Overexpression and Characterization of Carboxyl-terminal Processing Protease for Precursor D1 Protein

REGULATION OF ENZYME-SUBSTRATE INTERACTION BY MOLECULAR ENVIRONMENTS*

Yumiko YamamotoDagger , Noritoshi Inagaki§, and Kimiyuki SatohDagger ||

From the Dagger  Department of Biology, Faculty of Science, Okayama University, Okayama 700-8530, Japan and the § National Institute for Basic Biology, Okazaki 444-8585, Japan

Received for publication, September 28, 2000, and in revised form, November 14, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

CtpA, which is classified as a novel type of serine protease with a Ser/Lys catalytic dyad, is responsible for the C-terminal processing of precursor D1 protein (pD1) of the photosystem II reaction center, a process that is indispensable for the integration of water-splitting machinery in photosynthesis. In this study, overexpression in Escherichia coli and one-step purification of spinach CtpA were carried out to analyze the characteristics of this new type of protease and to elucidate the molecular interactions in the C-terminal processing of pD1 on the thylakoid membrane. The successful accumulation of functional CtpA in E. coli may argue against the possibility, based on homology to E. coli Tsp, that the enzyme is involved in the degradation of incomplete proteins in chloroplasts, e.g. by utilizing the ssrA-tagging system. Analysis using a synthetic pD1 oligopeptide demonstrated that the enzymatic properties (including substrate recognition) of overexpressed CtpA with an extra sequence of GSHMLE at the N terminus were indistinguishable from those of the native enzyme. CtpA was insensitive to penem, which has been shown to inhibit some Ser/Lys-type proteases, suggesting that the catalytic center of CtpA is quite unique. By using the substrate in different molecular environments (i.e. synthetic pD1 oligopeptide in solution and pD1 in photosystem II-enriched thylakoid membrane), we observed a dramatic difference in the pH profile and affinity for the substrate, suggesting the presence of a specific interaction of CtpA with a factor(s) that modulates the pH dependence of proteolytic action in response to physiological conditions.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The D1 protein is a membrane-spanning subunit constituting the core part of the photosystem II (PSII)1 reaction center (1, 2). In the predicted secondary structure, the D1 protein consists of five transmembrane alpha -helices and a C-terminal extension protruding into the luminal space of thylakoids (reviewed in Ref. 2). A curious feature of this protein is the extraordinarily high rate of metabolic turnover in vivo, despite its fundamental importance in the structure and function of PSII (3). In the dynamic process, the D1 protein, which is encoded by the psbA gene in the plastid genome in the case of eukaryotic organisms, is synthesized on membrane-associated ribosomes on the cytosolic or stromal surface of thylakoids in a precursor form with a short C-terminal extension consisting of 8-16 amino acids (4-6). This part of the protein is co-translationally translocated into the luminal space and then immediately removed by the action of an endopeptidase called the C-terminal processing protease (CtpA) (7-10). In eukaryotic organisms, the enzyme is nuclear encoded (8-10) and imported from the cytosol to thylakoidal lumen (11). When present on the D1 protein, this extension's removal is absolutely essential for the integration of the machinery for water oxidation, i.e. the manganese cluster in PSII (12-15). However, the absence of this extension by itself at least under the conditions examined has no serious effect on the viability of organisms such as Euglena gracilis, in which the C-terminal extension is absent in the psbA gene information (6), and genetically engineered mutants of Chlamydomonas (16, 17) and Synechocystis (15) in which the C terminus is truncated. Thus, at present, the physiological meaning behind the ubiquitous presence of the C-terminal extension in the D1 protein in a wide range of oxygenic photosynthetic organisms has not yet been established.

On the other hand, a protease involved in the C-terminal processing of precursor D1 protein (pD1) has been purified from spinach (18) and Scenedesmus (10, 19), and a gene coding for the protein named ctpA has been identified and sequenced (7-10). Based on recent site-directed mutational analysis of the catalytic center of the enzyme,2 given the fact that the proteolytic activity is resistant to any conventional protease inhibitor (8, 10, 19), CtpA has been classified as a novel type of serine protease that utilizes the Ser/Lys catalytic dyad instead of the Ser/His/Asp triad (20), characteristic of the thylakoidal processing peptidase (21), the leader peptidase (22), and the tail-specific protease (Tsp) of Escherichia coli (23, 24). Thus, the elucidation of the catalytic mechanism of this novel type of protease is receiving a lot of attention from enzymologists.

One of the unique features of CtpA is that the substrate specificity recognizing the sequence around the C terminus is very strict (25), although the site of cleavage by itself is rather simpler, i.e. the linkage between Aladown-arrow Ala (26, 27) or Aladown-arrow Ser (15). The interaction between enzyme and substrate is rather complicated in this system in terms of topographical organization. The substrate is a hydrophobic membrane-spanning protein integrated into the thylakoid (2), whereas the enzyme is a soluble protein loosely attached to the internal membrane surface (8, 18). Thus, the interaction between the enzyme and its protein substrate is substantially restricted by the integration of components compared with the conventional proteolytic system in cytosolic solution.

In previous studies, we analyzed the mechanism of the C-terminal processing of pD1 by utilizing partially purified protease from spinach chloroplasts and two types of substrate in solution: 1) synthetic oligopeptides corresponding to the C-terminal sequence of pD1 (25, 28) and 2) in vitro transcribed/translated full-length pD1 (18). An unexpected finding from these studies was that the proteolytic activity exhibited a strong pH dependence, with an optimum in the pH range of 7.5-8.0.3 Activity was quite low on the acidic side at pH 5.0-6.0, conditions that mimic the pH of the luminal space in the illuminated thylakoid, where the rate of C-terminal processing is expected to be extremely high under physiological conditions. In addition, the affinity of the protease for the substrate seemed to be too low to account for the expectedly high turnover rate (reviewed in Ref. 29).

This study was intended to further characterize the enzyme and to resolve these inconsistencies in pH dependence and affinity between the in vitro and in vivo systems, with the overall aim of understanding the mechanism of proteolytic processing of pD1 under physiological conditions. For the analysis, we utilized pD1 in the PSII complex embedded in the thylakoid membrane of chloroplasts obtained from the LF-1 mutant of Scenedesmus obliquus (12-14). For the purpose of enzymatic analysis, we have successfully developed an overexpression system for spinach CtpA in E. coli. By adjusting the temperature of IPTG induction, we have been able to obtain substantial amounts of processing enzyme in its active state. The results of analyses using these materials demonstrate that the mode of enzyme-substrate interaction in the C-terminal processing is largely modified by integrating the substrate into the thylakoid membrane. This suggests the presence of a specific molecular interaction(s) between a component(s) on the membrane surface and the soluble enzyme in the luminal space.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Experimental Organisms-- The competent BL21(DE3) strain of E. coli was purchased from Novagen (Madison, WI). The wild strain and the LF-1 mutant (12) of S. obliquus were gifts from Dr. N. I. Bishop (Oregon State University). Scenedesmus cells were heterotrophically grown at 25 °C in the dark in NGY liquid medium (30).

Construction of Expression Plasmids for CtpA-- A pair of polymerase chain reaction primers whose sequences are GTCTCTCGAGCTTTCTGAGGAGAATCGAAT (forward primer) and ATCGCTCGAGTCATCTTGAAAAGAGTTGTACGCC (reverse primer) were synthesized with a DNA synthesizer (Model 394, Applied Biosystems, Foster City, CA). These primers were designed for amplification of a gene fragment encoding the mature part of spinach CtpA protease, corresponding to the segment from Leu151 to the stop codon at position 540 (8). They have an external XhoI site at each 5' terminus for ligation into the expression vector pET-15b(+) (Novagen), which is underlined in the sequences above. In our expression strategy, the initiation codon, the N-terminal hexahistidine tag, and the subsequent epitope for thrombin cleavage encoded on pET-15b(+) were utilized (Fig. 1). Therefore, the XhoI site on the forward primer was arranged such that the desired reading frame was in accord with the strategy. The polymerase chain reaction products of ~1,200 base pairs were digested by XhoI and ligated into the XhoI site of pET-15b(+). These plasmids were then introduced into competent E. coli BL21(DE3) cells. Transformants suited to our strategy with regard to the direction of insertion, which had been assessed by mapping of the restriction sites, were sent to the next selection. Finally, a clone, pET-CTPA, whose insert had no error during the polymerase chain reaction amplification as confirmed by conventional sequencing, was isolated and used in this work.



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Fig. 1.   The N-terminal amino acid sequence of His-tagged mature CtpA. The hexahistidine tag and the thrombin-recognizable sequence are indicated by solid and dashed lines, respectively. The arrow shows the site of cleavage by thrombin. The sequence derived from the ctpA gene is boxed.

Overexpression of the ctpA Gene in E. coli-- The BL21(DE3) strain of E. coli carrying the expression plasmids for mature spinach CtpA was grown in LB medium containing 50 µg/ml ampicillin at the desired temperature. Growth was monitored by measuring the turbidity of culture at 600 nm (A600). When A600 reached 0.4, the expression of the ctpA gene was induced by the addition of IPTG at 1 mM. The culture was further incubated until A600 reached ~0.8. Cells were collected by centrifugation at 5,000 × g for 5 min and suspended in 0.1 culture volume of lysate buffer consisting of 50 mM Tris-HCl (pH 8.0) and 2 mM EDTA. After the addition of 0.01 volume of 10 mg/ml lysozyme and 0.1 volume of 1% Triton X-100, the cell suspension was incubated for 15 min at 30 °C and sonicated at high output (Model 250 sonifier, Branson Ultrasonics Corp., Danbury, CT) for 20 s at 4 °C. The soluble and insoluble fractions of the extracts were separated by centrifugation at 30,000 × g for 30 min at 4 °C.

Purification of CtpA-- The purification of overexpressed CtpA from E. coli was achieved by nickel-chelating affinity chromatography utilizing the His tag at its N terminus. In a typical experiment, 50 ml of soluble fraction from E. coli cells grown at 20 °C was used as the starting material. To remove EDTA, which strips Ni2+ from the chelating column, the soluble fraction was dialyzed against starting buffer (20 mM Tris-HCl (pH 7.9), 5 mM imidazole, and 0.5 M NaCl) prior to adsorption onto the column. After passing through a filter (0.45-µm pore size), the solution was loaded onto an AF-chelate TOYOPEARL 650M column (1 (inner diameter) × 6.5 cm; Tosoh, Tokyo, Japan) that was nickel-immobilized and equilibrated with starting buffer. The column was washed with 10 column volumes of starting buffer and 6 column volumes of wash buffer (20 mM Tris-HCl (pH 7.9), 60 mM imidazole, and 0.5 M NaCl). CtpA was eluted from the column with elution buffer (20 mM Tris-HCl (pH 7.9), 1 M imidazole, and 0.5 M NaCl). Purified CtpA was dialyzed against an appropriate buffer and stored at -20 °C until used.

Assay of Proteolytic Activity Using a Synthetic Oligopeptide as the Substrate-- In a typical experiment, the reaction mixture (33 µl) contained 300 µM synthetic oligopeptide corresponding to the C-terminal 19-amino acid sequence of spinach pD1 (S-19), 0.75 µg of purified CtpA, and 25 mM Hepes-KOH buffer (pH 7.7 at 25 °C). After incubation for 1.5 h at 25 °C, the reaction was terminated by the addition of 6 µl of 18% (w/v) trichloroacetic acid, and then the mixture was kept at room temperature for 20 min, followed by centrifugation at 20,000 × g for 20 min to remove denatured proteins. To 35 µl of the resultant supernatant was added 145 µl of 0.1% (v/v) trifluoroacetic acid, and then the mixture was passed through a filter (0.45-µm pore size). Peptides in 170 µl of filtrate were analyzed by reverse-phase HPLC as described previously (25).

Assay of Proteolytic Activity Using the PSII-enriched Membrane from the LF-1 Mutant of S. obliquus-- PSII-enriched membrane was prepared from logarithmic growth phase cells of the LF-1 mutant of Scenedesmus, in which CtpA activity is deficient (10), according to the modified method of Kuwabara and Murata (41) as described by Metz and Seibert (31). The PSII-enriched membrane containing pD1 was utilized as the substrate for CtpA. The processing activity was visualized by detecting the molecular mass shift of pD1 to mature D1 protein (mD1) by Western blot analysis using antisera raised against the AB-loop sequence of spinach D1 protein.

The standard reaction mixture (100 µl) contained 10 µg of chlorophyll from PSII-enriched membrane from LF-1, overexpressed CtpA (1.25 µg of protein), and 40 mM sodium/potassium phosphate buffer (pH 6.9). The proteolytic reaction was carried out at 25 °C for 20 min. After collecting the PSII-enriched membrane by centrifugation at 15,000 × g for 3 min at 0 °C, the membrane was solubilized with the sample buffer for electrophoresis (125 mM Tris-HCl (pH 6.8 at 25 °C), 5% (w/v) SDS, 20% (w/v) glycerol, and 10% (v/v) 2-mercaptoethanol), and then an aliquot of the extracts corresponding to 2.5 µg of chlorophyll from PSII-enriched membrane was subjected to SDS-PAGE with 15% acrylamide gel containing 6 M urea (32). Proteins separated on the acrylamide gel were electrophoretically transferred to nitrocellulose membrane. Western blot analysis was carried out using an enhanced chemiluminescence system (ECL, Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom) according to the manufacturer's protocol. Each D1 protein band on x-ray film was quantified with a densitometer (Personal Scanning Imager PD110, Molecular Dynamics, Inc., Sunnyvale, CA), and the processing activity was expressed as the ratio of processed D1 to total D1.

Amino Acid Sequencing-- The amino acid sequences of peptides separated by SDS-PAGE or HPLC were determined with a pulse liquid-phase automatic peptide sequencer (Model 476A, Applied Biosystems). The sequencing of peptide bands separated by SDS-PAGE was as described in detail by Inagaki et al. (8).

Chemicals-- Lysyl endopeptidase and thrombin were purchased from Wako (Tokyo) and Novagen, respectively. Penem (allyl-(5S,6S,1'R)-6-(1'-acetoxyethyl)penem-3-carboxylate, SB214357) was a gift of SmithKline Beecham Pharmaceuticals (Harlow, Essex, United Kingdom).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Expression of Spinach CtpA in E. coli

The expression of the spinach ctpA gene in E. coli was attempted in this study to obtain the C-terminal processing protease for pD1 in its active state. The expression plasmid (termed pET-CTPA) carrying a fragment of the ctpA gene encoding the mature part under a promoter for T7 phage RNA polymerase was constructed with pET-15b(+) and then transferred into the BL21(DE3) strain of E. coli, which contains a gene for T7 RNA polymerase under the control of the lacUV5 promoter, as described under "Experimental Procedures." Expression was induced by the addition of IPTG at 37, 30, or 20 °C (Fig. 2). SDS-PAGE analysis of whole cell proteins after the IPTG induction revealed distinct accumulation over time of a polypeptide with a mobility corresponding to ~47 kDa at each temperature. Edman degradation analysis demonstrated that the 47-kDa protein has a GSSHHHHHHSSG sequence at the N terminus, indicating that an N-terminal fMet was removed (data not shown). Thus, judging from both the sequence and the estimated molecular mass, we concluded that the mature form of CtpA was expressed in E. coli, as designed. At 37 °C, a temperature commonly used for cultivating E. coli cells, practically all of the 47-kDa protein was recovered in the insoluble fraction after high speed centrifugation of cell extracts, and the supernatant fraction was thus totally inactive with respect to the C-terminal cleavage of pD1. (A polypeptide of 47 kDa observed in the SDS-PAGE profile for the soluble fraction was shown not to be CtpA by N-terminal amino acid sequencing.) On the other hand, at 30 and 20 °C, we detected a polypeptide band of ~47 kDa with a His tag at the N terminus in the soluble fraction. By lowering the temperature of IPTG induction, the proportion of CtpA in the soluble fraction increased, whereas the overall amount of overexpressed CtpA in E. coli cells decreased on a protein basis. At 20 °C, CtpA in the soluble state was estimated by Coomassie Brilliant Blue staining to correspond to 10-20% of that in the bacterial cells. CtpA in the soluble fraction was active with respect to the C-terminal cleavage of the synthetic pD1 oligopeptide at the correct site, as demonstrated by amino acid sequence analysis of the cleavage products, despite the presence of an additional 22-amino acid sequence at the N terminus.



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Fig. 2.   Temperature dependence of ctpA gene expression. E. coli BL21(DE3) cells carrying expression plasmids for CtpA were grown at various temperatures (shown at the top of each panel in degrees Celsius) until the culture turbidity determined by A600 reached 0.4. After the addition of IPTG at 1 mM, the culture was further incubated for 10 h at 20 °C, for 4 h at 30 °C, or for 3 h at 37 °C until A600 reached ~0.8. Soluble and insoluble fractions were prepared by the method described under "Experimental Procedures" and subjected to SDS-PAGE analysis. The soluble (S) or insoluble (I) fractions loaded in each well corresponded to five times the amount of whole cell extracts (W) on the starting cell basis. Arrowheads indicate the positions of CtpA polypeptides. The magnitude of proteolytic activity in soluble fractions for the C-terminal oligopeptide (S-19) is indicated by plus and minus signs.

The purification of CtpA was achieved from the soluble fraction of cell extracts from E. coli grown at 20 °C by nickel-chelating affinity chromatography utilizing the His tag at the N terminus as described under "Experimental Procedures." In the purification process, most of the impurities were removed from the column with starting buffer or wash buffer (60 mM imidazole). CtpA was recovered almost at homogeneity in the fraction eluted by elution buffer (1 M imidazole) (Fig. 3A). After purification, the His tag at the N terminus was removed from CtpA by thrombin treatment (Fig. 3B), although the presence of the His tag at the N terminus has no apparent effect on the enzymatic activity of CtpA, at least under the conditions examined, as mentioned above. In a typical experiment, we obtained ~1 mg of CtpA from 50 ml of the soluble cell extract from E. coli. Purified CtpA was dialyzed against an appropriate buffer and stored at -20 °C until used. The enzymatic activity of CtpA was stable over a prolonged storage time. This may suggest that soluble CtpA expressed in E. coli, under the conditions described, is folded almost precisely.



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Fig. 3.   Purification of overexpressed CtpA by nickel-chelating chromatography. A, elution profile for polypeptides separated by SDS-PAGE and detected by Coomassie Brilliant Blue staining. CtpA was eluted with 1 M imidazole. S, starting material; Im, imidazole. The presence (+) or absence (-) of proteolytic activity in each fraction is indicated. B, thrombin digestion of purified CtpA. Before, non-thrombin-treated CtpA; After, thrombin-treated CtpA.

Cleavage of C-terminal Oligopeptides by CtpA Expressed in E. coli

Recognition of Substrate-- The properties of proteolytic action of purified CtpA expressed in E. coli for the C-terminal cleavage of pD1 were analyzed using a synthetic oligopeptide corresponding to the C-terminal 19 amino acids of spinach pD1, i.e. from Asn-335 to Gly-353 in pD1, as the substrate, which has been studied in detail (25, 28). When the oligopeptide was incubated with the purified enzyme, two products with the sequences AIEAPSTNG and NAHNFPLDLA, corresponding to the C-terminal extension and the C terminus of mD1, respectively, were eluted from the reverse-phase HPLC column (Fig. 4, Ala trace), demonstrating that thrombin-treated spinach CtpA expressed in E. coli with an extra N-terminal 6 amino acids (i.e. GSHMLE) recognizes the substrate and cleaves it at the correct site, i.e. between Ala-344 and Ala-345. However, a relatively high Km value of ~0.3 mM was estimated from the Lineweaver-Burk plot of the kinetic traces for overexpressed CtpA (Fig. 4, inset), as is the case for the native enzyme isolated from spinach chloroplasts (25).



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Fig. 4.   HPLC profiles for processing of C-terminal oligopeptides with or without substitution at the cleavage site (position +1). Each oligopeptide (in which Ala-345 was replaced by Gly, Val, or Pro) was subjected to enzymatic analysis as described under "Experimental Procedures." Peaks S-19, C-9, and N-10 correspond to the C-terminal 19-mer of pD1 with or without substitution, the C-terminal 9-mer of pD1, and the C-terminal 10-mer of mD1, respectively. The inset is the Lineweaver-Burk plot for the oligopeptide without substitution.

The efficiency of C-terminal cleavage of several synthetic oligopeptides with a substitution at the cleavage site was also examined using the enzyme overexpressed in E. coli for comparison with and verification of the results obtained from the analysis using native spinach enzyme for the recognition mechanism (25). Fig. 4 shows the HPLC elution profiles for a series of oligopeptides with a substitution at the cleavage site (position +1, Ala-345). The replacement of Ala by Gly, Val, or Pro served to reduce the rate of proteolysis to ~50, 30, and 0% of the control, respectively (Fig. 4), whereas replacement of Ala by Cys, Phe, or Ser did not affect the rate of cleavage (data not shown). This is consistent with the observation for the native enzyme in a previous analysis (25). Judging from the retention time of reaction products on the chromatogram, we concluded that CtpA expressed in E. coli cleaves the substituted oligopeptides at the normal site, i.e. after Ala-344. Thus, we also concluded that CtpA overexpressed in E. coli with an extra N-terminal GSHMLE sequence recognizes and cleaves the substrate, at least in vitro, in exactly the same manner as the native enzyme. This, on the other hand, suggests that the presence of an extra sequence with a positive charge at the N terminus has no serious effect on the enzyme-substrate interaction, although there is significant homology in the N-terminal sequence of CtpA among different organisms.

pH Dependence-- An unexpected finding for the C-terminal cleavage of pD1 analyzed in vitro is that the proteolytic reaction proceeds more slowly in the acidic region compared with that in the neutral or alkaline pH region,3 although the enzyme actually is demonstrated to be present in the thylakoidal luminal space, where the pH is estimated to be acidic under the functional conditions of illumination, as described in the Introduction. In this study, the pH profile for C-terminal cleavage of the synthetic oligopeptide was investigated in detail using CtpA overexpressed in E. coli. As shown in Fig. 5, this again demonstrated that the proteolytic activity of C-terminal cleavage of pD1 exhibits a strong pH dependence with a maximum around pH 7.5-8.0 and that the activity is quite low in the acidic region of pH 5.0-6.0. A similar pH profile has been obtained using in vitro translated full-length pD1 as the substrate in separate experiments (data not shown).



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Fig. 5.   pH dependence of the C-terminal processing of the pD1 oligopeptide. The synthetic oligopeptide corresponding to the C-terminal 19 amino acids of pD1 was incubated with thrombin-treated CtpA in each buffer shown at 100 mM. The maximum activity was normalized to 1.0. Mes, 2-(N-morpholino)ethanesulfonic acid; Pipes, piperazine-N,N'-bis(2-ethanesulfonic acid); Tricine, N-tris(hydroxymethyl)methylglycine; Ches, 2-(cyclohexylamino)ethanesulfonic acid; Caps, 3-(cyclohexylamino)propanesulfonic acid.

Inhibitor Specificity-- Thus far, no specific inhibitor has been reported for the C-terminal processing protease for pD1. Therefore, by taking advantage of the quantity of enzyme overexpressed in E. coli, we systematically examined a wide variety of protease inhibitors (including serine, cysteine, and aspartic protease and metalloprotease inhibitors) at different concentrations for the C-terminal cleavage of the synthetic oligopeptide by thrombin-treated CtpA overexpressed in E. coli. This included penem, which is unique in that it specifically inhibits some of the novel serine proteases possessing the Ser/Lys catalytic dyad (33, 34). However, as summarized in Table I, despite thorough investigation, the CtpA activity proved to be resistant to all of the protease inhibitors tested, including penem.


                              
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Table I
Inhibitor specificity

Processing of pD1 Integrated into the PSII Complex in the Thylakoid Membrane

To analyze the molecular mechanism of enzyme-substrate interactions in the C-terminal processing of pD1 integrated into the thylakoid membrane and to resolve the discrepancies between in vitro and in vivo results on pH dependence and affinity, we developed an assay system that mimics the in vivo system. It uses PSII-enriched membranes from the LF-1 mutant of S. obliquus lacking C-terminal processing activity in combination with spinach CtpA overexpressed in E. coli. The PSII-enriched membrane containing pD1 was utilized as the substrate for analysis. In this case, the processing activity was visualized by detecting the molecular mass shift from pD1 (34 kDa) to mD1 (32 kDa) by Western blot analysis using antiserum raised against the AB-loop sequence of spinach D1 protein as described under "Experimental Procedures." Fig. 6 shows the time course of C-terminal processing of pD1 by CtpA. pD1 of the Scenedesmus LF-1 mutant with a molecular mass of 34 kDa was converted to a smaller form with the same mobility as mD1 in the wild strain, but no further degradation could be observed, suggesting that spinach CtpA overexpressed in E. coli cleaved pD1 integrated into the thylakoid membrane of Scenedesmus at the correct site (Fig. 6). To confirm this, protease mapping analysis was conducted with lysyl endopeptidase by taking advantage of the fact that the D1 protein in Scenedesmus contains a single lysine residue at position 238.4 The PSII-enriched membrane from the LF-1 mutant of Scenedesmus was subjected to lysyl endopeptidase digestion before and after proteolysis by CtpA. The proteolytic products were detected by two kinds of anti-peptide antibodies that specifically recognize the AB-loop and C-terminal regions of D1, respectively.5 Comparison of the mapping patterns showed that the removal of a short peptide with a molecular mass of ~2 kDa from pD1 occurred at its C terminus, but not its N terminus, proving the validity of the above interpretation. The processing of pD1 by CtpA proceeded almost linearly with time up to 20 min.



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Fig. 6.   Time course of the C-terminal processing of pD1 in the PSII complex integrated into the thylakoid membrane. The PSII-enriched membrane from the LF-1 mutant of Scenedesmus was incubated with CtpA at 25 °C for the indicated time intervals. WT and LF-1 correspond to the untreated PSII-enriched membranes from the wild strain and the LF-1 mutant of Scenedesmus, respectively.

The relative affinity of CtpA for pD1 integrated into the thylakoid membrane was approximated by analyzing the competitive inhibitory effect of the synthetic oligopeptide on the processing of pD1 in the PSII-enriched membrane of Scenedesmus. By assuming 250 of chlorophyll molecules/PSII reaction center, the substrate concentration in the reaction mixture was estimated such that 0.1 mg/ml chlorophyll is equivalent to 0.4 µM pD1. The synthetic oligopeptide was added to the reaction mixture at concentrations ranging from 1- to 1,500-fold over pD1 integrated into the thylakoid membrane at a molar ratio (Fig. 7). The addition of oligopeptide at the 1-, 10-, and 100-fold concentrations exhibited no inhibitory effect on the proteolysis of pD1 by CtpA on the membrane. The concentration of oligopeptide required to inhibit the proteolytic processing of membrane-embedded pD1 was nearly 1,000 times higher than that of substrate pD1 in the membrane. From this, we infer that the affinity of the enzyme for pD1 is enhanced by the incorporation of the substrate into the thylakoid membrane.



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Fig. 7.   Competitive inhibition of C-terminal processing by a synthetic oligopeptide. The PSII membrane (0.4 µM pD1) was incubated with CtpA in the presence of a synthetic oligopeptide corresponding to the C-terminal 19 amino acids of spinach pD1. Concentrations of the oligopeptide in the reaction mixture ranged from 1- to 1,500-fold over pD1 at a molar ratio, as indicated.

In the experiment shown in Fig. 8, the pH profile for C-terminal processing of pD1 integrated into the thylakoid membrane of Scenedesmus by spinach CtpA overexpressed in E. coli was investigated. In contrast to the in vitro system using synthetic oligopeptide or in vitro translated full-length pD1 as described above, CtpA was remarkably active in this system in the acidic region, exhibiting an optimal processing activity at pH ~6.0, which is fairly consistent with the pH of the thylakoidal lumen under the functional conditions of illumination. However, under basic conditions, the proteolytic activity was quite low, in contrast to the results obtained with the in vitro system using synthetic oligopeptide or in vitro translated full-length pD1 as the substrate with native CtpA or CtpA expressed in E. coli.



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Fig. 8.   pH dependence of the C-terminal processing of pD1 integrated into the thylakoid membrane. A, the PSII-enriched membrane from the LF-1 mutant of Scenedesmus was incubated with CtpA in each buffer shown at 100 mM. Mes, 2-(N-morpholino)ethanesulfonic acid; Pipes, piperazine-N,N'-bis(2-ethanesulfonic acid); Tricine, N-tris(hydroxymethyl)methylglycine. B, the amount of each protein band was quantified by a densitometer, and the relative activity is given by the ratio of mD1 to total D1 (mD1 + pD1) (). For comparison, the pH profile for the C-terminal processing of the synthetic oligopeptide is shown (open circle ). The maximum activity was normalized to 1.0 in each pH profile.



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Under conventional conditions of growth at 37 °C, E. coli cells expressed spinach CtpA and accumulated it in large quantities in the insoluble fraction. However, upon lowering the temperature of cell growth to 20 °C, a substantial proportion of the protease was recovered in its active state in the soluble fraction of cell extracts. This probably is due to a decrease in misfolding of the induced protein as a result of the slower rate of synthesis at 20 °C, as generally anticipated. The success in fractionating CtpA into the supernatant fraction of cell extracts enabled us to establish a simple purification procedure based on the strategy of nickel-chelating affinity chromatography utilizing the His tag constructed at the N terminus.

E. coli has a tail-specific protease called Tsp in the periplasm and its counterpart in the cytoplasm (23). These proteases are reported to recognize nonpolar regions at the C terminus of proteins (23) or an ssrA-encoded peptide tag added to the C-terminal portion of nascent polypeptides translated from mRNA without a stop codon (35) and to degrade them at the site of a small residue, e.g. Aladown-arrow Ala, Aladown-arrow Leu, Aladown-arrow Lys, and Valdown-arrow Ser (23, 35, 36). The success in overexpressing CtpA in E. coli without affecting its viability suggests that the protease does not function in the bacterial cells, despite the fact that the enzyme has a significant homology to Tsp and that the cleavage site of the enzyme (Ala344down-arrow Ala345 or Ala344down-arrow Ser345) is rather similar to that of Tsp and its counterpart. This confirms the high degree of specificity of CtpA for its substrate and argues against the possibility that this enzyme is involved in the degradation of mistranslated proteins produced during biosynthesis in chloroplasts (37).

By taking advantage of the great quantity of protease overexpressed in E. coli, we thoroughly investigated the effect of different types of protease inhibitors on the catalytic activity of C-terminal processing of pD1 using synthetic C-terminal oligopeptide as the substrate. However, the proteolytic activity proved to be totally resistant to any of the standard inhibitors tested. This fact strongly supports the proposal, based on homology to Tsp, that CtpA should be classified as a novel type of serine protease. In fact, a recent study by our group, using site-directed mutagenesis of the ctpA gene in Synechocystis sp. PCC 6803, has revealed that Ser-282 and Lys-307, which correspond to Ser-291 and Lys-316, respectively, in the spinach enzyme (numbered from the N terminus of the mature protein), are possibly involved in the catalytic function of CtpA.2 This supports the view that this enzyme ought to be assigned to a novel type of protease group in which a hydroxyl/amine dyad is located in the catalytic center, which has been substantiated more clearly by a recent structural analysis (38). An important fact, however, is that the catalytic function of CtpA was not inhibited by penem, which has been shown to be a potent inhibitor of two kinds of serine protease with a Ser/Lys catalytic dyad, i.e. the E. coli leader peptidase (22, 33) and the thylakoidal processing peptidases from cyanobacteria and higher plants (34). This inhibitory effect of penem is reported to be due to covalent binding of the beta -lactam ring to the catalytic Ser residue (22). The tolerance of CtpA for penem demonstrated in this study suggests that the structure of the catalytic center in CtpA, which is responsible for the cleavage of the C-terminal extension of pD1, differs from that in other processing peptidases that are responsible for the N-terminal cleavage of translocated preproteins. Thus, at present, penem seems to be a specific inhibitor of type I signal peptidases. The successful preparation of a large amount of CtpA from E. coli cells in this study could provide us an opportunity to analyze the unique structure of the catalytic center of this new type of protease by crystallographic analysis, as exemplified by a recent study (38).

Purified spinach CtpA overexpressed in E. coli was demonstrated to recognize the C-terminal sequence of pD1 in a manner similar to that of the native protease purified from spinach chloroplasts (25), as shown by its selectivity for the +1 substituted C-terminal oligopeptides as the substrate, i.e. the replacement of Ala by Gly, Val, or Pro served to reduce the proteolysis, whereas replacement by Cys, Phe, or Ser did not affect the rate of cleavage. However, the analysis using overexpressed CtpA and pD1 oligopeptides confirmed the abnormalities in pH dependence and affinity, i.e. proteolysis revealed a pH optimum between 7.5 and 8.0 (but not between 5.0 and 6.0), and the affinity of the enzyme for the substrate was relatively weak (Km = 0.3 mM), despite its expected rapid rate of turnover in vivo in the damage repair cycle. Therefore, we had a suspicion that the processing of pD1 in vivo may not proceed by the mechanism elucidated by in vitro analyses using synthetic oligopeptides or artificially translated full-length pD1 as the substrate (39). However, a recent mixed-culture growth experiment using Chlamydomonas mutants strongly suggested that the recognition mechanism studied by using substituted synthetic oligopeptides in vitro also operates in the in vivo system (40). This convinced us of the existence of the additional interaction(s), between CtpA and pD1 in vivo integrated into the thylakoid membrane, that largely influences its pH dependence and its affinity for the C-terminal processing of pD1. Therefore, we developed an assay system that mimics the in vivo system, using PSII-enriched membranes from the LF-1 mutant of S. obliquus lacking a C-terminal processing protease due to early termination in the expression of the ctpA gene (10), to analyze the abnormalities mentioned above.

As shown in Fig. 7, the concentration of oligopeptide required to inhibit the proteolytic processing of membrane-embedded pD1 in the assay system was ~1,000 times greater than that of substrate pD1 in the membrane, putting the estimate for the Km value at ~0.3 µM for pD1 in the membrane. This can be taken to mean that the affinity of the protease for the substrate is greatly enhanced when the substrate is embedded in the thylakoid membrane. In other words, some other components on the membrane or in another region of the D1 protein (e.g. the AB- and/or CD-loop(s) protruding into the thylakoidal luminal space) greatly influence the interaction with the protease.

On the other hand, as shown in Fig. 8, the proteolytic processing of membrane-embedded pD1 in the assay system exhibited a pH optimum in the range of 5.5-6.0 (not 7.5-8.0), in accordance with the pH values of the functional site of this enzyme in vivo, i.e. the luminal space of illuminated thylakoids. This finding may also be explained by the presence of an additional component(s) on the membrane that influences the enzyme-substrate interaction. Trost et al. (10) analyzed the pH profile for purified Scenedesmus CtpA by enzyme-linked immunosorbent assay using the PSII core complex isolated from the LF-1 mutant as the substrate. They demonstrated a pH optimum of ~6.5, fairly consistent with our result using PSII-enriched membrane from the LF-1 mutant as the substrate. However, the pH profile they reported is unusual and greatly differs from ours, which exhibits an appreciably higher activity in the region of alkaline pH. Although this discrepancy may simply reflect differences in the respective assay systems, it may be explained by the presence of additional interactions between a component(s) on the membrane and the enzyme as discussed above. In our preliminary analysis, the PSII-enriched membrane of LF-1 was treated with reagents (e.g. lysyl endopeptidase and Triton X-100 at 4%) that could influence the structure of the thylakoid membrane. However, in any cases, the pH profile was not altered to a great extent, although a slight shift to the alkaline direction was detected. This may suggest the situation that several components contribute to the alteration of enzyme-substrate interactions observed in our study and that the crucial interaction(s) with the enzyme is provided by components in the PSII reaction center.

In the catalytic mechanism of this novel serine protease, which is proposed to employ Ser and Lys as the nucleophile and general base, respectively, the pertinent question is how the protease enables the epsilon -amino group of Lys to be maintained in the deprotonated state so that it acts as a base in the catalysis (20, 22). Since the pKa of Lys is 10.5, CtpA or the CtpA-pD1 interaction must alter the environment around the catalytic center to significantly lower the pKa of active Lys so that the C-terminal processing of pD1 may proceed under physiological conditions. The alteration in the pH profile for the CtpA activity that is dependent upon the condition of the substrate, as described above, may be correlated with the enzyme-substrate interaction lowering the pKa of the catalytic Lys residue.


    ACKNOWLEDGEMENTS

We thank Dr. Norman I. Bishop for providing the wild strain and the LF-1 mutant of Scenedesmus and SmithKline Beecham Pharmaceuticals for the gift of the penem inhibitor. We are also grateful to Dr. Tatsuya Tomo (RIKEN) for his technical support in determining the amino acid sequence of polypeptides.


    FOOTNOTES

* This work was supported by Grant-in-aid for Scientific Research (B) No. 09440268 from the Ministry of Education, Science, Sports, and Culture and by a grant from the Okayama Foundation for Science and Technology.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.

Present address: Lab. of Photosynthesis, National Inst. of Agrobiological Resources, Tsukuba 305-8602, Japan.

|| To whom correspondence should be addressed. Tel.: 81-86-251-7862; Fax: 81-86-251-7877; E-mail: kimiyuki@cc.okayama-u.ac.jp.

Published, JBC Papers in Press, November 30, 2000, DOI 10.1074/jbc.M008877200

2 N. Inagaki, R. Maitra, K. Satoh, and H. B. Pakrasi, manuscript in preparation.

3 Y. Yamamoto and K. Satoh, unpublished data.

4 J. R. Bowyer, personal communication.

5 Y. Yamamoto and K. Satoh, manuscript in preparation.


    ABBREVIATIONS

The abbreviations used are: PSII, photosystem II; pD1, precursor D1 protein; mD1, mature D1 protein; IPTG, isopropyl-beta -D-thiogalactopyranoside; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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