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J. Biol. Chem., Vol. 276, Issue 36, 33645-33651, September 7, 2001

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Identification and Characterization of SppA, a Novel Light-inducible Chloroplast Protease Complex Associated with Thylakoid Membranes^*

Martin Lensch, Reinhold G. Herrmann, and Anna Sokolenko

From the Botanisches Institut der Ludwig-Maximilians-Universität, Menzingerstrasse 67, D-80638 München, Germany

Received for publication, January 18, 2001, and in revised form, July 5, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A new component of the chloroplast proteolytic machinery from Arabidopsis thaliana was identified as a SppA-type protease. The sequence of the mature protein, deduced from a full-length cDNA, displays 22% identity to the serine-type protease IV (SppA) from Escherichia coli and 27% identity to Synechocystis SppA1 (sll1703) but lacks the putative transmembrane spanning segments predicted from the E. coli sequence. The N-terminal sequence exhibits typical features of a cleavable chloroplast stroma-targeting sequence. The chloroplast localization of SppA was confirmed by in organello import experiments using an in vitro expression system and by immunodetection with antigen-specific antisera. Subfractionation of intact chloroplasts demonstrated that SppA is associated exclusively with thylakoid membranes, predominantly stroma lamellae, and is a part of some high molecular mass complex of about 270 kDa that exhibits proteolytic activity. Treatments with chaotropic salts and proteases showed that SppA is largely exposed to the stroma but that it behaves as an intrinsic membrane protein that may have an unusual monotopic arrangement in the thylakoids. We demonstrate that SppA is a light-inducible protease and discuss its possible involvement in the light-dependent degradation of antenna and photosystem II complexes that both involve serine-type proteases.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Protein degradation is one of the major regulatory processes that allow the adaptation, repair, or removal of thylakoid proteins during environmental or developmental changes (reviewed in Refs. 1 and 2). However, our knowledge about chloroplast proteases remains limited. To date, the components of the degradation machinery that have been identified in the organelle include the stroma-located members of the Clp family, which are well conserved from prokaryotes to eukaryotes (3-7), the stromal (8, 9) and thylakoid (10) processing proteases that dissect imported protein precursors, the thylakoid-bound metalloprotease FtsH (11), and two lumenal components, the heat shock protease HtrA (DegP; Ref. 12) and CtpA, an enzyme involved in the C-terminal processing of the D1 protein (13, 14). However, the actual number of chloroplast-located proteases appears to be much higher as judged from the biochemical purification of numerous chloroplast proteolytic activities in chloroplast subfractions (15) and from the genome analysis of one chloroplast progenic ancestor, the Synechocystis genome, which displays about 40 different protease sequences. Biochemical studies have provided information about substrate and inhibitor specificity for some proteolytic activities (6), but a detailed knowledge of proteolytic processes requires that the enzymes themselves and the corresponding genes are characterized at the molecular level.

Two kinds of proteases can be operationally distinguished from a physiological point of view: constitutive proteases that, for instance, are involved in the degradation of abnormal or damaged proteins and inducible proteases that are produced or activated only under conditions where specific degradation processes are required. The latter include enzymes that are induced by high light regimes or under heat shock. Well known representatives of that category from plants and bacteria are the heat shock protease Lon (16, 17), the Clp family (7, 15, 18-21), HslUV (22), and HtrA proteases (12, 23, 24). In chloroplasts, several light-dependent degradation processes point to the possible existence of light-regulated proteases. For instance, the accumulation of early light-inducible proteins, a class of proteins that are induced at early stages of illumination, has been suggested to be controlled by a light-repressed protease (25, 26). Conversely, degradation of LHCII,¹ the major peripheral antenna protein of thylakoid membranes, is induced under high light and probably involves extrinsic (27) as well as thylakoid-intrinsic proteases (28). The best studied light-induced degradation process in chloroplasts is that of photosystem II. Photosystem II repair under light stress requires the specific removal and degradation of photodamaged D1 (29-31) and of the chlorophyll a core antenna protein CP 43 (32).

We describe here a new chloroplast-located protease encoded by a nuclear gene in Arabidopsis that fulfills the requirements for participating in light-induced degradation processes in the organelle. This protease displays significant homology to prokaryotic SppA proteases.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Plant Growth-- Arabidopsis and spinach seedlings were grown for 3-4 weeks in a greenhouse at 25 °C under a 12-h light period with a moderate light regime (150 µE m² s¹). To monitor the effect of light on RNA or protein levels, the plants were transferred to darkness or high intensity light (700 µE m² s¹) for periods of 0-52 h. Plants exposed to different light regimes were harvested and used for preparation of total cellular RNA and protein fractions. For heat shock treatment plants were transferred from 25 °C to 42 °C for 30 min and harvested immediately.

cDNA Library Screening and Nucleotide Sequence Analysis-- The Arabidopsis expressed sequence E8A10T7 (accession number AA042706), which displayed homology to prokaryotic SppA proteases, was obtained from the Arabidopsis Biological Resource Center (Ohio State University). A full-length SppA cDNA was isolated from an Arabidopsis cDNA library (33) using radiolabeled E8A10T7 insert as a probe. Nucleotide sequences were determined by energy transfer fluorochrome dideoxy nucleotide chain termination (34, 35) with the 377 system from Applied Biosystems or by fluorochrome primer labeling with the LI-COR 4200IR2 two-laser system (MWG-BIOTECH, Ebersberg, Germany). Sequence analysis was performed using the BLASTN and BLASTP programs on HUSAR Service. Protein localizations and transit peptide cleavage sites were predicted by PSORT (psort.nibb.ac.jp/), ChloroP (www.cbs.dtu.dk/services/ChloroP/), and Predotar (www.inra.fr/Internet/Produits/Predotar) programs, respectively.

DNA/RNA Analysis, Protein Gel Electrophoresis, and Immunodetection of Proteins-- Isolation of total genomic DNA and RNA from Arabidopsis and Southern and Northern analysis were performed as described in Ref. 36. For Northern analysis 5 µg of total RNA were loaded per lane. Gel electrophoresis of proteins under denaturing conditions was performed according to Ref. 37. Immunodetection was performed using ECL and ¹²⁵I-labeled protein A according to the manufacturer's recommendations (Amersham Pharmacia Biotech).

Overexpression of SppA in Escherichia coli and Antisera Production-- The C- and N-terminal segments of SppA cDNA were separately inserted into pQE-31 and pRSET5b vectors (Qiagen, Hilden, Germany) and expressed in the E. coli strains M15 and BL21 (Qiagen and Promega) after induction with 1 mM isopropyl-1-thio--D-galactopyranosides. Soluble and membrane proteins of E. coli cells were fractionated according to Qiagen protocols. Fractions containing overexpressed protein were subjected to polyacrylamide gel electrophoresis (37). The component of interest was excised from the gel, and the gel slice was homogenized in 0.025 M Tris-HCl, pH 8.5, 0.9 M glycine, 0.1% SDS and used for antibody production in rabbits. Antisera were prepared according to Ref. 38.

Preparation and Fractionation of Etioplasts and Chloroplasts; Protein Extraction-- Intact chloroplasts were isolated from Arabidopsis thaliana (var. Landsberg erecta) and spinach (var. Monatol), fractionated into stroma, thylakoid membranes, and lumenal proteins as described in Ref. 39. Etioplasts from spinach were isolated according to Ref. 40. Thylakoids were separated from stroma proteins after osmotic shock of chloroplasts (1 mg/ml of chlorophyll) with 10 volumes of 10 mM Tricine, pH 8.0, and following centrifugation for 10 min at 5.000 × g. The membranes were washed in buffer containing 10 mM Tricine-HCl, pH 8.0, 0.1 M sucrose, 5 mM MgCl₂ and used for the separation of lumenal and thylakoid membrane fractions as described in Ref. 6. The major thylakoid complexes were isolated after partial, controlled solubilization of thylakoid membranes by -dodecyl-maltoside and separation in linear 0.1-1.0 M sucrose gradients (41). Grana and stroma stacks were isolated from thylakoids after lysis with 0.2% digitonin as described in Ref. 42. To characterize protein topology, thylakoids (0.5 mg/ml of chlorophyll) were incubated with solutions of chaotropic salts (2 M NaBr, 2 M NaSCN) or alkaline solutions (0.1 M Na₂CO₃, 0.1 M NaOH) as described in Ref. 43. For protease protection assays thylakoids were resuspended in 10 mM HEPES-KOH, pH 8.0, 0.1 M sucrose at a concentration of 0.5 mg/ml chlorophyll, and aliquots were incubated with thermolysin (150 mg/ml), trypsin (10 mg/ml), or proteinase K (15 mg/ml) for 30 min at 20 °C. The reactions were stopped by the addition of protease inhibitors: 20 mM EDTA for thermolysin, 50 mg/ml of soybean trypsin inhibitors for trypsin, and 2 mM phenylmethylsulfonyl fluoride for proteinase K. In organello import experiments were performed according to Ref. 39. Thylakoid proteins were analyzed by SDS-PAGE.

Partial Purification of SppA and Assay for Proteolytic Activity-- SppA protease was separated from -dodecyl-maltoside-solubilized thylakoid membrane fractions (0.2 mg of chlorophyll/run) by size exclusion chromatography on Superose 6 columns (3.2 × 30 cm; Pharmacia) pre-equilibrated with buffer containing 10 mM Tris-HCl, pH 6.8, 10 mM MgCl₂, 20 mM KCl and 0.06% -dodecyl-maltoside. The proteins were eluted at a flow rate of 0.3 ml/min, and 1-ml fractions were collected. The column was calibrated with Dextran Blue (2000 kDa), thyroglobulin (669 kDa), apoferritin (443 kDa), catalase (240 kDa), and bovine serum albumin (66 kDa). The fractions were analyzed in gelatin-containing (0.2%) gels (6) under nondenaturing conditions in a Tris-borate system (45 mM Tris, pH 8.5, 45 mM boric acid). After electrophoresis the gels were incubated for 3 h at 37 °C in 10 mM Tris-HCl, pH 8.0, to activate proteases and then stained with 0.1% Coomassie Blue R-250, 40% ethanol, 10% acetic acid for 30 min followed by destaining in 40% ethanol, 10% acetic acid. The protease activity became visible as a white zone in a Coomassie Blue-stained background.

Identification of Hybridizing DNA Fragments in the A. thaliana Institut für Genbiologische Forschung-BAC Library-- The library of BACs used was constructed at the Institut für Genbiologische Forschung Berlin GmbH (Instit für Genbiologische Forschung; Ref. 44). Individual recombinant BACs of this library are currently being ordered into contigs that at present represent more than 90% of the Arabidopsis genome. Additionally, representative DNA probes with known map positions were hybridized to BAC clones anchoring the available contigs on the genetic map of molecular markers. Taking advantage of these data (www.mpimp-golm.mpg.de/101/igf_bac_cont.html), sppA was mapped by hybridization to library filters of 9216 BAC clones spotted in duplicate in 3 × 3 arrays. The filters were obtained from the Resorcezentrum im Deutschen Humangenomprojekt, (Berlin, Germany; www.rzpd.de) and hybridized according to Legen et al. (45).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification of the SppA Protease from A. thaliana and Characterization of the Corresponding cDNA-- A search of the Arabidopsis data base revealed a DNA sequence exhibiting homology to the prokaryotic protease SppA. An expressed sequence tag (E8A10T7, accession number AA042706) of 835 base pairs was used as a probe for isolating the full-length cDNA by screening of an appropriate A. thaliana cDNA library. Five positive clones were selected. The recombinant DNA with the largest insert, 2196 base pairs (accession number AF114385), was sequenced. The presence of an in-frame stop codon at position 15, a typical translation initiation sequence ⁶GAAACAATGGCA⁺⁶ (46) and a poly(A) tail at the 3' end suggested that the insert comprised a full-length cDNA. It contained a single open reading frame of 2068 nucleotides, which corresponds to a polypeptide of 680 amino acid residues (Fig. 1) and a calculated molecular mass of 74.8 kDa. The coding region is preceded by a 28-base pair 5'-untranslated region and is trailed by an untranslated stretch of 119 base pairs. The N-terminal 68-residue domain of the protein displays typical features of a chloroplast stroma-targeting transit peptide (47); it initiates with MetAla characteristic of such presequences (48, 49) and contains positively charged (Arg) and hydrophilic short side chain residues (Ser, Ala) and a predicted amphiphilic -sheet (residues 62-67). PSORT and ChloroP programs predicted two potential cleavage sites following Ala residues at positions 60 and 69, respectively (see arrowheads in Fig. 1).

View larger version (84K): [in this window] [in a new window]   Fig. 1.   Amino acid alignment of SppA proteases from A. thaliana (At), Synechocystis (Syn), and E. coli (Ec). Amino acid residues identical between Arabidopsis and one of the other sequences are marked by shaded gray boxes, and identical residues between all sequences are indicated by black boxes. The three purported transmembrane domains in the E. coli sequence are underlined. Predicted cleavage sites for the stroma-targeting transit peptide in the Arabidopsis sequence are marked by arrowheads. Possible "catalytic" amino acid residues are indicated by asterisks (see "Discussion"). The amino acid residues in the Arabidopsis and E. coli sequences, respectively, are numbered above and below the alignments.

SppA Is a Single Copy Gene in the Arabidopsis Genome-- Southern analysis of total Arabidopsis genomic DNA as well as a search through the A. thaliana Data Base (Stanford, CA) provided no evidence for a second sppA gene in the Arabidopsis genome. High density filters of BAC contigs covering almost the entire Arabidopsis genome (44) were used to substantiate the gene copy number and to determine the chromosomal localization of sppA. A signal consistent with a single gene copy was found on a single BAC (number F2P9) originating in chromosome I (data not shown). The recent sequencing of the Arabidopsis genome (internet information at mips.gsf.de/proj/thal/) confirmed the presence of a single sppA gene on that chromosome (accession number AC016662).

SppA Is a Chloroplast Protein Located in Thylakoid Membranes-- To evaluate the chloroplast location of SppA, we conducted in organello import experiments with radiolabeled precursor protein made in vitro from the SppA cDNA. The sample displayed various premature termination products together with the precursor form of SppA, labeled p in Fig. 2 (lane TP). The radiolabeled SppA precursor protein was successfully imported into intact chloroplasts from spinach and processed to the mature form (labeled m in Fig. 2). The imported mature SppA was resistant to proteolytic attack upon incubation of intact spinach chloroplasts with thermolysin or trypsin for 30 min (experiment not shown). Subfractionation of the organelles into thylakoid (Fig. 2, lane Th) and soluble stroma fractions (Fig. 2, lane S) showed that the radiolabeled SppA mature protein was recovered exclusively in the thylakoid fraction (Fig. 2, lane Th). The thylakoid location of SppA within the organelle was confirmed by a serological approach with stromal, thylakoid membrane and lumenal proteins prepared from intact Arabidopsis chloroplasts using an antiserum elicited against the C-terminal part of the protein (amino acid residues 493-680; Fig. 3A). SppA could be detected as a 68-kDa polypeptide that was exclusively associated with thylakoid membranes (Fig. 3A).

View larger version (100K): [in this window] [in a new window]   Fig. 2.   Import of SppA precursor into isolated intact spinach chloroplasts. Chloroplasts were incubated with in vitro translated 35S-radiolabeled SppA pre-protein (TP) and fractionated into stroma (S) and thylakoids (Th). p, precursor protein; m, mature protein.

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Localization of SppA protease within chloroplasts. A, intact chloroplasts were isolated from Arabidopsis plants and fractionated into stroma (S), thylakoid membranes (TM), and lumenal (L) proteins. The proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and immunodecorated with an antiserum against the C-terminal part of SppA. B, thylakoids were partially lysed by digitonin and fractions enriched in grana (G), grana margins (M), and stroma lamellae (S) were separated by differential centrifugation. Antisera against SppA and the 33-kDa subunit of oxygen-evolving complex of photosystem II (control) were used for immunodetection in fractionated thylakoid membranes.

We further fragmented thylakoid membranes by digitonin treatment and subsequent differential centrifugation. This treatment allowed separate fractions enriched in grana membranes, grana margins, and stroma lamellae (for details see "Experimental Procedures"). As illustrated by Fig. 3B, the immunodetection of the 33-kDa subunit of the oxygen-evolving system of photosystem II showed a preferential location in the grana membranes and grana margins (lanes G and M in Fig. 3B) in agreement with the well known distribution of that photosystem in the thylakoid system (50). In contrast, SppA was highly enriched in stroma lamellae. Its presence in the grana fraction can be probably attributed to a cross-contamination inherent in the available procedures.

To estimate the nature of the association of SppA with the stroma lamellae, purified thylakoids were treated with solutions of chaotropic salts (NaBr and NaSCN) or with alkaline solutions (Na₂CO₃ and NaOH). The protein was partially released from the membranes only after treatment with the strong chaotropic reagent NaSCN (Fig. 4). That the membrane association of SppA resisted alkaline treatments as well as NaBr treatment suggests that SppA behaves rather as an integral than as a peripheral membrane protein.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Association of SppA with thylakoid membranes. The nature of association of SppA with the membrane was monitored by extracting thylakoids with solutions of chaotropic salts or alkaline solutions (see text) for 30 min at 20 °C. The samples were centrifuged for 15 min at 10,000 × g to fractionate peripheral and integrated membrane proteins and separated into supernatant (S) and pellet (P).

The topology of SppA within thylakoid membranes was further investigated by protease protection assays. Freshly prepared intact thylakoids or thylakoids permeabilized by sonication were treated with thermolysin, trypsin, or proteinase K (Fig. 5). As a control experiment, we first assayed the thylakoid samples with an antibody against the 33-kDa protein of the oxygen-evolving system that is peripherally located at the lumenal face of thylakoid membranes (51). In intact thylakoids (Fig. 5, left bottom panel), the 33-kDa protein remained fully protected against proteolytic attack, even using the harshest treatment with proteinase K. Upon thylakoid sonication, the 33-kDa protein became mildly sensitive to thermolysin and produced a degradation product of low molecular mass seen at the gel bottom. It was fully degraded by trypsin and proteinase K under the same conditions. Thus, the externally added proteases got full access to the lumenal membrane surface only upon sonication of the thylakoids.

View larger version (71K): [in this window] [in a new window]   Fig. 5.   SppA topology within thylakoid membranes. Arabidopsis intact thylakoids and thylakoids disrupted by sonication were incubated with buffer (), thermolysin (Tl), trypsin (Tr), or proteinase K (Pr). Incubation was carried at 20 °C for 30 min. Proteolysis was stopped by addition of EDTA, soybean trypsin inhibitor, and phenylmethylsulfonyl fluoride, respectively. Thylakoids were reisolated and analyzed by SDS-PAGE and Western analysis using monospecific polyclonal antisera elicited against SppA and the 33-kDa polypeptide of the oxygen-evolving complex that served as a control.

In intact thylakoids, SppA was partially digested by thermolysin or trypsin, and it was extensively digested by proteinase K (Fig. 5, left upper panel). Thus, SppA was readily accessible from the stromal surface to externally added proteases. Sonicated thylakoids that were also accessible to proteases from the lumenal surface displayed the same SppA proteolytic patterns as those observed with intact thylakoids. Thus, we conclude that SppA is exposed at the stromal face of thylakoids and that it has no notable protease-sensitive domains exposed to the thylakoid lumen.

SppA Is a Proteolytically Active Enzyme-- To study the proteolytic activity of SppA we first attempted to overexpress precursor and mature forms of the protein in E. coli. Overexpression of the precursor form proved unsuccessful, whereas that of the mature form, starting from Phe⁶⁰, resulted in low yield production of two truncated products (data not shown). These results suggest that the heterologous expression of chloroplast SppA was detrimental to the bacteria most likely by virtue of its homology to the bacterial SppA protease (59). Therefore, the proteolytic activity of SppA was further analyzed by gel exclusion chromatography after mild solubilization of the major protein complexes from the thylakoid membranes. SppA was part of a high molecular mass complex of ~270 kDa that eluted in fractions 15-17 marked in Fig. 6A, consistent with the homotetrameric structure of SppA from E. coli (59). The fractions collected from size exclusion chromatography were analyzed both for the presence of SppA by immunodetection (Fig. 6B) and for proteolytic activity (Fig. 6C). The SppA-containing fractions, when run on nondenaturing gels containing gelatin, showed high proteolytic activity as visualized by the white band on a dark background (Fig. 6C). The apparent molecular mass deduced from the electrophoretic migration of this proteolytically active band, ~270 kDa, corresponded well with that of the complex eluted by size exclusion chromatography. Similar data were obtained by sucrose gradient centrifugation of thylakoid lysates according to Ref. 41 (data not shown). The proteolytic activity of SppA was significantly inhibited by the specific antiserum (~90%; no suppression occurred with preimmune serum), by phenylmethylsulfonyl fluoride but not iodoacetamide consistent with a serine-type protease (data not shown).

View larger version (32K): [in this window] [in a new window]   Fig. 6.   Partial purification of SppA by size exclusion chromatography and assay for proteolytic activity. Solubilized thylakoid membranes were separated on a Superose 6 column, and 1-ml fractions were collected. A, absorbance profile (280 nm) of eluates. The molecular masses of the proteins used for column calibration are given on the upper x-scale: 2000 (blue dextran), 670 kDa (thyroglobulin), 443 kDa (apoferritin), 240 kDa (catalase), and 66 kDa (bovine serum albumin). The hatched area marks the protease activity. B, immunodetection of SppA in the collected fractions under denaturing conditions with a specific antiserum raised against the protease (only the relevant and flanking fractions 13-19 are shown). The protein molecular mass standards (in kDa) are shown on the left. C, assay for the proteolytic activity. The fractions were loaded onto substrate-containing activity gels, the proteins were separated under nondenaturing conditions, and the gels were incubated at 37 °C for 3 h. After incubation the gel was stained with Coomassie Brilliant Blue and destained, and the protease detected as a white band by degraded substrate within the dark background (as in B, fractions 13-19 are shown). Molecular mass markers, catalase (240 kDa) and bovine serum albumin (66 kDa), were loaded in large amounts (15-20 mg of each protein) to allow their detection in the blue gelatin background.

SppA Is a Light-inducible Protease in Arabidopsis-- RNA filter hybridization was employed to investigate the expression of SppA at the transcript level (Fig. 7). When Arabidopsis plants were grown under moderate light (150 mE m² s¹), they displayed no detectable SppA transcript (Fig. 7, lane 0). However, the more sensitive combined the reverse transcriptase-polymerase chain reaction technique on a Light Cycler machine (Roche Molecular Biochemicals) allowed to detect minute amounts of the transcripts (data not shown). SppA transcripts remained below detection using conventional Northern procedures in plants after heat shock treatment (data not shown). However, when Arabidopsis plants were transferred to higher light intensities (e.g. 700 µE m² s¹), a 2.2-kilobase pair SppA transcript became detectable after 10 h of exposure. The stationary mRNA concentrations increased about five times during the next 40 h under high light (Fig. 7, lanes 10-52 h). This light-induced transcription was accompanied by a substantial increase in the steady-state levels of the corresponding protein (Fig. 8). In this experiment Arabidopsis plants grown under moderate light for 6 days were either kept under the same light regime or transferred to darkness or to higher light intensities for 52 h. A serological identification of SppA using radiolabeled ¹²⁵I-protein A (Fig. 8A) or the ECL method (Fig. 8B) revealed that the protein was already present in significant amounts in plants grown under moderate light conditions, although the corresponding transcript was hardly visible in Northern experiments (Fig. 7). Quantification using ¹²⁵I-protein A showed that the SppA protein increased approximately four times after transfer of Arabidopsis plants to higher light intensities. The amount of cytochrome f, used here as a control, remained virtually unchanged under these conditions (Fig. 8C). Using the nonquantitative but more sensitive ECL method (Fig. 8B), we observed a lower molecular mass product that accumulated in much higher amounts after transfer of the plants from moderate light to darkness but became less abundant upon transfer to high light. These observations indicate that mature SppA is slowly converted to a truncated product, especially in low light or darkness. On the other hand, the steady-state fraction of mature SppA increases drastically at higher light intensities in parallel with the light induction of SppA gene expression.

View larger version (51K): [in this window] [in a new window]   Fig. 7.   Expression of sppA in A. thaliana. Total genomic RNA was isolated from Arabidopsis plants grown in a greenhouse at 150 µE m2 s1 and subsequently placed at 700 µE m2 s1 for different periods (0.5-52 h). Lower panel, 18 and 16 S rRNA, respectively (loading control).

View larger version (21K): [in this window] [in a new window]   Fig. 8.   SppA protein levels under different light conditions. Spinach plants were grown under moderate light (ML; 150 µE m2 s1) for 6 days. Part of the plants was retained under moderate light (ML), and the rest were transferred to darkness (D) and high light regimes (HL; 700 µE m2 s1) for 52 h. The thylakoids were isolated from intact chloroplasts, and the membrane proteins were separated on SDS-PAGE and transferred onto nitrocellulose filters. The SppA protein was detected with a SppA antiserum using 125I-labeled protein A (A) or strongly overexposed ECL detection (B). The truncated product of SppA seen in the overexposed B is marked by an arrowhead (see text). Anti-cytochrome f serum was applied to the same protein fractions as a loading control (C).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We provide evidence for a novel, light-regulated protease, designated SppA, that is located in chloroplast thylakoids. This protease appears to be ubiquitous because it possesses homologues in eubacteria, archaebacteria, and DNA viruses (MEROPS, peptidase data base; www.merops.co.uk/). SppA was identified as a bona fide chloroplast protease in Arabidopsis, because (i) sequence analysis predicted a transit peptide typical of a stroma-targeting presequences, (ii) in vitro expressed SppA was successfully imported into intact spinach chloroplast, and (iii) SppA could be detected immunologically as well as functionally active among the proteins from isolated chloroplasts.

Organelle subfractionation demonstrated that SppA is a membrane-bound protease that is heterogeneously distributed along thylakoid membranes. On a total protein basis, it was highly enriched in stroma lamellae and grana margins rather than in grana. Purification of SppA by size exclusion chromatography after limited detergent solubilization of the thylakoid membranes showed that the protease is part of a high molecular mass complex. This complex possesses a similar molecular mass as SppA from E. coli, which was found to form a tetrameric structure (60). Thus, chloroplast SppA most likely undergoes a comparable oligomerization as in bacteria. The SppA complex showed proteolytic activity in gelatin-containing gels, implying that it constitutes a new chloroplast protease of the SppA family.

Interesting aspects emerge from comparisons of topology predictions for SppA in chloroplasts (thylakoid membranes) and E. coli, which may relate to different localizations and the relatively low homology between the corresponding enzymes. In thylakoids, SppA was largely exposed at the stromal surface of the thylakoid membrane because it was extensively digested from the stromal side of the membrane by proteinase K. However, its limited accessibility to trypsin and thermolysin suggests that the stroma-exposed domains of SppA are tightly folded. We found no evidence for the exposure of protein domains at the lumenal face of the membrane, indicating that the protease is a monotopic component. This is intriguing because the association of SppA with the membranes resisted treatments with alkaline or NaBr solutions that release most of the peripheral membrane proteins. Thus, SppA behaved rather as an integral membrane protein. Monotopic integral membrane proteins with comparable properties have been described previously (for reviews see Refs. 52 and 53). The best characterized monotopic integral membrane protein is prostaglandin synthase, whose crystal structure has revealed an amphiphilic helix that is most likely lying at the junction between the polar head groups and the fatty acid chains of the membrane lipids (54).

To further understand the nature of the interaction of SppA with membranes, we analyzed the SppA sequences from three sources in greater detail, notably from Arabidopsis, Synechocystis, and E. coli (Fig. 1). The SppA protease from the Arabidopsis chloroplast displays 27% identity to Synechocystis SppA1 (sll1703) and 22% to E. coli SppA. Highest conservation was found in the central domain of the protein (Fig. 1; residues 418-485 of Arabidopsis sequence), with a homology exceeding 50% within two weakly hydrophobic stretches (underlined in the E. coli sequence included in Fig. 1). These stretches, together with an N-terminal hydrophobic segment (also underlined in Fig. 1), have been previously suggested to contribute to the transmembrane binding of the E. coli enzyme to the plasmalemma (55, 56). However, the purported N-terminal transmembrane helix of the bacterial protease, which is the most reasonable candidate for the membrane anchoring of the E. coli protease, is absent in the Arabidopsis sequence (Fig. 1). Therefore, the chloroplast-located SppA protease should interact with the lipid bilayer through one or both of the other hydrophobic stretches that are homologous to the corresponding E. coli sequence (in the domain defined by amino acid residues 398-441 in E. coli). As a serine-type protease (55), part of this SppA domain is likely to contribute to the catalytic site of the enzyme because it contains the four serine residues (Ser⁴²⁶, Ser⁴⁵³, Ser⁴⁶⁰, and Ser⁴⁸²) that are strictly conserved among all SppA proteases known so far. Studies of catalytic domains of the serine-type proteases (57, 58) have indicated that serine residues involved in catalysis should be found close to histidine or lysine residues to form the so-called catalytic dyad. Only the two first serine residues (Ser⁴²⁶ and Ser⁴⁵³) are located at an appropriate distance from highly conserved lysines (positions 418 and 448). Because the two subsequent serines (positions 460 and 482) are placed within a hydrophobic stretch without lysine or histidine residues in their vicinity, we infer that Ser⁴²⁶ and Ser⁴⁵³ are involved in the catalytic function of SppA, whereas the latter may be part of a membrane anchor region. The hydrophobic interaction of the protease with the membrane may then be based on the domain encompassing residues 454-492 in Arabidopsis. However, there are no hydrophobic stretches that are long enough to span the membrane in this region. Instead, a wheel presentation of this region from the three sources (Fig. 9A) shows the occurrence of an amphiphilic helix. Its more polar face (Fig. 9A, domains I-IV) could allow an interaction with the polar head groups of the lipids, whereas the more hydrophobic face (Fig. 9A, domains V-VII) could interact with the fatty acid chains. This proposal is fully consistent with the biochemical and topological analysis that we have discussed above. A schematic view of the monotopic positioning of a single SppA molecule with respect to the thylakoid membrane surface is given in Fig. 9B. The catalytic domains of the protease could thus be held close to the thylakoid surface, in an appropriate position for an interaction with membrane protein substrates (Fig. 9B).

View larger version (26K): [in this window] [in a new window]   Fig. 9.   Possible topology of a monomeric form of SppA protease within thylakoid membranes. A, helical wheel projection of the hydrophobic stretch, spanning residues 449-492 of the Arabidopsis SppA protease. The most hydrophylic residues are marked in bold type. Domains I-VII represent the segmented surfaces of the helices. Ec, E. coli; S, Synechocystis; At, A. thaliana. B, model of the monomeric SppA polypeptide in thylakoid membranes. K (Lys) and S (Ser) indicate hypothetical catalytic amino acid residues.

A most remarkable finding of our study is that SppA is a light-inducible protease. Although the substrates of the chloroplast SppA enzyme are unknown at present, it is conceivable that this protease participates in the light-dependent turnover of thylakoid membrane proteins or of peptides released from such proteins under light stress conditions. If so, SppA is another example of a light-induced protease, besides the metallo-protease FtsH (11), which may be involved in the second step of D1, a photosystem II core protein, degradation (60). SppA appears as a reasonable candidate for participating in the light-induced degradation of photosystem II (61-63) and/or LHCII antenna proteins (27, 64) as well. Both degradation processes were reported to involve serine-type endoproteases (27, 65). Two candidate enzymes for LHC degradation have been described: a membrane-bound protease (28, 66) and an extrinsic component that can be reversibly removed from thylakoids (27). Our observation that SppA is firmly attached to the membranes and enriched in stroma lamellae strongly argues for its involvement in LHC degradation through the proteolytic activity described in Refs. 28 and 66. This suggestion is supported by the kinetics of the light-induced LHC degradation process. Acclimation of thylakoid membranes, leading to the disposal of part of the LHCII complexes, is a slow process and requires an induction phase of ~48 h after transferring the plants from low to higher light intensities (27, 64). Maximal induction of SppA mRNA was reached between 26 and 52 h of illumination with high light intensities, a period that corresponds with the maximum degradation of LHC protein during light adaptation (28). LHCII was shown to become highly accessible to proteolytic digestion upon its dephosphorylation in nonappressed membranes. Thus, SppA should be considered as a potential candidate for participating in the LHC degradation process, because of its light induction, thylakoid localization in nonappressed membrane domains, and serine content in the purported catalytic domains. The physiological role of SppA in the light-induced degradation processes of core and antenna complexes of photosystem II is currently under study.

The outlined findings bear on an intriguing phylogenetic aspect. Whereas the light induction of SppA points to its possible role in the degradation of transmembrane proteins, knock-out mutants in E. coli have suggested that SppA functions in the degradation of signal peptides once they are released from the precursor proteins (67, 68). A similar role has been suggested for SppA from Bacillus subtilis (69). It will be interesting to see whether the functional differences between bacterial and chloroplast SppAs, if correct, reflect a phylogenetic change in substrate usage and correlate with the limited sequence conservation that is restricted to the predicted catalytic core domain of the protein, whereas other parts involved in substrate recognition may have diverged during evolution.

	FOOTNOTES

^* This work was supported by Deutsche Forschungsgemeinschaft Grant DFG SO448/2-1 (to A. S.) and SFB 184 (to R. H.) and the Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 49-89-17861242; Fax: 49-89-1782274; E-mail: anna@botanik.biologie.uni-muenchen.d.

Published, JBC Papers in Press, July 6, 2001, DOI 10.1074/jbc.M100506200

	ABBREVIATIONS

The abbreviations used are: LHC, light harvesting complex; PAGE, polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; contig, group of overlapping clones; BAC, bacterial artificial chromosome.

	REFERENCES

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