harvesting complexes in the thylakoids are rich in photosynthetic pigments (chlorophyll, carotenoids, and xanthophylls) and contain a unique set of prenylquinol lipids (tocochromanol/vitamin E, plastoquinol, and phylloquinol/vitamin K1). In this chapter, methods for the isolation and quantification of chloroplast and leaf glycerolipids and prenylquinol lipids are presented. Glycerolipids are separated by thin-layer chromatography prior to conversion of the fatty acids into methyl esters. Fatty acid methyl esters are subsequently quantified by gas chromatography. Prenylquinol lipids are separated by HPLC and quantified by UV absorption (plastoquinol) or fluorescence (tocochromanol, phylloquinol).

Phytol, the prenyl side chain of chlorophyll, is derived from geranylgeraniol by reduction of three double bonds. Recent results demonstrated that the conversion of geranylgeraniol to phytol is linked to chlorophyll synthesis, which is catalyzed by protein complexes associated with the thylakoid membranes. One of these complexes contains light harvesting chlorophyll binding like proteins (LIL3), enzymes of chlorophyll synthesis (protoporphyrinogen oxidoreductase, POR; chlorophyll synthase, CHLG) and geranylgeranyl reductase (GGR). Phytol is not only employed for the synthesis of chlorophyll, but also for tocopherol (vitamin E), phylloquinol (vitamin K) and fatty acid phytyl ester production. Previously, it was believed that phytol is derived from reduction of geranylgeranyl-diphosphate originating from the 4-methylerythritol-5-phosphate (MEP) pathway. The identification and characterization of two kinases, VTE5 and VTE6, involved in phytol and phytyl-phosphate phosphorylation, respectively, indicated that most phytol employed for tocopherol synthesis is derived from reduction of geranylgeranylated chlorophyll to (phytol-) chlorophyll. After hydrolysis from chlorophyll, free phytol is phosphorylated by the two kinases, and phytyl-diphosphate employed for the synthesis of tocopherol and phylloquinol. The reason why some chloroplast lipids, i.e. chlorophyll, tocopherol and phylloquinol, are derived from phytol, while others, i.e. carotenoids and tocotrienols (in some plant species) are synthesized from geranylgeraniol, remains unclear.

Cyanobacteria are complex prokaryotes, incorporating a Gram-negative cell wall and internal thylakoid membranes (TMs). However, localization of proteins within cyanobacterial cells is poorly understood. Using subcellular fractionation and quantitative proteomics, we produced an extensive subcellular proteome map of an entire cyanobacterial cell, identifying ~67% of proteins in Synechocystis sp. PCC 6803, ~1000 more than previous studies. Assigned to six specific subcellular regions were 1,712 proteins. Proteins involved in energy conversion localized to TMs. The majority of transporters, with the exception of a TM-localized copper importer, resided in the plasma membrane (PM). Most metabolic enzymes were soluble, although numerous pathways terminated in the TM (notably those involved in peptidoglycan monomer, NADP+, heme, lipid, and carotenoid biosynthesis) or PM (specifically, those catalyzing lipopolysaccharide, molybdopterin, FAD, and phylloquinol biosynthesis). We also identified the proteins involved in the TM and PM electron transport chains. The majority of ribosomal proteins and enzymes synthesizing the storage compound polyhydroxybuyrate formed distinct clusters within the data, suggesting similar subcellular distributions to one another, as expected for proteins operating within multicomponent structures. Moreover, heterogeneity within membrane regions was observed, indicating further cellular complexity. Cyanobacterial TM protein localization was conserved in Arabidopsis (Arabidopsis thaliana) chloroplasts, suggesting similar proteome organization in more developed photosynthetic organisms. Successful application of this technique in Synechocystis suggests it could be applied to mapping the proteomes of other cyanobacteria and single-celled organisms. The organization of the cyanobacterial cell revealed here substantially aids our understanding of these environmentally and biotechnologically important organisms.