Two mutants affected in lipid metabolism of Chlamydomonas reinhardtii were isolated by treating cells with ultraviolet light. Both mutants showed high chlorophyll fluorescent yields, as compared with parents, and were designated as hf-2 and hf-9 (for high fluorescence). hf-2 was shown to be defective in the synthesis of a chloroplast-specific lipid, sulfoquinovosyl diacylglycerol. hf-9 was shown to be defective in desaturation at the omega 6 position of fatty acids of monogalactosyl diacylglycerol, digalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol and phosphatidylglycerol. The mutants exhibited alterations in photosynthetic activity and chloroplast ultrastructure.

Euglena complex chloroplasts evolved through secondary endosymbiosis between a phagotrophic trypanosome host and eukaryotic algal endosymbiont. Cytoplasmically synthesized chloroplast proteins are transported in vesicles as integral membrane proteins from the ER to the Golgi apparatus to the Euglena chloroplast. Euglena chloroplast preprotein pre-sequences contain a functional N-terminal ER-targeting signal peptide and a domain having characteristics of a higher plant chloroplast targeting transit peptide, which contains a hydrophobic stop-transfer membrane anchor sequence that anchors the precursor in the vesicle membrane. Pulse-chase subcellular fractionation studies showed that 35S-labeled precursor to the light harvesting chlorophyll a/b binding protein accumulated in the Golgi apparatus of Euglena incubated at 15°C and transport to the chloroplast resumed after transfer to 26°C. Transport of the 35S-labeled precursor to the chlorophyll a/b binding protein from Euglena Golgi membranes to Euglena chloroplasts and import into chloroplasts was reconstituted using Golgi membranes isolated from 15°C cells returned to 26°C. Transport was dependent upon extra- and intrachloroplast ATP and GTP hydrolysis. Golgi to chloroplast transport was not inhibited by N-ethylmaleimide indicating that fusion of Golgi vesicles to the chloroplast envelope does not require N-ethylmaleimide-sensitive factor (NSF). This suggests that N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are not utilized in the targeting fusion reaction. The Euglena precursor to the chloroplast-localized small subunit of ribulose-1,5-bisphosphate carboxylase was not imported into isolated pea chloroplasts. A precursor with the N-terminal signal peptide deleted was imported, indicating that the Euglena pre-sequence has a transit peptide that functions in pea chloroplasts. A precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase with the hydrophobic membrane anchor and the pre-sequence region C-terminal to the hydrophobic membrane anchor deleted was imported localizing the functional transit peptide to the Euglena pre-sequence region between the signal peptidase cleavage site and the hydrophobic membrane anchor. The Euglena precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase and the deletion constructs were not post-translationally imported into isolated Euglena chloroplasts indicating that vesicular transport is the obligate import mechanism. Taken together, these studies suggest that protein import into complex Euglena chloroplasts evolved by developing a novel vesicle fusion targeting system to link the host secretory system to the transit peptide-dependent chloroplast protein import system of the endosymbiont.

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Two mutants affected in lipid metabolism of Chlamydomonas reinhardtii were isolated by treating cells with ultraviolet light. Both mutants showed high chlorophyll fluorescent yields, as compared with parents, and were designated as hf-2 and hf9 (for high fluorescence). hf-2 was shown to be defective in the synthesis of a chloroplast-specific lipid, sulfoquinovosyl diacylglycerol. hf-9 was shown to be defective in desaturation at the 0 6 position of fatty acids of monogalactosyl diacylglycerol, digalactosyl diacylglycerol, sulfoquinovosyl diacylglycerol and phosphatidylglycerol. The mutants exhibited alterations in photosynthetic activity and chloroplast ultrastructure.Keywords. Chlamydomonas reinhardtii; chlorophyll fluorescence ; desaturation; sulfoquinovosyl diacylglycerol ; thylakoid membranes.Biomembranes are constructed from specific lipids and proteins which are responsible for their special functions. They facilitate partitioning, keeping materials at proper sites, transporting special substances or producing bioenergy. Chloroplasts contain three distinct membrane systems, i.e. thylakoid, inner envelope and outer envelope membranes. Thylakoid membranes convert light energy to ATP and NADPH, while the two types of envelope membranes are the sites of functions such as lipid biosynthesis [I].Major lipid constituents of thylakoid membranes are monogalactosyl diacylglycerol (Gal-acyl,Gro), digalactosyl diacylglycerol (Gal,-acyl,Gro), sulfoquinovosyl diacylglycerol (SQuiacyl,Gro), and phosphatidylglycerol (PtdGro). These lipids are ubiquitous in prokaryotic and eukaryotic oxygenic photosynthetic organisms [2-41. The former three lipids are specific to chloroplasts, and PtdGro of chloroplasts contains a unique monounsaturated fatty acid, 3-trans-hexadecenoic acid [16 : 1 (3t)l. In addition, Gal-acy1,Gro and Gal,-acy1,Gro contain substantial amounts of polyunsaturated fatty acids such as a-linolenic acid [I8 :3(9, 12, IS)]. Since thylakoid membranes possess photosynthetic electron transport and photophosphorylation activities, how these unique lipids contribute to the functions of thylakoid membranes is an intriguing question. Thylakoid membrane proteins such as the CF,-CF, and cytochrome b,lf complexes were co-purified with specific lipids [5, 61. It was demonstrated that ATPase activity of the CF,-CF, complex was stimulated by exogenously added Gal-acy1,Gro containing highly unsaturated Correspondence to A. Kawaguchi, Department of Biology, College of Arts and Sciences, The University of Tokyo, Komaba, Meguro-ku, Tokyo 153, JapanAbbreviations. Gal,-acyl,Gro, digalactosyl diacylglycerol ; acy1,GroMe3Hse, diacylglyceryl trimethylhomoserine ; Gal-acyl,Gro, monogalactosyl diacylglycerol; SQui-acyl,Gro, sulfoquinovosyl diacylglycerol; fatty acyl groups are denoted by the numbers of carbon atoms and double bonds, e.g. 16:1(7) = palmitoleoyl. fatty acids, and plastoquinol-plastocyanin oxidoreductase activity of the cytochrome b,lf complex was restored by Gal,-acy1,-Gro, PtdGro and phosphatidylcholine (PtdCho) [7, 81....