Chloroplast Import of Four Carotenoid Biosynthetic Enzymes In Vitro Reveals Differential Fates Prior to Membrane Binding and Oligomeric Assembly

Abstract

The precursor proteins of the carotenogenic enzymes geranylgeranyl diphosphate synthase, phytoene synthase, phytoene desaturase and lycopene cyclase were imported into isolated pea chloroplasts. Geranylgeranyl diphosphate synthase remained soluble in the stroma in a free form and phytoene synthase associated to thylakoid membranes upon import, both as expected. Surprisingly, phytoene desaturase and lycopene cyclase, which strongly depend on membrane association for enzymatic activity, also remained soluble in the chloroplast stroma. The soluble forms of these enzymes were, however, still competent for membrane-association, e.g. with protein-free liposomal membranes. Indeed the soluble forms of phytoene synthase, phytoene desaturase and lycopene cyclase occurred as ATP-and cold-sensitive high-molecularmass complexes. Gel-filtration experiments and blue native-PAGE plus autoradiography and western blot analysis indicated a participation of the chloroplast 60-kDa chaperonin (Cpn60) in the soluble highmolecular-mass complexes of imported carotenogenic enzymes. Finally, it was inferred that a membranebound regulatory factor plays a decisive role in membrane-binding.Keywords: carotenoid; chloroplast import; membrane binding; chaperone ; daffodil.In plants, carotenoids are synthesized in plastids by nuclearencoded enzymes. Those participating in the formation of isopentenyl diphosphate are still obscure and presumably follow an alternative reaction sequence (Liitke-Brinkhaus and Kleinig, 1987;Schwender et al., 1996), different from the so-called mevalonate pathway operating in the cytoplasm and transiently in immature chloroplasts (Heintze et a]., 1994). The five subsequent enzymes, catalyzing the reactions from isopentenyl diphosphate to p-carotene, have been molecularly identified by several groups (for review, see Bartley and Scolnik, 1995). Of these, geranylgeranyl diphosphate synthase, phytoene synthase, phytoene desaturase and lycopene cyclase were investigated here. Only some of the plant enzymes functioning later in the pathway forming xanthophylls have been identified, such as zeaxanthine epoxidase (Marin et al., 1996) and capsanthin capsorubin synthase (Bouvier et al., 1994).Carotenoid biosynthesis is not an independent pathway, having instead numerous off-branching prenyllipid-forming activities competing for intermediates. At the stage of geranyl diphosphate, monoterpenes are formed (Mettal et al., 1988) for the geranylgeranyl diphosphate substrate. Many of these prenyllipid products are vitally important, so the metabolite flow must be reliably regulated. For example, carotenoids and chlorophylls must be synthesized in a quantitatively and qualitatively coordinated manner to form and maintain a functional photosynthetic apparatus. Constitutive overexpression of phytoene synthase leads to withdrawal of geranylgeranyl diphosphate from gibberellin biosynthesis and thus to deleterious effects on plant growth (Fray et al., 1995), and interference with quinone biosynthesis results in bleaching effects due to inh...

Phytoene Desaturase Is Localized Exclusively in the Chloroplast and Up-Regulated at the mRNA Level during Accumulation of Secondary Carotenoids in Haematococcus pluvialis (Volvocales, Chlorophyceae),

“…Fractionation of cells with increased PDS amounts resulted in four fractions, of which only the chloroplast membranes gave a signal in westernblot analysis. This confirms previous data on PDS localization in different plants and sequence information of a chloroplast import signal that is found in all PDS (Bonk et al, 1997).…”

The Maize Phytoene Synthase Gene Family: Overlapping Roles for Carotenogenesis in Endosperm, Photomorphogenesis, and Thermal Stress Tolerance

“…Chloroplast envelope membranes were shown to contain a unique carotenoid profile compared to thylakoid membranes (Jeffrey et al, 1974;Siefermann-Harms et al, 1978); chloroplast envelope membrane extracts also exhibited enzyme activity for conversion of zeaxanthin to violaxanthin (Costes et al, 1979). More recent reports have localized PSY and other pathway enzymes to thylakoid membranes in chloroplasts (Linden et al, 1993;Bonk et al, 1997;Welsch et al, 2000). Therefore, the carotenoid pathway may actually be two pathways from a topological standpoint, potentially existing on envelope and thylakoid membranes.…”

Synergistic Interactions between Carotene Ring Hydroxylases Drive Lutein Formation in Plant Carotenoid Biosynthesis

“…The pathway likely functions as a multienzyme complex(es) to facilitate metabolite channeling, as predicted by the absence of pathway intermediates and the presence of complexes containing carotenoid biosynthetic enzymes (Maudinas et al, 1977;Camara et al, 1982;Kreuz et al, 1982;AlBabili et al, 1996;Bonk et al, 1997;Lopez et al, 2008). In this study, we examined an intriguing portion of the pathway, where hydroxylation of rings in carotenes is catalyzed by two structurally distinct enzymes, P450 heme (CYP97) and nonheme diiron (HYD) enzymes DellaPenna, 2001, 2004;Kim and DellaPenna, 2006;Quinlan et al, 2007).…”