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Transorganellar Complementation Redefines the Biochemical Continuity

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Transorganellar Complementation Redefines the Biochemical Continuity Transorganellar complementation redefines the biochemical continuity of endoplasmic reticulum and chloroplasts Payam Mehrshahia, Giovanni Stefanob,c, Joshua Michael Andaloroa, Federica Brandizzib,c, John E. Froehlicha,c, and Dean DellaPennaa,1 Departments of aBiochemistry and Molecular Biology and bPlant Biology and cMichigan State University-Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI 48824 Edited* by Chris R. Somerville, University of California, Berkeley, CA, and approved June 11, 2013 (received for review April 3, 2013) Tocopherols are nonpolar compounds synthesized and localized in cell-wall development in vascular tissues, and photoassimilate plastids but whose genetic elimination specifically impacts fatty translocation from source leaves (4–8). In tocopherol-deficient acid desaturation in the endoplasmic reticulum (ER), suggesting Arabidopsis mutants, these phenotypes are inducible by low a direct interaction with ER-resident enzymes. To functionally temperature, allowing assessment of their timing and causality probe for such interactions, we developed transorganellar comple- (5, 6, 8). Surprisingly, before low-temperature induction, linoleic mentation, where mutated pathway activities in one organelle are acid desaturation in tocopherol-deficient mutants is decreased experimentally tested for substrate accessibility and complemen- specifically in endoplasmic reticulum (ER)-synthesized but not in tation by active enzymes retargeted to a companion organelle. plastid-synthesized membrane lipids, the organelle that synthe- Mutations disrupting three plastid-resident activities in tocopherol sizes and contains tocopherols. This ER-membrane lipid phe- and carotenoid synthesis were complemented from the ER in this notype is exacerbated by low-temperature treatment, followed by fashion, demonstrating transorganellar access to at least seven the full suite of other tocopherol-deficient phenotypes and can be nonpolar, plastid envelope-localized substrates from the lumen completely suppressed by introducing mutant alleles of the ER- of the ER, likely through plastid:ER membrane interaction do- resident oleic desaturase (6, 8). These data clearly demonstrate, PLANT BIOLOGY mains. The ability of enzymes in either organelle to access shared, but do not explain how, chloroplast-synthesized and localized nonpolar plastid metabolite pools redefines our understanding tocopherols specifically impact ER fatty acid desaturation. of the biochemical continuity of the ER and chloroplast with pro- One possibility is that tocopherols directly interact with and found implications for the integration and regulation of organ- influence ER-resident desaturases from within the chloroplast, elle-spanning pathways that synthesize nonpolar metabolites which, if true, also predicts that tocopherols and their bio- in plants. synthetic intermediates should be directly accessible by enzymes in the ER. To test this hypothesis, we designed experiments in hemifusion | PLAM | vitamin E | MAM | metabolism Arabidopsis thaliana to test whether null mutations eliminating plastid-localized pathway activities could be functionally com- n addition to meeting cellular energy needs through photo- plemented by corresponding wild-type (WT) enzymes retargeted Isynthesis, chloroplasts are centers of anabolic metabolism that to the ER, an approach we term “transorganellar complemen- contain complete biosynthetic pathways (e.g., for de novo syn- tation.” The data from this study demonstrate that nonpolar thesis of fatty acids, amino acids, tocopherols, and carotenoids) substrates, including those involved in tocopherol biosynthesis, and participate in numerous pathways that span multiple sub- are accessible from within the lumen of the ER. We propose cellular compartments (e.g., for synthesis of membrane lipids, a mechanism that allows the two organelles bidirectional access monoterpenes, diterpenes, and photorespiration). Such metab- to nonpolar compounds without necessarily involving trans- olism requires exchange of a multitude of polar and nonpolar porters. This would explain the paucity of nonpolar transporters metabolites with the extraplastidic environment and consistent in the envelope proteome and has far-reaching implications for with this, proteomic and bioinformatic analysis of the chloroplast the regulation and integration of organelle-spanning pathways envelope identified 102 transporter candidates (Dataset S1). for the synthesis of nonpolar metabolites in plants. Sixty-six have recognized functions as ion or metabolite trans- porters, but only one transports nonpolar metabolites. This ap- Results parent paucity of nonpolar metabolite transporters in the envelope, Retargeting of Tocopherol Cyclase from the Chloroplast to the ER despite the large numbers of nonpolar metabolites synthesized Allows Transorganellar Complementation of the vte1 Mutant. Ini- by plastids, highlights a significant gap in our understanding of tial transorganellar complementation experiments were per- plant metabolism. formed with tocopherol cyclase (TC) (encoded by VTE1; Fig. 1), Tocochromanols are one well-studied group of nonpolar and all retargeting constructs were transformed into a TC-null compounds synthesized and localized in plastids that include the vitamin e-deficient 1 (vte1) mutant background (9). The native, biosynthetically related tocopherols, tocotrienols, and plasto- plastid-localized TC (plastid:TC) was engineered for retargeting chromanol-8 (PC8) (Fig. 1). Tocochromanol biosynthesis has and retention in the ER (ER:TC). Fusion of yellow fluorescent been fully elucidated, null mutants with well-defined biochemical phenotypes are available for each reaction, and with the excep- p tion of -hydroxyphenylpyruvate dioxygenase, all biosynthetic Author contributions: P.M. and D.D. designed research; P.M., G.S., J.M.A., and J.E.F. per- activities localize to the plastid inner envelope where synthesis formed research; P.M., G.S., F.B., J.E.F., and D.D. contributed new reagents/analytic tools; occurs (1, 2). Because tocochromanols are only present in P.M., G.S., J.E.F., and D.D. analyzed data; and P.M. and D.D. wrote the paper. chloroplast membranes (1, 3), it was assumed that their functions The authors declare no conflict of interest. would be restricted to this organelle; however, many tocopherol- *This Direct Submission article had a prearranged editor. deficient mutant phenotypes are, instead, consistent with impacts 1To whom correspondence should be addressed. E-mail: [email protected]. on extraplastidic processes. These include alterations in mem- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. brane lipids, formation of secretory pathway-derived vesicles, 1073/pnas.1306331110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1306331110 PNAS Early Edition | 1of6 Downloaded by guest on September 29, 2021 HPP membranes, as reported previously (12). Analysis of leaves and seed from the three homozygous, single-insert ER:TC lines HPPD solanesyl-PP showed that complementation in both tissues paralleled immu- nologically detectable TC levels (Fig. 3A) with ER:TC-line1 << HGA phytyl-PP ER:TC-line2 ≤ ER:TC-line3 (Table 1). Plastid:TC-YFP also com- HPT HST plemented with average α-tocopherol, γ-tocopherol, and PC8 levels 52%, 67%, and 54% of WT, respectively, whereas ER:TC- MPBQ MSBQ YFP complementation averaged 17%, 35%, and 20% of WT for α γ A MPBQ/MSBQ MT -tocopherol, -tocopherol, and PC8, respectively (Fig. S2 ). Demonstration of Transorganellar Complementation with Additional DMPBQ PQ-9 Tocopherol and Carotenoid Biosynthetic Enzymes. To determine vte1 Tocopherol Cyclase (TC) whether the accessibility of ER:TC to its three plastid envelope- localized substrates is indicative of a fundamental biochemical δ-tocopherol γ-tocopherol PC8 process in plants, we attempted transorganellar complementation for two additional chloroplast envelope activities: γ-tocopherol vte4 γTMT methyltransferase (γTMT) and α-carotene e-ring hydroxylase [LUTEIN DEFICIENT1 (LUT1)]. γTMT is encoded by VTE4 β-tocopherol α-tocopherol and catalyzes the final step in tocopherol synthesis (Fig. 1 and Fig. 1. Tocochromanol biosynthesis in Arabidopsis. Enzymes catalyzing refs. 1 and 2), with null mutants accumulating γ-andδ-tocopherols synthesis of the four tocopherols and PC8 include: p-hydroxyphenylpyruvate (13). LUT1 encodes a cytochrome P450 required for synthesis of (HPP) dioxygenase (HPPD), homogentisate phytyl (or solanesyl) transferases the most abundant leaf carotenoid lutein (14), with null mutants (HPT or HST, respectively), 2-methyl-6-phytyl (or solanesyl)-1,4-benzoquinol accumulating the monohydroxy precursor, zeinoxanthin (Fig. γ fi methyltransferase (MPBQ/MSBQ MT), TC, and TMT. The vitamin e-de cient S3A). Because LUT1 is one of four Arabidopsis genes encoding 1 and 4 null mutations (vte1 and vte4) used in this study are indicated in red italics. plastid-localized carotenoid hydroxylases (15), transorganellar complementation was performed in a triple-mutant background also null for two nonheme monooxygenase carotenoid hydrox- protein (YFP) to the C terminus of plastid:TC (plastid:TC-YFP) ylases, BCH1 and BCH2. The b1b2lut1 triple mutant retains or upstream of the ER:TC ER-retention signal (ER:TC-YFP) allowed intracellular localization by confocal microscopy. Lines expressing plastid:TC-YFP or ER:TC-YFP were exclusively Fluorescence chloroplast- or ER-localized, respectively (Fig. 2). Localization YFP controls Merged
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