Transorganellar complementation redefines the biochemical continuity of and

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 -wall development in vascular tissues, and photoassimilate 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 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. -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, -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 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 of ER:TC was also confirmed by bimolecular fluorescence com- plementation (Fig. S1 A–F). Tissue from WT and three independent, homozygous ER:TC transgenic lines with a range of immuno- logically detectable TC levels (ER:TC-line1, -line2, and -line3) TC-YFP were fractionated to yield chloroplast- and microsome-enriched fractions. Immunoblots showed that ER luminal binding protein (BiP) (an ER luminal marker) was only present in microsome Plastid: fractions (Fig. 3A, ER lanes). Probing WT fractions with a TC antibody showed TC only in the chloroplast fraction (Fig. 3A, Chl lane), consistent with reported localization data from cell fractionation and proteomic studies (1, 2, 10). In ER:TC lines,

TC was only detected in microsome fractions (Fig. 3A,ER ER:TC-YFP lanes), where it was resistant to short-term protease digestion in the absence, but not the presence, of detergent (Fig. 3B, top blot), as expected for an ER-luminal protein. Conclusive demonstration of ER:TC localization allowed us to test the hypothesis that plastid envelope-localized TC substrates are accessible from within the ER lumen. WT leaves accumulate, in order of abundance, α-tocopherol, PC8, and γ-tocopherol as the major tocochromanols, whereas vte1 mutants are devoid of Plastid: γ TMT-YFP tocochromanols (Fig. 4A and Table 1) and, instead, accumulate the TC substrates 2-methyl-6-phytyl-1,4-benzoquinol (MPBQ), 2,3-dimethyl-6-phytyl-1,4-benzoquinol (DMPBQ), and plasto- quinone (PQ)-9 (Fig. 1 and refs. 9 and 11). As expected, plastid: vte1 α γ

TC strongly complemented with average - and -tocoph- ER: γ TMT-YFP erol levels 75% and 86% of WT, respectively (Fig. 4A). Re- markably, transformation with ER:TC also led to substantial Fig. 2. Subcellular localization of plastid- and ER-targeted biosynthetic complementation with average α- and γ-tocopherol levels 52% of enzymes. Plastid:TC-YFP and plastid:γTMT-YFP (yellow, YFP column) and WT, demonstrating that ER:TC can access the three tocochro- chlorophyll fluorescence (red, fluorescence control column) colocalize (merged manol substrates in the plastid envelope nearly as well as plastid: column), confirming chloroplast localization of the native enzymes. ER:TC- YFP and ER:γTMT-YFP (YFP column) localize to reticulate ER networks that TC. Notably, average PC8 levels for ER:TC and plastid:TC were fl A do not overlap with (red) chlorophyll uorescence signals in the merged 28% and 695% of WT, respectively (Fig. 4 ). This is consistent channel, indicating accurate retargeting to the ER. As an additional control, with ER localization restricting TC access to the smaller pool of ER:TC-YFP and ER:GFP (blue) were also shown to colocalize (merged image). PQ-9 substrate in envelope membranes, whereas plastid-local- (Insets) Enlargements of the same region in each channel. (Scale bars: larger ized TC can additionally access the large PQ-9 pool of thylakoid images, 5 μm; Insets,1μm.)

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1306331110 Mehrshahi et al. Downloaded by guest on September 29, 2021 fi ABTC Antibody BiP Antibody ER:TC-line2 bidirectionality of substrate accessibility is evident by the ef - cient methylation of γ-tocopherol to α-tocopherol in both organ- Total Chl ER Total Chl ER Protease - ++ elles. This is true whether γ-tocopherol is produced in the ER by WT Triton - - + ER:TC and methylated in the plastid (by endogenous plastid: TC ER:TC- γTMT; Fig. 4A) or when γ-tocopherol is produced in the plastid line1 by endogenous plastid:TC and methylated in the ER (by ER:γ ER:TC- BiP B line2 TMT; Fig. 4 ). The maintenance of distinctive membrane lipid ER:TC- and protein compositions by organelles is well documented (2, SMT1 line3 17), and this remains true in transorganellar complemented lines. Complemented products do not accumulate in the ER and Fig. 3. Characterization of ER:TC localization by subcellular fractionation fi and immunoblot analysis. (A) Immunoblots of microsome- and chloroplast- must presumably be ef ciently returned to the plastid (Table 2). At enriched fractions from WT and homozygous ER:TC lines. Immunoblots are least two different models could explain these data, a transporter of proteins from total cellular extracts (Total) (45 μg), chloroplast-enriched (Chl) (100 μg), and microsome-enriched (ER) (10 μg) fractions of WT and three ER:TC lines probed with TC and ER luminal BiP antibodies. (B) Ten micrograms of protein from the ER-enriched fraction of ER:TC-line2 were α γ treated with 200 ng of thermolysin with or without Triton X-100, and A -Tocopherol -Tocopherol PC8 immunoblots were probed with antibodies to TC, BiP, or sterol methyl- 140 300 1600 transferase (SMT)1, an ER integral membrane protein. 120 1400 250 1200 100 a single functional carotenoid hydroxylase, LUT5 (CYP97A3), 200 1000 a second plastid envelope cytochrome P450 that can hydroxylate β α β 80 800 the -rings of - and -carotene (15). Retargeting and localiza- 150 tion experiments analogous to those for TC were undertaken for 60 600 γ native (plastid-targeted) TMT and LUT1 or versions engi- Percent WT 100 150 neered for ER targeting. The respective YFP-tagged constructs 40 100 PLANT BIOLOGY showed each enzyme was targeted to the intended organelle (Fig. 20 50 2 and Fig. S3B, respectively). 50 When biochemical complementation was assessed, plastid:γ 0 0 0 TMT and plastid:LUT1 strongly complemented their null mutants, with average α-tocopherol and lutein levels 90% and ER:TC ER:TC 57% of WT, respectively (Fig. 4 B and C). Plastid:γTMT-YFP ER:TC Plastid:TC Plastid:TC and plastid:LUT1-YFP also complemented, with average α- Plastid:TC tocopherol and lutein levels 117% and 55% of WT, respectively B α-Tocopherol γ-Tocopherol C Lutein B C γ (Fig. S2, and ). Transorganellar complementation with ER: 140 6,000 120 TMT and ER:γTMT-YFP also strongly complemented with α B -tocopherol levels 40% and 107% of WT, respectively (Fig. 4 120 5,000 100 and Fig. S2B). ER:LUT1 showed a low but highly significant (P < 100 0.0016) average complementation of lutein to 2% that of WT 4,000 80 C (Fig. 4 ), whereas ER:LUT1-YFP failed to complement (Fig. 80 S2C). ER:LUT1 lines with the highest complementation levels 3,000 60 (7% that of WT) also partially rescued the whole-plant pheno- 60 type of the b1b2lut1 mutant background (Fig. S4). The relatively 2,000 40 Percent WT low level of ER:LUT1 transorganellar complementation may be 40 1,000 attributable to poor interaction of the enzyme with the ER cy- 20 20 tochrome P450 reductase or the inability of ER-localized LUT1 to form complexes with other plastid-resident carotenoid bio- 0 0 0 synthetic enzymes (15, 16). TMT TMT γ γ TMT Discussion ER:LUT1 ER: γ TMT ER: In this study, we developed transorganellar complementation as Plastid:LUT1 Plastid: an experimental approach to directly probe for accessibility of Plastid: γ substrates in the chloroplast envelope by ER-lumen localized Fig. 4. Transorganellar complementation of null mutations in chloroplast- enzymes. Null mutations for three enzymatic steps in plastid- resident pathways by ER-targeted enzymes. Leaves from 4-wk-old primary transformants expressing plastid- or ER-targeted enzymes were analyzed for localized pathways were complemented in this fashion, demon- tocopherols, PC8, and lutein (black circles). All results are expressed as per- strating luminal access to seven lipid-soluble, chloroplast enve- centages of WT. Black dotted lines and red triangles indicate compound lope-localized substrates (MPBQ, DMPBQ, and PQ-9 for ER: levels in WT and null mutants, respectively, and red lines indicate the aver- TC; δ- and γ-tocopherols for ER:γTMT; and α-carotene and age complementation level of primary transgenics. (A) Complementation of zeinoxanthin for ER:LUT1). These combined results suggest the vte1 by plastid:TC and ER:TC. vte1 lacks α-tocopherol, γ-tocopherol, and PC8, existence of a general mechanism that provides ER-resident whereas WT contains 18, 0.7, and 0.7 pmol/mg fresh weight, respectively. enzymes access to a range of nonpolar metabolites located in the Note the split y axis used for PC8. (B) Complementation of vte4 by plastid:γ γ α plastid envelope. Any mechanistic explanation for transorganellar TMT and ER: TMT. vte4 lacks -tocopherol and accumulates its substrate γ-tocopherol at levels 48 times that of WT. (C) Complementation of b1b2lut1 complementation must take into account that enzymes have bi- by plastid:LUT1 and ER:LUT1. Lutein levels in b1b2lut1 are 0.5% of WT. directional access to structurally diverse nonpolar compounds of Significance levels were determined using Student t test relative to the re- − the other organelle without disrupting the distinctive protein and spective mutants. For all transgenic lines and compounds in A, P < 2 × 10 5; nonpolar compound compositions of each organelle (2, 17). The for B, P < 0.0006 and; for C, P < 0.0016.

Mehrshahi et al. PNAS Early Edition | 3of6 Downloaded by guest on September 29, 2021 Table 1. ER:TC complements vte1 in both leaf and seed tissue Leaf tocochromanols, pmol/mg fresh weight Seed tocochromanols, pmol/mg dry weight

Genotype α-Tocopherol δ-Tocopherol γ-Tocopherol PC8 α-Tocopherol δ-Tocopherol γ-Tocopherol PC8

WT 21.9 ± 2.0 ND 0.4 ± 0 1.7 ± 0.3 18.8 ± 0.8 47.1 ± 2.5 1124.4 ± 47.8 141.2 ± 5.6 vte1 ND ND ND ND ND ND 2.6 ± 0.2 ND ER:TC-line1 2.7 ± 1.1 (13) ND 0.2 ± 0.1 (46) 0.1 ± 0 (7) 0.9 ± 0.4 (5) ND 12.7 ± 9.4 (1) ND ER:TC-line2 23.8 ± 1.1 (109) ND 0.4 ± 0 (100) 0.6 ± 1.1 (38) 17.8 ± 0.6 (95) 6.7 ± 0.3 (14) 902.2 ± 18.1 (80) 51.1 ± 1.1 (36) ER:TC-line3 23.9 ± 1.8 (109) ND 0.3 ± 0 (93) 1.5 ± 1.1(90) 20.1 ± 1.1 (107) 7.5 ± 0.3 (107) 962.3 ± 23.7 (86) 59.5 ± 1.7(42)

Tocopherols and PC8 were analyzed in 4-wk-old leaf and dried seed of WT, vte1, and homozygous ER:TC-line1, -line2, and -line3. Values are averages ± SD, with n = 5. Values in parentheses indicate complementation as a percentage of WT. ND, not detected.

based model and a model based on membrane hemifusion be- contact sites in yeast and animals, ascribing functions to PLAMs tweenthetwoorganelles. has been elusive. We speculate that one function of PLAMs is to A transporter-based model (Fig. S5) would require four trans- facilitate the formation of hemifused bilayers between the ER porters for each complemented compound: one pair to transport and plastid envelope membranes and that it is at these sites substrate down its concentration gradient into the ER and, because where transorganellar complementation occurs (Fig. 5). products do not accumulate there (Table 2), a second pair for Hemifused bilayers are an intermediate step in membrane returning product against its concentration gradient to the fusion, during which the outer membrane leaflets have merged plastid. Because the outer envelope membrane lacks a proton and the inner leaflets of the two membranes form a bilayer (31). gradient (18), product return would likely be directly coupled to Because flipping of proteins and lipids between bilayer leaflets is ATP hydrolysis, most likely through ATP-binding cassette (ABC) enzyme-mediated, in the absence of such activities, the inner transporters. Consistent with this idea, the only known envelope leaflets of each membrane forming the hemifused bilayer still transporter of nonpolar compounds is a phosphatidate-binding retain their distinctive nonpolar compound and protein identi- ABC transporter complex required for the import of membrane ties, a key feature of transorganellar complementation (Fig. 5). lipids from the ER into the plastid (19). However, this mem- Bilayer fusion involves recruitment of protein complexes, such as brane lipid ABC transporter is not involved in transorganellar the N-ethylmaleimide–sensitive factor (NSF) or soluble NSF complementation because introduction of mutant alleles for this attachment protein receptors (SNAREs), that allow the hydra- transporter complex into the ER:TC-line2 background had no tion barrier of the lipid bilayer to be overcome, leading to for- impact on transorganellar complementation levels (Fig. S6). The mation of a hemifusion state that can subsequently proceed to Arabidopsis envelope proteome contains nine other uncharac- terized ABC transporters (Dataset S1), seven of which are full fusion of membranes (32). Extended hemifused membranes occur in vivo during egg fertilization, enveloped viral infection, expressed in leaf tissue and may be involved in other endogenous – plant pathways that span the plastid and ER (e.g., fatty acid, and synaptic transmission (31, 33 35) and have recently been membrane lipid, and gibberellin synthesis). Their potential par- demonstrated in vitro with liposomes and mutated SNAREs ticipation in transporting tocopherol and carotenoid pathway (36). Although stable hemifused membranes have not yet been intermediates and products to and from the ER for transorgan- visualized in plants, the presence of high levels of ER-derived fi ellar complementation (Fig. 4 A–C) would imply a significant phosphatidylcholine membrane lipids speci cally in the outer degree of promiscuity in their binding and transport of nonpolar leaflet of the plastid outer envelope (37) is consistent with outer metabolites. Such activities would also be cryptic because the leaflet mixing with the ER due to hemifusion. To stabilize the native tocopherol and carotenoid enzymes and metabolites are hemifusion interface and maintain the overall lipid and protein plastid envelope localized (1, 17) and do not involve the ER. identities of the outer membrane leaflets at hemifused sites, the Indeed, such characteristics could explain why envelope trans- porters for nonpolar compounds have been so challenging fi to identify. Table 2. Quanti cation of tocochromanols in chloroplast- and Although a mechanism involving promiscuous ABC transporters microsome-enriched fractions with cryptic transport activities is possible, a growing body of Fraction Tocochromanols Lutein Chlorophyll evidence supports a fundamentally different mechanism involv- ing membrane contact sites that could enable the bidirectional Microsome fraction interorganellar access to nonpolar chloroplast compounds demon- WT 3.7% (0.6) 0.2% (0.5) 0.1% (0.2) strated by transorganellar complementation. Physical contact ER:TC-line2 3.2% (0.5) 0.2% (0.5) 0.1% (0.2) sites between the chloroplast and ER membranes, termed plas- Chloroplast fraction tid-associated membranes (PLAMs), have been observed by WT 96.3% (16.4) 99.8% (190.5) 99.9% (362.5) transmission electron microscopy in many plant species (20–23) ER:TC-line2 96.8% (15.4) 99.8% (253.5) 99.9% (423.7) and are also the likely sites of discrete regions of contiguous ER Microsomal and chloroplast fractions were isolated from WT and and plastid outer membrane leaflets (hemifused membranes) homozygous ER:TC-line2 seedlings, and the amount of compound in each observed in freeze-fracture scanning electron microscopy (24, fraction was determined and expressed as a percentage of the total (in bold) 25). PLAMs have also been characterized in live cells using and as micrograms of tocochromanols or absorbance units for lutein and optical manipulation and are distinct regions of the ER physi- chlorophyll per fraction (in parentheses). Lutein and chlorophyll are bound cally associated with the plastid through protein:protein inter- to light-harvesting chlorophyll a/b complexes in the thylakoid and are actions (26). Precedent exists for involvement of membrane markers for thylakoid contamination. Their low abundance in the micro- contact sites in organelle-spanning pathways, because the ma- some fraction indicates the near absence of thylakoid contamination in this fraction. Tocochromanols are present in thylakoids and outer and inner jority of phosphatidylethanolamine and phosphatidylcholine syn- envelope membranes (17). The 3.2–3.7% tocochromanols present in the mi- thesis in yeast and mammals requires mitochondria:ER contact crosome fraction is attributable to low-level contamination with chloroplast sites (27, 28), although how these membrane lipids traverse the inner and outer envelopes as a result of their similar densities (43). Results two organelles remains unclear (29, 30). As with membrane shown are the average of two experiments.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1306331110 Mehrshahi et al. Downloaded by guest on September 29, 2021 Plastid

Interface Interface Stabilizing Stabilizing complex FAD complex ER:TC Enzyme

ER Lumen

Fig. 5. Hemifusion-based model for transorganellar complementation and interorganellar regulation of enzyme activities. Membrane lipids synthesized by the plastid and ER pathways are shown in green and blue, respectively. Red single- and double-ring structures depict TC substrates and products, respectively, and gray four-ring structures indicate sterols. This model postulates an interface-stabilizing complex that facilitates formation and stabilization of a hemi- fused bilayer composed of the inner leaflets of the ER and plastid envelope membranes and limits mixing of some lipid classes in the fused outer-membrane leaflets. The model depicted is consistent with ER-synthesized phosphatidylcholine being present at high levels in the outer leaflet of the chloroplast outer envelope (37) and the presence of ER-synthesized sterols in the outer envelope membrane (44). Plastid synthesized galactolipids are not present in the ER under phosphate-replete conditions (as shown) but are present in extraplastidic membranes in response to phosphate deficiency (17). A hemifused bilayer would allow enzymes in either organelle to directly access and use metabolites from either compartment, thereby allowing transorganellar complementation of plastidic mutations by ER-targeted enzymes (e.g., ER:TC complementation of vte1) and also enable regulation of enzymes across organelles by compounds present in either organelle inner leaflet [e.g., regulation of ER fatty acid desaturase (FAD) enzymes by tocopherols].

rate and specificity of nonpolar compound mixing could be shown that a variety of nonpolar compounds previously thought PLANT BIOLOGY regulated by proteinaceous complexes and/or by compound- restricted to a single organelle are accessible by enzymes in specific transporters (e.g., the phosphatidate transporter in the a companion organelle, results that are surprising and challenge envelope membrane). In the context of transorganellar com- our current understanding of nonpolar compound metabolism in plementation, a hemifused bilayer between the ER and chloroplast plants. As additional transorganellar complementation experi- membranes would fulfill a second key feature of transorganellar ments are carried out for other pathways and organelle pairs, an complementation by providing enzymes in both compartments expanding array of nonpolar plant metabolites once thought bidirectional access to nonpolar compounds of both organelles. restricted to a single organelle will likely be found accessible by In this fashion, the interorganellar substrate:enzyme interactions and able to influence metabolism in other organelles. needed for transorganellar complementation and enzyme regu- lation across organelles can occur (e.g., regulation of ER fatty Materials and Methods Growth Conditions and Genotyping. Plants were grown in a 16-h day at 22 °C/ acid desaturation by tocopherols; Fig. 5). − − Although the hemifusion and transporter models both provide 8-hdarkat18°Cand100μmol·m 2·s 1.Homozygousvte1, vte4,andb1b2lut1 a mechanistic explanation for transorganellar complementation, mutants used for transorganellar complementation studies were genotyped as the hemifusion model is particularly appealing because it can described previously (5, 11, 15). readily accommodate the immense chemical diversity that has Arabidopsis Chloroplast and Microsome Preparation. Arabidopsis chloroplast evolved in the plant kingdom. Unlike animals and yeast, as a and microsome fractions were isolated from 4-wk-old seedlings, as described group, plants synthesize thousands of different nonpolar com- previously (39) and detailed further in the SI Materials and Methods. For all pounds by pathways spanning multiple organelles (e.g., the plastid experiments, chloroplast samples were diluted to 1 mg chlorophyll/mL, and and ER). For example, diterpenes, such as gibberellins and taxol, microsome samples were diluted to 1 mg/mL total protein. For protease and monoterpenes, such as those in mint, can account for several treatment experiments, the crude microsome fraction was treated briefly dozen compounds in an individual plant species and collectively with thermolysin as described previously (40) and detailed further in the SI for >10,000 distinct chemistries in the plant kingdom. A large Materials and Methods. family of plastid-localized terpene synthases catalyzes the initial cyclization reactions generating structurally diverse, and often Immunoblot Analysis. Protein extracts were separated by SDS/PAGE and transferred to nitrocellulose membranes (Bio-Rad) following standard pro- multiple, products, whereas later oxidation steps are catalyzed by tocols. Immunoblots were incubated with the following primary antibodies ER-localized P450s (38). Although the synthesis of many mon- (sources and dilutions used are indicated): TC antibody [1:500; a gift from oterpenes and diterpenes is now well understood, envelope Peter Dörmann (University of Bonn, Bonn, Germany)]; BiP antibody (1:5,000; transporters for pathway intermediates have never been repor- Santa Cruz Biotechnology); SMT1 antibody (1:200; Agrisera); and TOC75 ted. Unlike the transporter-based model, hemifusion would pro- (1:3,000), TIC110 (1:3,000), and TOC159 (1:2,000) antibodies [provided by vide the biochemical platform to accommodate the evolution of John E. Froehlich (Michigan State University)]. membrane-spanning pathways that catalyze this extreme level of nonpolar compound chemical variation in the plant kingdom with- Tocochromanol and Carotenoid Analysis. Tocochromanols were extracted out a requirement to also coevolve membrane transporters with from 50 mg of leaf tissue or 12 mg of dry seed and analyzed as described (11). novel activities for each new cyclized compound or compound class. Carotenoids were extracted as described previously (14) and analyzed by HPLC (Shimadzu Scientific) equipped with a Kinetex 2.6-μm, 100 × 4.6 mm Transorganellar complementation provides a direct, functio- reverse-phase column (Phenomenex) at 40 °C with 2 mL/min acetonitrile/ nal assay for probing compound accessibility between the chlo- water/triethylamine (85:15:0.1, vol/vol/vol; buffer A) and ethyl acetate roplast and ER and will likely prove equally useful for probing (buffer B) using the following gradient: 0–3 min, 100% (vol/vol) buffer A; 3– interorganellar metabolism between other organelles in both 9.2 min buffer B increased to 100% (vol/vol) and held at 100% (vol/vol) for plant and nonplant systems. By using this approach, we have an additional 0.8 min; and 10–12 min reequilibration with 100% (vol/vol)

Mehrshahi et al. PNAS Early Edition | 5of6 Downloaded by guest on September 29, 2021 buffer A. Quantification of carotenoids by spectra and retention time were constructs were also generated without a YFP tag and cloned into the binary performed as described previously (14). Standard curves were constructed with vector pCAMBIA1300 (TC and γTMT constructs) or pMLBART (LUT1 con- commercial standards or those prepared in the laboratory. A γ-tocopherol structs). Both native and YFP constructs were assessed for functional com- standard curve was used for PC8 quantification (11). plementation by HPLC.

Molecular Cloning and Plant Transformation. For localization experiments, Sampling and Imaging. Confocal imaging was conducted with an inverted γ plastid:TC-YFP, plastid: TMT-YFP, and plastid:LUT1-YFP were generated by Zeiss LSM 510 Meta confocal microscope (Zeiss). Simultaneous imaging of YFP amplifying the full-length coding sequence of each gene using the primers and chlorophyll fluorescence was achieved with 514-nm argon and 594-nm listed in Table S1: TC-F1 and TC-R1 for VTE1 and TMT-F1 and TMT-R1 for HeNe lasers, with emission detected at 530–600 nm for YFP and 625–750 nm VTE4, which were then subcloned in-frame and upstream of YFP in the bi- for chlorophyll fluorescence. Simultaneous excitation of GFP, YFP, and nary vector pVKH18En6 (41), and for LUT1, LUT1-F1, and LUT1-R1 were used chlorophyll fluorescence was performed using argon laser lines 458 and for subcloning in-frame and upstream of YFP in a synthesized pUC57 vector, 514 nm, and emission was detected at 475–525, 560–615, and 660–750 nm, which was subsequently cloned into the pMLBART binary vector (42). ER respectively. Image J and Adobe Photoshop were used to process the images. targeting of TC, γTMT, and LUT1 was achieved by removing the predicted chloroplast transit peptides (57, 53, and 46 amino acids, respectively) and placing the truncated coding sequence in-frame and between the sporamin ACKNOWLEDGMENTS. We thank Sabrina Gonzalez-Jorge, Maria Magal- lanes-Lundback, and other members of the laboratory of D.D. for technical signal peptide and YFP-HDEL in a synthesized pUC57 vector. For ER:TC-YFP, γ assistance and critical review of the manuscript. J.E.F. is supported by US ER: TMT-YFP, and ER:LUT1-YFP, this was accomplished using the following Department of Energy Grant DE-FG02-91ER20021, F.B. is supported by primers: ER:TC-F1 and ER:TC-R1; ER:TMT-F1 and ER:TMT-R1; and ER:LUT1-F1 National Science Foundation Grant MCB0948584 and National Institute of and ER:LUT1-R1, respectively. All ER-targeting constructs were subsequently Health Grant R01 GM101038-01, and D.D. is supported by the Michigan State subcloned into the pMLBART binary vector (42). All aforementioned University Foundation.

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