Three Thioredoxin Targets in the Inner Envelope Membrane of Chloroplasts Function in Protein Import and Chlorophyll Metabolism
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Three thioredoxin targets in the inner envelope membrane of chloroplasts function in protein import and chlorophyll metabolism Sandra Bartsch*, Julie Monnet†, Kristina Selbach†, Franc¸oise Quigley†, John Gray‡, Diter von Wettstein§¶, Steffen Reinbothe†, and Christiane Reinbothe*†¶ *Lehrstuhl fu¨r Pflanzenphysiologie, Universita¨t Bayreuth, Universita¨tsstrasse 30, D-95447 Bayreuth, Germany; †Unite´Mixte de Recherche 5575, Centre d’Etudes et de Recherches sur les Macromole´cules Organiques, Universite´Joseph Fourier et Centre National de la Recherche Scientifique, BP53, F-38041 Grenoble Cedex 9, France; ‡Department of Biological Sciences, University of Toledo, 2801 West Bancroft Street, Toledo, OH 43606; and §Department of Crop and Soil Sciences and School of Molecular Biosciences, Washington State University, Pullman WA 99164-6420 Contributed by Diter von Wettstein, January 16, 2008 (sent for review December 11, 2007) Thioredoxins (Trxs) are ubiquitous small proteins with a redox- of Trxs in dark/light regulation of protein translocation and active disulfide bridge. In their reduced form, they constitute very chlorophyll (Chl) metabolism and provoke a scenario of deter- efficient protein disulfide oxidoreductases. In chloroplasts, two rence to pigment-sensitized oxidative stress in chloroplasts. types of Trxs (f and m) coexist and play central roles in the regulation of the Calvin cycle and other processes. Here, we Results identified a class of Trx targets in the inner plastid envelope Affinity Purification of Plastid Envelope Proteins Interacting with Trx membrane of chloroplasts that share a CxxC motif Ϸ73 aa from f and Trx m. Chloroplast Trxs contain two neighboring Cys their carboxyl-terminal end. Members of this group belong to a residues in a conserved motif, Trp-Cys-Gly-Pro-Cys (in some superfamily of Rieske iron–sulfur proteins involved in protein cases Trp-Cys-Pro-Pro-Cys), that is referred to as the Trx translocation and chlorophyll metabolism. These proteins include signature (summarized in refs. 1–3). We began by designing the protein translocon protein TIC55, the precursor NADPH:proto- mutant Trx f and Trx m molecules in which the buried Cys was chlorophyllide oxidoreductase translocon protein PTC52, which exchanged by a Ser or Ala residue. These were bound on operates as protochlorophyllide a-oxygenase, and the lethal leaf Sepharose 4B and tested for their capability to bind known Trx spot protein LLS1, which is identical with pheophorbide a oxygen- target proteins present in stromal extracts of barley chloroplasts. ase. The role of these proteins in dark/light regulation and oxida- Fig. 1A (lane 1) shows proteins that could be eluted with DTT. tive control by the Trx system is discussed. Protein mass spectrometry verified the presence of sedoheptu- lose-1,7-bisphosphate bisphosphatase (75 kDa), phosphoribu- protein translocation ͉ tetrapyrrole biosynthesis ͉ Tigrina d12 ͉ lokinase (80 kDa), and NADP-dependent malate dehydrogenase FLU mutant ͉ photooxidative stress (85 kDa), which are known to be Trx targets in spinach chloro- plasts (1, 3). hioredoxins (Trxs) are small multifunctional redox-active In a next step, outer and inner plastid envelope membranes Tproteins that are widely, if not universally, distributed among were isolated from barley chloroplasts and solubilized in buffer living organisms (1–3). In chloroplasts and other plastid types, containing 3% SDS. Each protein extract in turn was passed over two main types of Trxs (f and m) operate in disulfide/dithiol the Trx affinity column, and protein was bound to Trx with interchange and thereby act as powerful regulators of enzyme eluted with DTT. The outer plastid envelope fraction gave rise activity. Trxs catalyze two successive one-electron transfer re- to three main and a few minor bands (Fig. 1A, lane 2). Mass actions. Themselves, they are reduced via ferredoxin with elec- spectrometry identified the 55-kDa band as a protein related to trons provided by photosynthetic electron transport. Trx targets Erv1 and Erv2, enzymes implicated in disulfide bridge formation involve enzymes of the Calvin cycle and oxidative pentose in mitochondria and bacteria (16–19). The role of this protein needs to be established. For the inner plastid envelope protein phosphate cycle that often contain redox-sensitive C(x)nC pairs (n ϭ 2–8, or longer spacing between the two Cys residues; see fraction, five major bands were obtained, of which the 52-kDa ref. 1 for review). The mechanism requires the formation of a band was by far more abundant than the other bands (Fig. 1A, transient heterodisulfide bond between the reduced Trx and the lane 3). Several additional minor bands appeared that were not oxidized enzyme before complete reduction of the target disul- characterized further. Upon 2D separation of proteins in the 50- fide (4–7). Affinity purification procedures take advantage of to 52-kDa range, six spots of different isoelectric points and molecular masses were found (Fig. 1B). Microsequence analysis the fact that mutation in one of the redox-active cysteines in the identified three of the spots to be caused by TIC55, PTC52, and Trx active site (the one buried in the molecule) stabilizes the PAO (Fig. 1B, spots d-f). To confirm their role as potential Trx normally transient heterodisulfide, thereby covalently linking targets, the eluates were reduced by Trx, and the change in redox the target protein to Trx via a bond that can be cleaved by DTT state was measured by fluorescence coupled with gel ele- (e.g., refs. 4, 6, 8, and 9). ctrophoresis. Free SH groups were first blocked with Using this approach, we identified three inner plastid envelope N-ethylmaleimide, then the eluates were treated with the NADP/ membrane proteins from barley chloroplasts as novel Trx tar- gets: the protein translocon protein TIC55 (10), the precursor NADPH:protochlorophyllide (Pchlide) oxidoreductase A Author contributions: C.R. designed research; S.B., J.M., K.S., F.Q., S.R., and C.R. performed (pPORA) translocon protein PTC52 (11), and pheophorbide a research; J.G., D.v.W., and S.R. analyzed data; and C.R. wrote the paper. oxygenase [PAO; originally named lethal leaf spot protein The authors declare no conflict of interest. (LLS1) (12, 13)]. All three proteins contain redox-active CxxC ¶To whom correspondence may be addressed. E-mail: [email protected] or christiane. motifs and interacted with Trxs f and m. By contrast, the related [email protected]. Rieske nonheme oxygenase family member chlorophyllide a- This article contains supporting information online at www.pnas.org/cgi/content/full/ oxygenase (CAO) (14), which lacks the CxxC motiv (15), was 0800378105/DC1. PLANT BIOLOGY found not to be a Trx target. Together, our findings reveal a role © 2008 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0800378105 PNAS ͉ March 25, 2008 ͉ vol. 105 ͉ no. 12 ͉ 4933–4938 Downloaded by guest on October 2, 2021 Fig. 2. Expression of PTC52 and PAO. (A) Identification of PTC52 and PAO among the messenger products of 5-day-old dark-grown barley plants that had been exposed to light for 24 h, by hybrid-selected translation in the presence of [35S]methionine and immunoprecipitation using corresponding antisera and preimmune sera (PIS). (B) Expression of PTC52 and PAO in 5-day-old etiolated barley plants (lane a), 5-day-old dark-grown plants that had been exposed to light for 24 h (lane b), and 5-day-old light-grown plants (lane c), as revealed by RT- PCR, Northern hybridization, and Western blotting. As control, expression of actin was assayed. PTC52, and PAO share CxxC motifs at Ϸ73 aa from their COOH-terminal ends (Fig. 1E). Effect of Trx on PTC52 and PAO Activity. Given that no full-length cDNAs were available for PTC52 and PAO of barley, we used corresponding EST clones for barley PTC52 and a partial cDNA encoding lls1 of Zea mays for hybrid-selected translation and subsequent activity measurements. Fig. 2A shows that PTC52 and PAO could be detected in the mRNA of light-grown barley plants. The size of the detected precursors was slightly different and presumably reflected differences in the length of their transit peptides. Immunoprecipitations using monospecific (PTC52) (11) or monoclonal (PAO) (15) antibodies confirmed the iden- tity of the different bands. Northern blotting revealed differ- ences in the abundance of ptc52 and pao transcripts in dark- grown, light-exposed, and light-grown plants. Whereas ptc52 Fig. 1. Identification of outer and inner plastid envelope membrane pro- mRNA was most abundant in dark-grown seedlings, the pao teins interacting with Trx. (A) Identification of Trx targets in stromal extracts mRNA was present at similar levels in all samples (Fig. 2B). (lane 1) and solubilized outer (lane 2) and inner (lane 3) plastid envelope membranes of barley chloroplasts. The bands marked a, b, and c represent PTC52 is normally active in the pPORA translocon where it NADP-dependent malate dehydrogenase (85 kDa), phosphoribulokinase (80 operates as Pchlide a oxygenase (11). In contrast to this anabolic kDa), and sedoheptulose-1,7-bisphosphate bisphosphatase (75 kDa), respec- role of PTC52 in the C5 pathway, PAO acts catabolically in Chl tively. (B) Re-electrophoresis of the inner plastid envelope membrane proteins breakdown (13). The reaction catalyzed by PAO is coupled to of Ϸ52 kDa obtained as in A. Protein spots were subjected to mass spectrom- that of red Chl catabolite (RCC) reductase, leading to the loss etry and identified by sequence comparisons to be caused by TIC55 (d), PTC52 of pigment color during leaf senescence. In vitro, the interme- (e), and PAO (f). (C) Monobromobimane labeling of TIC55 (d), PTC52 (e), and diate of the coupled reaction, RCC, does not accumulate to PAO (f) before reduction with bacterial Trx. (D) Monobromobimane labeling substantial amounts such that pFCC1 is the only detectable of TIC55 (d), PTC52 (e), and PAO (f) after reduction with bacterial Trx.