Identification of plastid envelope proteins required for import of protochlorophyllide oxidoreductase A into the chloroplast of barley

Abstract

Chloroplasts synthesize an abundance of different tetrapyrrole compounds. Among them are chlorophyll and its precursor protochlorophyllide (Pchlide), which accumulate in light- and dark-grown plants, respectively. Pchlide is converted to chlorophyllide by virtue of the NADPH:Pchlide oxidoreductase (POR), which, in angiosperms, is the only known light-dependent enzyme of the chlorophyll biosynthetic pathway. In etiolated barley plants, two closely related POR proteins exist termed PORA and PORB, which are nuclear gene products. Here we identified plastid envelope proteins that interact with the cytosolic PORA precursor (pPORA) during its posttranslational chloroplast import. We demonstrate that pPORA interacts with several previously unreported components. Among them is a Pchlide a oxygenase, which provides Pchlide b as import substrate for pPORA, and a tyrosine aminotransferase thought to be involved in the synthesis of photoprotective vitamin E. Two other constituents were found to be orthologs of the GTP-binding proteins Toc33/34 and of the outer plastid envelope protein Oep16.

Fig. 4.

Detection of Ptc52 and Pchlide b in OM-IM junction complexes. (A) An antiserum was raised against a fingerprint amino acid sequence of Ptc52 (see Materials and Methods). This antiserum was used to determine the distribution of Ptc52 in outer membranes (OM, fractions 1 and 2), inner membranes (IM, fractions 8-10), and OM-IM junction complexes (fractions 3-7). As a control, membrane fractions of chloroplasts that had not been incubated with ³⁵S-pPORA-(His)₆ (-precursor) were probed identically. Pchlide levels determined in parallel are indicated. (B) Import and processing of the envelope-bound ³⁵S-pPORA-(His)₆. ³⁵S-pPORA-(His)₆ was bound to chloroplasts in the presence of 0.1 mM Mg-ATP and 0.1 mM Mg-GTP, and the plastids were reisolated. The plastid-bound precursor then was chased into the chloroplasts by raising the Mg-ATP concentration to 2 mM and supplementing the assay mixtures with 5-aminolevulinic acid (0.5 mM final concentration). At time zero (0) and 2.5 min after the beginning of the incubation, protein bound to the precursor was recovered from OM-IM junction complexes containing ³⁵S-pPORA-(His)₆ by Triton X-100 solubilization, as described in Fig. 2, and analyzed by SDS/PAGE and Coomassie staining. The positions of the envelope-bound pPORA and processed PORA are indicated. The copurifying 52-, 33-, and 16-kDa proteins are marked. (C) HPLC analysis of pigments recovered after 0 and 2.5 min of import. Pigments were extracted with 100% acetone containing 0.1% (vol/vol) diethylpyrocarbonate from the import reactions described in B and separated by HPLC on a C18 column.

Fig. 3.

Identification of Ptc52 as a member of the Lls1, Tic55, and Cao superfamily. Ptc52 was purified and sequenced. Three partial amino acid sequences were obtained which matched with the conserved Rieske center-binding and mononuclear iron-binding motifs of Lls1 of maize (AAC49676) and Arabidopsis (AAC49679), Tic55 of pea (CAA04157) and Arabidopsis (AAD23030), and Cao of Arabidopsis (BAA82484). A protein likely to represent Ptc52 was deduced from EST BF266467 that covers both sequence domains. Staphylococcus aureus V8 protease- and endoproteinase Lys C-derived peptide sequences are overlined and underlined, respectively.

Fig. 1.

Generation of the ³⁵S-pPORA-(His)₆ import intermediate. Isolated barley chloroplasts were incubated with bacterially expressed, urea-denatured ³⁵S-pPORA-(His)₆ in the presence of 0.1 mM Mg-ATP and 0.1 mM Mg-GTP. After 15 min in darkness, the plastids were diluted into ice-cold import buffer lacking ATP and GTP and reisolated. The chloroplasts were then lysed under hypertonic conditions, and total envelope membranes were subfractionated by flotation into linear sucrose gradients (20-38%). (A) Distribution of Oep37, Iep36, Oep16, and the ³⁵S-pPORA-(His)₆ in outer membranes (OM, fractions 1 and 2), inner membranes (IM, fractions 8-10), and OM-IM junction complexes (fractions 3-7) obtained after subfractionation. (B) Same as in A, but showing, in addition, distribution of Toc86, Toc75, Oep24 (lanes 1-3), and Oep16 (lanes 4-6) in gradient fractions 2 (lanes 1 and 4), 5 (lanes 2 and 5), and 8 (lanes 3 and 6), respectively (+pPORA). Lanes 7-9 show the distribution of Oep16 in a gradient of crude envelopes of chloroplasts that had been incubated in the absence of ³⁵S-pPORA-(His)₆. Each lane contained 10 μg of protein. Note that the larger blot (lanes 1-3) showing the different outer- and inner-envelope proteins was probed with a heterologous antiserum against Oep16 of pea, whereas the two smaller blots were developed with a homologous antiserum against barley Oep16. (C and D) Immunoblot of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (SSU) and light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCP) with protein from crude envelope membranes (ME, lanes 10 and 13) before their separation on sucrose gradients, and stromal (Stroma, lanes 11 and 14) and thylakoid (Thyl., lanes 12 and 15) fractions.

Fig. 2.

Purification on Ni²⁺-NTA-agarose of envelope proteins bound to ³⁵S-pPORA-(His)₆. Import intermediates were prepared as described in Fig. 1 and solubilized from reisolated envelope membranes with 2% Triton X-100 (TX100). ³⁵S-pPORA-(His)₆-containing protein complexes were then purified on Ni²⁺-NTA-agarose and analyzed by SDS/PAGE. (A) Coomassie stain of recovered envelope proteins. The positions of marker proteins are indicated. Ptc stands for Pchlide-dependent translocon protein; the number denotes the relative molecular mass. (B) Autoradiogram of ³⁵S-pPORA-(His)₆ (arrow) in gradient fractions that should contain OM-IM complexes. (C) Coomassie stain of two-dimensionally separated Ptc proteins. Protein equivalent to 150 μg of BSA was resolved by isoelectric focusing (IEF) in the first dimension (left to right, pH 5-9) and SDS/PAGE (SDS, top to bottom) in the second dimension.

Fig. 5.

Ptc47 is an inner plastid envelope membrane protein which interacts with Ptc52. (A) Import intermediates were prepared in the presence of ³⁵S-pPORA-(His)₆ (+pPORA, lanes 1-3), and total membranes were fractionated on sucrose gradients as described in Fig. 1. As a control, total membranes were prepared from chloroplasts that had not been incubated with ³⁵S-pPORA-(His)₆ (-pPORA, lanes 4-6). The Western blot shows the distribution of Ptc47 in the indicated gradient fractions, which correspond to those shown in Fig. 2. (B) Chloroplasts were isolated from light-grown barley plants and fractionated into stroma (lane 8) and thylakoids (lane 9). As a control, OM-IM junction complexes containing pPORA-(His)₆ were prepared and solubilized as described in Figs. 1 and 2 (OM-IM, lane 10). For comparison, a total leaf extract was used (lane 7). Protein (10 μg) found in each of these different fractions was resolved by SDS/PAGE and blotted, and the filters were probed with a heterologous antiserum against a tyrosine aminotransferase of Arabidopsis.(C) Isolated chloroplasts (lanes 11-13) or inner-envelope membranes (lanes 14-17) prepared from isolated intact barley chloroplasts were treated with thermolysin (Thl) and trypsin (Trp) as indicated (lanes 11-13) or extracted with 1 M NaCl or 0.1 M Na₂CO₃ (pH 11, lanes 14-17). Each assay was centrifuged, and protein found in the resulting pellet (P, lanes 14 and 16) and supernatant (S, lanes 15 and 17) were fractions detected by Western blotting as described. In lane 13, 50 times the amount of protein loaded in lanes 11 and 12 was used. (D) Coimmunoprecipitation of proteins solubilized from OM-IM junction complexes containing pPORA-(His)₆ (see Fig. 2) with the indicated anti-Ptc52 and anti-Ptc47 antisera and respective preimmune sera (PIS). After SDS/PAGE, the blots of the immunoprecipitates were developed with the anti-Ptc52, anti-Ptc47, and anti-pPORA antisera marked to the right.

Fig. 6.

Label transfer crosslinking of envelope polypeptides at different stages of import. (A) Detection of APDP-pPORA (lane 1) and its mature form (lane 2) generated in an in vitro processing experiment. (B) Crosslinking of envelope polypeptides with APDP. Energy-depleted barley chloroplasts were incubated at 23°C in the dark with APDP-pPORA in the presence of Mg-GTP and Mg-ATP (in millinolar concentrations). After 15 min, the samples were irradiated at 312 nm for 20 min on ice. Chloroplasts were reisolated and fractionated, and total envelope protein was solubilized with SDS and analyzed by reducing SDS/PAGE and autoradiography. Note that part of the envelope-bound pPORA is artificially processed as a result of UV-light treatment. (C) Identification of crosslink products by immunoprecipitation. Aliquots of the samples described in B were subjected to immunoprecipitation by using the indicated anti-Ptc130 (lane 6, corresponding to lane 3), anti-Ptc33 (lane 7, corresponding to lane 4), anti-Ptc16 (lane 7, corresponding to lane 5), and anti-Ptc52 (lane 9, corresponding to lane 5) antisera. Lanes 10-13 show that no precipitates are formed with respective preimmune sera (PIS).