Transorganellar complementation redefines the biochemical continuity of endoplasmic reticulum and chloroplasts

Abstract

Tocopherols are nonpolar compounds synthesized and localized in plastids but whose genetic elimination specifically impacts fatty acid desaturation in the endoplasmic reticulum (ER), suggesting a direct interaction with ER-resident enzymes. To functionally probe for such interactions, we developed transorganellar complementation, where mutated pathway activities in one organelle are experimentally tested for substrate accessibility and complementation by active enzymes retargeted to a companion organelle. Mutations disrupting three plastid-resident activities in tocopherol and carotenoid synthesis were complemented from the ER in this fashion, demonstrating transorganellar access to at least seven nonpolar, plastid envelope-localized substrates from the lumen of the ER, likely through plastid:ER membrane interaction domains. The ability of enzymes in either organelle to access shared, nonpolar plastid metabolite pools redefines our understanding of the biochemical continuity of the ER and chloroplast with profound implications for the integration and regulation of organelle-spanning pathways that synthesize nonpolar metabolites in plants.

Keywords: MAM; PLAM; hemifusion; metabolism; vitamin E.

Fig. 1.

Tocochromanol biosynthesis in Arabidopsis. Enzymes catalyzing synthesis of the four tocopherols and PC8 include: p-hydroxyphenylpyruvate (HPP) dioxygenase (HPPD), homogentisate phytyl (or solanesyl) transferases (HPT or HST, respectively), 2-methyl-6-phytyl (or solanesyl)-1,4-benzoquinol methyltransferase (MPBQ/MSBQ MT), TC, and γTMT. The vitamin e-deficient 1 and 4 null mutations (vte1 and vte4) used in this study are indicated in red italics.

Fig. 2.

Subcellular localization of plastid- and ER-targeted biosynthetic enzymes. Plastid:TC-YFP and plastid:γTMT-YFP (yellow, YFP column) and chlorophyll fluorescence (red, fluorescence control column) colocalize (merged column), confirming chloroplast localization of the native enzymes. ER:TC-YFP and ER:γTMT-YFP (YFP column) localize to reticulate ER networks that do not overlap with (red) chlorophyll fluorescence signals in the merged channel, indicating accurate retargeting to the ER. As an additional control, ER:TC-YFP and ER:GFP (blue) were also shown to colocalize (merged image). (Insets) Enlargements of the same region in each channel. (Scale bars: larger images, 5 μm; Insets, 1 μm.)

Fig. 3.

Characterization of ER:TC localization by subcellular fractionation and immunoblot analysis. (A) Immunoblots of microsome- and chloroplast-enriched fractions from WT and homozygous ER:TC lines. Immunoblots are 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 treated with 200 ng of thermolysin with or without Triton X-100, and immunoblots were probed with antibodies to TC, BiP, or sterol methyltransferase (SMT)1, an ER integral membrane protein.

Fig. 4.

Transorganellar complementation of null mutations in chloroplast-resident pathways by ER-targeted enzymes. Leaves from 4-wk-old primary transformants expressing plastid- or ER-targeted enzymes were analyzed for tocopherols, PC8, and lutein (black circles). All results are expressed as percentages of WT. Black dotted lines and red triangles indicate compound levels in WT and null mutants, respectively, and red lines indicate the average complementation level of primary transgenics. (A) Complementation of vte1 by plastid:TC and ER:TC. vte1 lacks α-tocopherol, γ-tocopherol, and PC8, whereas WT contains 18, 0.7, and 0.7 pmol/mg fresh weight, respectively. Note the split y axis used for PC8. (B) Complementation of vte4 by plastid:γTMT and ER:γTMT. vte4 lacks α-tocopherol and accumulates its substrate γ-tocopherol at levels 48 times that of WT. (C) Complementation of b1b2lut1 by plastid:LUT1 and ER:LUT1. Lutein levels in b1b2lut1 are 0.5% of WT. Significance levels were determined using Student t test relative to the respective mutants. For all transgenic lines and compounds in A, P < 2 × 10⁻⁵; for B, P < 0.0006 and; for C, P < 0.0016.

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 hemifused 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].