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Plant Physiol. (1994) 106: 617-624 ldentification of Surface-Exposed Domains on the Reducing Side of Photosystem I' Qiang Xu, James A. Cuikema, and Parag R. Chitnis* Division of Biology, Kansas State University, Manhattan, Kansas 66506-4901 centers, A,, A,, and Fx. PsaC binds two [4Fe-4S] clusters, FA and FB(Oh-oka et al., 1988). PsaD provides an essential Fddocking site on the reducing side of PSI (Zanetti and Merati, 1987; Zilber and Malkin, 1988; Lelong et al., 1994; Xu et al., 1994a) and is also required for the stable assembly of PsaC and PsaE into the PSI complex (Li et al., 1991; Chitnis and Nelson, 1992). PsaE is required for Fd reduction (Rousseau et al., 1993; Sonoike et al., 1993; Strotmann and Weber, 1993; Xu et al., 1994a) and may be involved in cyclic electron flow around PSI (Yu et al., 1993). PsaL is essential for the formation of PSI trimers (Chitnis and Chitnis, 1993). PsaF is exposed to the p-side (lumenal) of the photosynthetic membranes and can be cross-linked with plastocyanin or Cyt c6 (Hippler et al., 1989; Wynn et al., 1989a, 1989b), but is not essential for the photooxidation of Cyt c6 (xu et al., 199413). Other subunits, such as PsaJ, PsaK, PsaI, and PsaM, are much smaller ( c 5 . 5 kD) and are conserved from cyanobacteria to higher plants, but their functions are not known (Ikeuchi et al., 1993; Xu et al., 1994b). The recent*x-raydiffraction analysis of cyanobacterial PSI crystals at 6-A resolution (Krauss et al., 1993), in combination with biochemical studies (Golbeck, 1993), provides a framework for understanding the overall architecture of PSI. PsaC, PsaD, and PsaE are peripheral subunits, located on the nside (stromal in chloroplasts and cytoplasmic in cyanobacteria) of photosynthetic membranes, with PsaC positioned in the center of each monomeric PSI on a local pseudo-2-fold axis of symmetry (Krauss et al., 1993). Besides structural studies, the subunit interactions in PSI are also deciphered from biochemical experiments and characterization of PSI mutants. For example, the availability of subunit-deficient PSI complexes and improved Tricine-urea-SDS-PAGE has revealed structural interactions among PsaE, PsaF, and/or PsaJ (Xu et al., 1994b) or between PsaD and PsaL (Q. Xu, P.R. Chitnis, unpublished data). These findings are corroborated by chemical cross-linking of these subunits (T.S. Armbrust, J.A. Guikema, unpublished data). The detailed architecture of PSI is yet to be determined. Protease accessibility studies have provided insights regarding organization and topology of PSI subunits (Zilber and Malkin, 1992). In higher plant PSI, PsaA, PsaB, PsaD, PsaE, PsaH, PsaK, and PsaL have stroma-exposed regions (Zilber and Malkin, 1992). Mapping of the proteolytic fragments of PsaD and PsaE indicates that N-terminal extensions of PsaD and PsaE are accessible to proteolysis (Lagoutte and Vallon, Photosystem I (PSI) i s a multisubunit enzyme that catalyzes the light-driven oxidation of plastocyanin or cytochrome c6 and the concomitant photoreduction of ferredoxin or flavodoxin. To identify the surface-exposed domains in PSI of the cyanobaderium Synechocystis sp. PCC 6803, we mapped the regions in PsaE, PsaD, and PsaF that are accessible to proteases and N-hydroxysuccinimidobiotin (NHS-biotin). Upon exposure of PSI complexes to a low concentration of endoproteinase glutamic acid (CIu)-C, PsaE was cleaved to 7.1- and 6.6-kD N-terminal fragments without significant cleavage of other subunits. C W and Clu6', located near the C terminus of PsaE, were the most likely cleavage sites. At higher protease concentrations, the PsaE fragments were further cleaved and an N-terminal 9.8-kD PsaD fragment accumulated, demonstrating the accessibility of Clu residue(s) in the C-terminal domain of PsaD to the protease. Besides these major, primary cleavage products, severa1 secondary cleavage sites on PsaD, PsaE, and PsaF were also identified. PsaF resisted proteolysis when PsaD and PsaE were intact. Clu' and C I U ' ~ of~PsaF became susceptible to endoproteinase CIu-C upon extensive cleavage of PsaD and PsaE. Modification of PSI proteins with NHS-biotin and subsequent cleavage by endoproteinase CIu-C or thermolysin showed that the intact PsaE and PsaD, but not their major degradation produds lacking C-terminal domains, were heavily biotinylated. Therefore, lysine74 at the C terminus of PsaE was accessible for biotinylation. Similarly, lysine-107, or lysine-118, or both i n PsaD could be modified by NHS-biotin. PSI in cyanobacteria and chloroplasts is a Chl-binding membrane-protein complex that catalyzes light-dependent electron transfer from reduced plastocyanin or Cyt c6 to oxidized Fd or flavodoxin (Chitnis and Nelson, 1991; Bryant, 1992; Golbeck, 1993). PSI contains at least 11 polypeptides in cyanobacteria (Miihlenhoff et al., 1993) and three additional proteins, PsaG, PsaH, and PsaN, in higher plants (Okkels et al., 1989, 1992; Knoetzel and Simpson, 1993). PsaA and PsaB form a heterodimeric core that harbors the primary electron donor, P700, and a chain of electron transfer ' This work was supported in part by grants from the National Science Foundation (MCB#9202751 to P.R.C.),the U.S. Department of Agriculture-National Research Initiative Competitive Grants Program (USDA-NRICGP) (92-37306-7661 to P.R.C. and 93-373069147 to J.A.G.),and the National Aeronautics and Space Administration (NAGW 2328 to J.A.G.).We also achowledge an equipment grant from the USDA-NRICGP (93-3731 1-9456 to P.R.C.).This is contribution 94-538-J from the Kansas Agricultura1 Experiment Station. * Corresponding author; fax 1-913-532-6653. Abbreviations: Glu-C, endoproteinase Glu-C; NHS-biotin, N-hydroxysuccinimidobiotin. 61 7 Downloaded from on August 2, 2017 - Published by www.plantphysiol.org Copyright © 1994 American Society of Plant Biologists. All rights reserved. Xu et at. 61 8 1992; Zilber and Malkin, 1992). However, these N-terminal extensions are absent in cyanobacterial PsaD and PsaE. Although the three-dimensional structure of cyanobacterial PsaE in solution has recently been proposed from multidimensional NMR analysis (Falzone et al., 1994a, 1994b), data regarding accessibility of cyanobacterial PsaE to proteases are not available. Hence, the positioning of PsaE relative to the photosynthetic membranes has not been determined. Clearly, the interactions among subunits of PSI and surface topology of the complex remain largely unknown. Here we treated PSI complexes with Glu-C and mapped the proteolytic peptides by N-terminal amino acid sequencing. We also labeled the PSI complexes with NHS-biotin and mapped the biotinylation sites on PsaD and PsaE. Based on these data, we identified the surface-exposed domains of PsaD and PsaE in cyanobacterial PSI. MATERIALS A N D METHODS Materials Prestained protein molecular mass standards were from GIBCO-BRL. The centrifugal filtration apparatus (Centricon100 with 100-kD cutoff membrane) was from Amicon (Beverly, MA) and the polyvinylidine difluoride (Immobilon-P) membrane was from Millipore (Bedford, MA). Avidin-peroxidase and NHS-biotin were from Sigma. Sequencing-grade Glu-C (protease V8 from Staphylococcus aureus; EC 3.4.21.19) was purchased from Boehringer Mannheim, and thermolysin (protease type X from Bacillus thermoproteolyticus; EC 3.4.24.4) was from Sigma. The enhanced chemiluminescence reagents for westem blotting were purchased from Amersham. Other chemicals were from Sigma or Fisher. Plant Physiol. Vol. 106, 1994 The reaction was terminated with 20 m~ EDTA. After protease treatment, the association of cleavage products with the PSI core could be examined by incubating the complexes with Na1 (Xu et al., 1994a), followed by diluion with an excess amount of 10 mM Mops-HC1 (pH 7.C9 containing 0.05% Triton X-100 and filtration with the Centricon-100. This procedure desalts the PSI complexes and separates them from the proteins released during incubation with NaI. Modification of the PSI Subunits with NHS-Biotin PSI complexes at 150 pg Chl/mL were incubated with 58 NHS-biotin, 10 mM Mops (pH 7.0), 0.05% ‘Triton X-100, and 0.05% DMSO for 30 min at room temperature. The reaction was quenched with 50 m~ ammonium bicarbonate (pH 7.8). The labeled PSI subunits were separatcmd by Tricineurea-SDS-PAGE and electroblotted to Immobilon-P membranes. The blot was probed with an avidin-peroxidase conjugate and developed with hydrogen peroxide and 4chloro-1-naphthol (Frankel and Bricker, 1992). p~ Analytical Gel Electrophoresis and lmmunodelection PSI complexes were solubilized with 1% SDS and 0.1% 2mercaptoethanol at room temperature for 1 h. Polypeptides were resolved by Tricine-urea-SDS-PAGE (Xu et al., 1994a). After electrophoresis, gels were stained with Coomassie blue or silver nitrate. Altematively, proteins were tlectro-transferred to Immobilon-P membranes. Immunodetection was performed using enhanced chemiluminescencereagents. The antibody against PsaD of Synechococcus sp. PCC 7002 was a gift from Dr. John H. Golbeck at the University of Nebraska. Other antibodies were raised against Synechocystis sp. PCC 6803 proteins. Isolation and Characterization of PSI Complexes Synechocystis sp. PCC 6803 culture was grown in BG-11 under a light intensity of 21 pmol m-’ s-’ and harvested at late exponential phase of growth. The cells were suspended in 0.4 M SUC,10 m~ NaC1,200 m~ PMSF, 5 m~ benzamidine, and 10 mM Mops-HC1 (pH 7.0), and photosynthetic membranes were isolated using a mini bead-beater (Xu et ai., 1994b). The membranes were treated with 10 mg Triton/mg Chl, and PSI complexes were purified by ion-exchange chromatography and Suc gradient ultracentrifugation (Xu et al., 1994b). PSI isolated by this method can function as a lightdriven Cyt c6-Fd oxidoreductase (Xu et al., 1994b). Chl concentrations were determined in 80% (v/v) acetone (Amon, 1949). Treatment of PSI Complexes with Proteases Purified PSI was adjusted to 150 pg Chl/mL prior to proteolytic cleavage. For Glu-C cleavage, PSI preparations were incubated with O to 100 pg Glu-C/mg Chl in the presence of 50 m~ ammonium bicarbonate (pH 7.8) at 15OC. Under these conditions, Glu-C cleaves the peptide bond at the carboxyl side of glutamyl residues (Houmard and Drapeau, 1972). The reaction was terminated with 10 m~ PMSF. For cleavage by thermolysin, PSI was incubated with the protease (20 pg/mg Chl) and 5 mM CaC& at 37OC for 40 min. N-Terminal Amino Acid Sequencing Peptides were blotted to Immobilon-P membranes, stained with Coomassie blue containing 1% acetic acid for 2 min, destained with 50% methanol, and rinsed extensively with deionized water. N-terminal sequences were determined on an Applied Biosystems (Foster City, CA) 477A sequencer at the Biotechnology Core Facility of Kansas State University. RESULTS Accessibility of PSI Subunits to GIu-C We treated PSI complexes with dfferent concentrations of Glu-C and subsequently examined PSI subunits by SDSPAGE to estimate their relative accessibility to proteolysis (Fig. 1).Upon exposure of PSI complexes to 1 p.g Glu-C/mg Chl, most of the PsaE subunit was degraded without a significant cleavage of other subunits and with s,imultaneous appearance of 7.1- and 6.7-kD peptides, termed E1 and E11 (Fig. 1A). A polyclonal antibody against PsaE of Synechocystis sp. PCC 6803 recognized both E1 and E11 fragments (data not shown). At Glu-C concentrations of 5 pg/mg Chl or higher, additional PSI subunits were degraded. Notably, a 9.8-kD fragment (labeled DI) accumulated, with a concomitant decrease in the relative amount of PsaD. This fragment was Downloaded from on August 2, 2017 - Published by www.plantphysiol.org Copyright © 1994 American Society of Plant Biologists. All rights reserved. Topology of PSI Subunits ^ Glu-C/mg Chl 0 1 5 20 50 100 Figure 1. Accessibility of PSI subunits to Clu-C. A, Purified PSI complexes were treated with 0, 1, 5, 20, 50, and 100 fig Glu-C/mg Chl at 15°C for 20 min. The polypeptides in PSI complexes containing 5 ng of Chl were separated by Tricine-urea-SDS-PACE and visualized by silver staining. The subunits labeled in the figure have been identified using immunodetection and N-terminal amino acid sequencing (Xu et al., 1994b), and their molecular masses were determined from migration of the following protein markers: insulin (2.9 kD), bovine trypsin inhibitor (6.2 kD), lysozyme (14.3 kD), /3lactoglobulin (18.4 kD), carbonic anhydrase (29 kD), ovalbumin (43 kD), and BSA (68 kD). The PsaA-PsaB, PsaD, PsaF, PsaL, PsaE, PsaC, PsaK, PsaJ, Psal, and PsaM of Synecriocyst/s sp. PCC 6803 migrated on a Tricine-urea-SDS-polyacrylamide gel with apparent molecular masses of 66, 18.2, 15.7, 14.2,8.9,8.0, 5.2, 3.4, 3.1, and 2.9 kD. B, The PSI complexes were treated with 0 and 20 ^g Clu-C/mg Chl as in A (lanes 0 and 20). An identical sample of protease-treated PSI complexes was ultrafiltered using a Centricon-100. The retentate was resuspended with 12% Sue, 0.05% Triton X-100, and 10 ITIM Mops-HCI, pH 7.0 (lane 20*). The samples containing 10 ^g of Chl were solubilized and proteins were separated by Tricine-urea-SDSPACE and visualized by staining the gel with Coomassie blue. recognized by an anti-PsaD polyclonal antibody (data not shown). PsaA-PsaB remained largely intact at Glu-C concentrations up to 20 ng/mg Chl. At higher protease concentrations, more fragments were observed; most of these were detected only after silver staining. PsaL, PsaK, Psa], Psal, and PsaM were resistant to Glu-C cleavage at all protease concentrations tested (Fig. 1A). The results in Figure 1A would not distinguish between proteolytic fragments that were released from PSI and those that remained as peripheral PSI components. The El, EH, and DI fragments were prominent species during cleavage of PSI complexes with 20 ng Glu-C/mg Chl (Fig. IB). To determine their relative association with the PSI core, the proteasetreated PSI complexes were filtered using the Centricon-100, and retentates were analyzed by Tricine-urea-SDS-PAGE. The El and EII cleavage products of PsaE were more tightly associated with the PSI complexes than was the 9.8-kD DI fragment of PsaD (Fig. IB, lane 20*). The stability of these associations was further examined by incubation with 1 M Nal prior to filtration. The El and EII fragments were relatively resistant to removal by Nal (data not shown). When in solution, both PsaE (Rousseau et al., 1993) and PsaD (data not shown) are completely cleaved by Glu-C. Therefore, the data in Figure 1 demonstrated that specific Glu residues of PsaD and PsaE are shielded from the protease after their assembly into PSI complexes. These results prompted identification of El, EII, and DI peptides by Nterminal amino acid sequencing (Table I). Both El and EII fragments had N termini that matched the 2ALNRG terminus of PsaE (Chitnis et al., 1989). Therefore Glu-C cleaved PsaE at the Glu residues 1.8 and 2.3 kD from the C terminus to generate the El and EII fragments. The N terminus of the DI fragment had a sequence that matched the 2TELSG of PsaD (Reilly et al., 1988) (Table I). Thus, a 6-kD C-terminal domain of PsaD was cleaved by Glu-C. A 6-kD polypeptide was observed on the polyacrylamide gel (Fig. 1A) but did not accumulate to the same extent as the DI fragment. This fragment may be more rapidly cleaved to smaller fragments. These data showed that the Glu residues in the C-terminal domains of PsaD and PsaE were accessible to Glu-C and thus were exposed to the aqueous phase. To identify secondary sites of Glu-C cleavage, we subjected PSI complexes to extensive proteolysis using 100 jtg Glu-C/ mg Chl. Increased protease concentration yielded numerous proteolytic fragments from PSI subunits, including FI, FII, DII, Dili, DIV, DV, DVI, and EHI, with apparent molecular masses of 13.3, 9.0, 6.4, 6.2, 4.4, 3.8, 3.5, and 2.5 kD, respectively (Fig. 2). Although some of these fragments were initially identified by western blotting (data not shown), we determined amino acid sequences of their N termini for accurate identification and mapping (Table H). These studies revealed that all sequenced cleavage products were derived from PsaD, PsaE, and PsaF. In contrast, PsaL and PsaK did not decrease in intensity after extensive proteolysis, and none of the peptides identified in this study (Tables I and II) were derived from these subunits. This suggested that Glu residues in PsaL and PsaK were not easily accessible to the protease. The pattern of fragment appearance upon increasing proteolysis suggested a sequence of subunit accessibility to Glu- Table I. N-terminal sequence analysis of prominent PSI fragments produced using Clu-C as described in Figure / Proteolytic Fragment Apparent Mass" El Ell DI 7.1 6.7 9.8 Sequencing Cycle Identification and Position of the First Amino Acid kD ALNRC ALNRC TELSC 619 2 ALNRC of PsaE (Chitnis et al., 1989a) ALNRC of PsaE (Chitnis et al., 1989a) 2 TELSC of PsaD (Reilly et al., 1988) 2 ' Average of three independent estimates from the electrophoretic mobility. Downloaded from on August 2, 2017 - Published by www.plantphysiol.org Copyright © 1994 American Society of Plant Biologists. All rights reserved. 620 Xu et al. Plant Physiol. Vol. 106, 1994 Hg Glu-C/mg Chl 0 ^g Glu-C/mg Chl 100 0 1 5 20 50 100 PsaA-PsaB Anti-PsaD \nti-PsaF EIII Figure 2. Extensive cleavage of PSI complexes with Clu-C. The PSI complexes (lane 0) were treated with 100 Mg Glu-C/mg Chl at 37°C for 1 h (lane 100). The complexes containing 15 v% of Chl were solubilized. The proteins were separated by Tricine-urea-SDS-PAGE and electroblotted on Immobilon-P membranes. The membranes were stained with Coomassie blue in 1% acetic acid. C. For example, PsaE was readily cleaved at Glu-C concentrations that have minimal impact on PsaD (Fig. 1). When levels of PsaD and PsaF were immunodetected after cleavage of PSI with increasing concentrations of Glu-C (Fig. 3), PsaF fragments were observed only after extensive degradation of PsaA-PsaB, PsaD, and PsaE, which may expose Glu-C-sensitive sites on PsaF. Figure 3. Immunodetection of PsaD and PsaF after Glu-C cleavage of PSI. PSI was treated with 0, 1, 5, 20, 50, and 100 ^g Glu-C/mg Chl as in Figure 1A. Proteins in the PSI complexes containing 5 ^g of Chl were separated by Tricine-urea-SDS-PAGE, blotted on Immobilon-P membranes, and sequentially probed with antibodies against PsaD (top) and PsaF (bottom). Modification of PSI Subunits with NHS-Biotin Analyses of the deduced amino acid sequences revealed that PsaD and PsaE contain 12 and 4 lysyl residues, respectively (Reilly et al., 1988; Chitnis et al., 1989). NHS-biotin reacts with the N terminus and the t-amino group of lysyl residues (Bayer and Wilchek, 1990). Modification with NHSbiotin has been used to examine exposure of protein surfaces to the aqueous phase (e.g. Frankel and Bricker, 1992). To identify the surface-exposed lysyl residues in PSI polypeptides, we biotinylated the PSI complexes and detected the protein modification using avidin peroxidase and a chromo- Table II. N-terminal sequence analyses of the proteolytic fragments produced by extensive cleavage of PSI complexes by Clu-C as described in Figure 2 Proteolytic Apparent Fragment Mass" Sequencing Cycles Identification and Position of the First Amino Acid kD Fl 13.3 DDFANL Dl Fll" 9.8 9.0 TELSG DDFAN DM 6.4 6.2 4.4 3.8 3.5 2.5 KYAIT VQYLH GENLL AQGTK QCLAL ALNRG DIM DIV DV DVI Elllc 'DDFANL of mature PsaF (Chitnis and Nelson, 1991) 2 TELSG of PsaD (Reilly et al., 1988) 'DDFANL of mature PsaF (Chitnis and Nelson, 1991) "KYATT of PsaD (Reilly et al., 1988) '"VQYLH of PsaD (Reilly et al., 1988) "GENLL of PsaD (Reilly et al., 1988) ""AQGTK of PsaD (Reilly et al., 1988) "QCLAL of PsaD (Reilly et al., 1988) 2 ALNRG of PsaE (Chitnis et al., 1989a) b ' Average of three independent estimates from the electrophoretic mobility. To facilitate proper identification of Fll, PSI complexes from a PsaE-less mutant strain (Chitnis et al., 1989a) were c digested under the same condition as for the wild-type PSI. The fragment EIII migrated below 2.9-kD insulin marker and its apparent mass was calculated by extrapolation of the calibration curve. Downloaded from on August 2, 2017 - Published by www.plantphysiol.org Copyright © 1994 American Society of Plant Biologists. All rights reserved. 621 Topology of PSI Subunits DISCUSSION PsaA-PsaB PuDPsaFPsaL- El P«aK- Figure 4. PsaE modification by NHS-biotin and cleavage with CluC. The biotinylated PSI complexes (lane 1) were cleaved with CluC at concentration of 2 /ig/mg Chl for 20 min to yield the PsaE fragments lacking the C terminus (lane 2). The preparations equivalent to 10 Mg of Chl were solubilized and the proteins were separated by Tricine-urea-SDS-PACE, electroblotted on Immobilon-P membranes, and stained with Coomassie blue. A replica blot of lanes 1 and 2 was probed with avidin-peroxidase conjugate (lanes 3 and 4). The immunoreaction was visualized using hydrogen peroxide and 4-chloro-l-naphthol. genie substrate for peroxidase. PsaA-PsaB, PsaD, PsaF, PsaL, and PsaE showed significant amounts of biotin incorporation (Fig. 4). When biotinylated PSI was subjected to GIu-C cleavage to generate El and EII fragments (Fig. 4, lanes 1 and 2), most the biotin-avidin peroxidase-generated signal from PsaE was lost upon its cleavage (Fig. 4, lane 4). There is a lysyl residue (Lys74) at the C terminus of PsaE (Chitnis et al., 1989). Based on the difference in biotinylation intensity of PsaE and its proteolytic products El and EII, Lys74 is the most likely NHS-biotin reactive site on PsaE. The treatment of biotinylated PSI with higher concentration of Glu-C produced the DI fragment of PsaD (Fig. 5). A comparison of the biotinylation-dependent signal and the Coomassie blue staining intensity showed that most of the biotinylation signal in the intact PsaD was not present in the DI fragment (Fig. 5, lane 4). Four lysyl residues (Lys107, Lys118, Lys132, and Lys136) in the C-terminal 6-kD domain of PsaD (Reilly et al., 1988) are removed by Glu-C to generate the DI fragment (Table III). Therefore, one or more of these residues are exposed to the aqueous phase and can be biotinylated. To identify modified lysyl residues in PsaD more accurately, we treated the biotinylated PSI with thermolysin. A 13.8-kD fragment was generated by thermolysin, albeit to a lesser level than the Glu-C-generated fragments (Fig. 6). Thermolysin cleaves PsaD toward its C terminus to generate the 13.8-kD fragment containing Lys107 and Lys118 (Xu et al., 1994a). This fragment was labeled with NHS-biotin (Fig. 6, lane 4), indicating that Lys107, Lys118, or both could react to NHS-biotin. Chemical cross-linking, protease accessibility, and in vitro reconstitution experiments have revealed that PsaD, PsaE, and PsaC are exposed on the n-side of photosynthetic membranes and that a considerable part of PsaC is buried under PsaD and PsaE (Oh-oka et al., 1989; Zilber and Malkin, 1992), PsaF is substantially exposed on the p-side of the photosynthetic membranes (Wynn and Malkin, 1988; Hippler et al., 1989; Wynn et al., 1989a, 1989b). Here we examined the surface exposure of PsaD, PsaE, and PsaF in a topological assessment of PSI. For this purpose we used proteolytic cleavage and chemical modification experiments that probe different reactive groups with different levels of accessibility from the aqueous phase. Glu-C, a 34-kD protease, recognizes peptide bonds that are C terminal to glutamyl residues, and NHS-biotin, a smaller molecule, recognizes primary amines in a protein. The solution conformation of Synechococcus sp. PCC 7002 PsaE, proposed from NMR analysis, reveals that the protein has an antiparallel five-stranded /3-sheet structure (Falzone et al., 1994a, 1994b). Because of high sequence identity among cyanobacterial PsaE, the structure of Synechococcus sp. PCC 7002 PsaE provides a reliable model for Synechocystis sp. PCC 6803 PsaE. There are five glutamyl residues at positions of 15, 29, 63, 65, and 67 in PsaE of Synechocystis sp. PCC 6803 (Chitnis et al., 1989). Among them, glutamyl residues at positions 15, 63, 65, and 67 are conserved between the PsaE subunits from the two cyanobacterial species. The peptide mapping of the major PsaE fragments (Table I) and 1 PsaA-PsaB Figure 5. PSI modification by NHS-biotin and cleavage with CluC. The biotinylated PSI complexes (lane 1) were cleaved with GluC (20 fig protease/mg Chl) for 20 min to yield the PsaD fragments lacking the C terminus (lane 2). The preparations equivalent to 10 ^g of Chl were solubilized and the proteins were separated by Tricine-urea-SDS-PACE, electroblotted on Immobilon-P membranes, and stained with Coomassie blue. A replica blot of lanes 1 and 2 was probed with avidin-peroxidase conjugate (lanes 3 and 4). The immunoreaction was visualized using hydrogen peroxide and 4-chloro-1-naphthol. Downloaded from on August 2, 2017 - Published by www.plantphysiol.org Copyright © 1994 American Society of Plant Biologists. All rights reserved. Xu et al. 622 Plant Physiol. Vcd. 106, 1994 Table 111. Predicted fragments of PsaE, PsaD, and PsaF after complete digestion with Clu-C These analyses are based on t h e deduced amino acid sequences of psaE ((Chitniset al., 1989a), psaD (Reilly et al., 1988), and psaF (Chitnis and Nelson, 1991). Predicted CIu-C Fragment PsaE fragments 'ALN RC DKVSlKRT' 5E 16SYWYG DVCTVASVZ9E "KSG I LYPVIVRFDRVNY NC FSCSASGVNTNNFA63E 6 4 ~ 6 5 ~ 6 6 ~ 6 7 ~ 68LVQAAA74K PsaD Fragments 'T3E 4LSCQPPKFGCSTGCLLSKAN RZ5E 26E 27KYAITWTSAS37E 38QVF4'E 42M PTCGAAIMNs2E 53GE54 55NLLYLARK63E MQCLALGTQLRTKFKPKlQDYKIYRVYPSG93E 94VQYLHPADCVFPlo6E 1 0 7 ~ ~ ~ 1 1 0 ~ 111GR113E 114AQGTKTRRICNP127E PVTlKFSGKAPY14"E 1 4 1 v PsaF Fragments DDFANLTPTSE" 1ZNPAYLAKSKNFLNTTNDPNSGKIRAE37 "RYASALCC PE47 48CYPH LlVDGRFTHACDFLlPSILFLYIAGW IGWVGRSYLIEB8 891RE91 92SKNPEq6 97MQEq9 '%I NVPLAlKKMLCCFLW PLAAVC E' 24 '25YTSCKLVMKDSE' 36 '371PTSPR142 predicted Glu-C fragments of PsaE (Table 111) indicate that the primary Glu-C cleavage sites may be in a cluster of Glu residues at positions 63, 65, and 67. Examination of the structure of Synechococcus sp. PCC 7002 PsaE revealed that the C-terminal peptide bonds of Glu"' and G1d6(corresponding to G l d 3 and G l d 7 in Synechocystis sp. PCC -6803 PsaE), but not of G l d 4 (corresponding to G l d 5 in Synechocystis sp. PCC 6803 PsaE), are accessible from the surface. Therefore, GW3 and G l d 7 in Synechocystis sp. PCC 6803 PsaE are the most likely sites of primary cleavage by Glu-C. The remaining Glu residues in PsaE are not readily accessible to Glu-C and therefore may be shielded by the other PSI subunits. The loop of PsaE, located opposite the surface-exposed Gld3GhP7 region, varies in length depending on the source of PsaE and may anchor PsaE in the PSI complex. Interestingly, a 2.5-kD PsaE fragment with N termini of PsaE was accumulated when the PSI complexes were extensively degraded by Glu-C. This implies that the Glu15, which is near the Nterminal domain of PsaE, may be relatively shielded from Mass Mass from the N Terminus kD kD 1.585 1.531 3.690 0.261 0.260 0.699 1.585 3.116 6.806 7.067 7.327 8.026 0.248 2.199 O. 147 1.255 0.521 1.O90 0.204 1.118 3.541 0.248 2.447 2.594 3.849 4.370 5.460 5.664 6.782 10.323 1.470 0.488 0.360 1.554 1.435 0.117 11.793 12.281 12.641 14.195 15.630 15.747 1.210 2.861 1.O65 4.659 0.41 6 0.573 0.406 2.633 1.356 0.669 1.210 4.071 5.136 9.795 10.21 1 10.784 11.190 13.823 15.179 15.848 Proposed Identification i IDV Dlll } FII FI aqueous phase. It is possible that proteolytic cleavage of the C-terminal domain of PsaD exposed the G h z 9of PsaE. Glutamyl residues in the C-terminal domain of PsaD are the primary cleavage sites for Glu-C (Table I). The C-terminal domain of PsaD is also accessible to thermolyfiin (Xu et al., 1994a). Similarly, the proteolytically cleaved P5;aD fragment with an N terminus identical to that of intact PsaD was found after chaotrope-induced release of peripheral proteins from purified PSI of Synechococcus sp. PCC 6301 (Li et al., 1991). When PSI complexes were extensively cleaved with Glu-C, Glu5' and G l d 3 were accessible. The peptide m,ipping of the observed PsaD fragments (Tables I and 11) and fragments predicted from the deduced amino acid sequerte (Table 111) indicated that G1ulo6,Glu"", and Glu113were readily accessible to Glu-C. Interestingly, Lys'07 of PsaD in Synechocystis sp. PCC 6803 can be cross-linked with Glu9,' of Fd by a hydrophilic, zero-length cross-linker, N-ethyl-3-(3-dimethylaminopropy1)carbodiimide (Lelong et al., 1994). The glutamyl residues in the C-terminal domain of PsaF Downloaded from on August 2, 2017 - Published by www.plantphysiol.org Copyright © 1994 American Society of Plant Biologists. All rights reserved. Topology of PSI Subunits 1 2 3 PuA-PsaB PsaD PsaF PsaL PsaD fragment Figure 6. PSI modification by NHS-biotin and cleavage with thermolysin. The biotinylated PSI complexes (lane 1) were cleaved with thermolysin (20 jig protease/mg Chl) for 20 min to yield the PsaD fragments lacking the C terminus (lane 2). The preparations equivalent to 10 Mg of Chl were solubilized and the proteins were separated by Tricine-urea-SDS-PACE, electroblotted on Immobilon-P membranes, and stained with Coomassie blue. A replica blot of lanes 1 and 2 was probed with avidin-peroxidase conjugate (lanes 3 and 4). The immunoreaction was visualized using hydrogen peroxide and 4-chloro-1-naphthol. were also accessible to Glu-C. The 13.3- and 9.0-kD PsaF fragments with intact N termini gradually accumulated when PsaE and PsaD had been cleaved by Glu-C. Based on the apparent masses of these proteolytic fragments and deduced amino acid sequence (Chitnis et al., 1991), Glu124 and GIu88 were the likely cleavage sites. A hydrophobic domain preceding Glu124 is 20 amino acids in length and may span a membrane. Although chemical cross-linking and functional reconstitution studies have demonstrated that PsaF is exposed to the lumenal side (Hippler et al., 1989; Wynn et al., 1989a, 1989b), several lines of evidence suggest that PsaF is an integral membrane protein. First, the amino acid sequences of PsaF deduced from the gene sequences reveal two moderately hydrophobic regions in this protein (Chitnis et al., 1991). Second, PsaF resists removal from wild-type PSI by chaotropic agents (Xu et al., 1994a, 1994b). However, it could be released from PSI core upon exposure to a high concentration of detergent (Bengis and Nelson, 1977). Third, a crosslinked product that is recognized by both anti-PsaE and antiPsaF antibodies can be obtained by treatment of PSI with glutaraldehyde, showing that PsaE and PsaF are in close proximity (T.S. Armbrust, J.A. Guikema, unpublished data). PsaF is synthesized as a precursor protein with a transit sequence typical of proteins targeted to the thylakoid lumen (Chitnis et al., 1991), suggesting that PsaF is positioned in the membranes with its N terminus on the lumenal side and its C-terminal hydrophilic domain on the n-side of the membranes. Thus, it is possible that extensive cleavage of the nside components of PSI, most notably PsaE and PsaD, expose 623 the C-terminal domain of PsaF. Alternatively, the C-terminal domain of PsaF may be shielded by PsaA-PsaB loops that are degraded after extensive proteolysis. There is an apparent difference in the sensitivity of PSI subunits to proteolysis. PsaE was selectively cleaved by GluC before detectable degradation of PsaD, PsaC, or other PSI subunits (Fig. 1). A similar sensitivity pattern of the n-side components of PSI to extraction by Nal has been observed (Xu et al., 1994a, 1994b). Upon treatment of PSI complexes with Nal, a significant amount of PsaE was released before removal of PsaD and PsaC. The absence of PsaE leads to an enhanced cleavage of PsaD by thermolysin (Xu et al., 1994a). Both PsaD and PsaE are required for efficient Fd-mediated NADP+ photoreduction by PSI (Xu et al., 1994a). PsaD can also be chemically cross-linked with Fd (Merati and Zanetti, 1987; Zilber and Malkin, 1988; Lelong et al., 1994). Thus, PsaD and PsaE may partially overlap and form the docking site for the electron acceptors of PSI. These functionally important docking domains of PsaD and PsaE are expected to be surface exposed. Hence, the domains of PsaD and PsaE that were identified in this study may form the Fd-docking region on the reducing side of PSI. ACKNOWLEDGMENTS We acknowledge John Tomich and Gary Radke for their valuable advice about N-terminal amino acid sequencing. We thank Vaishali P. Chitnis for expert technical assistance and William R. Odom for critically reading the manuscript. We also thank Juliette T.J. Lecomte for providing structural coordinates of PsaE before publication. Received May 23, 1994; accepted July 1, 1994. Copyright Clearance Center: 0032-0889/94/106/0617/08. LITERATURE CITED Arnon D (1949) Copper enzyme in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol 24: 1-14 Bayer EA, Wilchek M (1990) Protein biotinylation. Methods Enzymol 184: 138-152 Bengis C, Nelson N (1977) Subunit structure of chloroplast photosystem I reaction center. J Biol Chem 252: 4564-4569 Bryant D (1992) Molecular biology of photosystem I. In} Barber, ed, The Photosystems: Structure, Function and Molecular Biology. Elsevier, Amsterdam, pp 501-549 Chitnis PR, Nelson N (1991) Photosystem I. In L Bogorad, IK Vasil, eds, The Photosynthetic Apparatus: Molecular Biology and Operation. 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Plant Physiol 103 171-180 Zanetti G, Merati G (1987) Interaction between photosystem I and ferredoxin:identification by chemical cross-linking(of the polypeptide which binds ferredoxin. Eur J Biochem 169 143-146 Zilber A, Malkin R (1988)Ferredoxin cross-links to a 22 kD subunit of photosystem I. Plant Physiol 88: 810-814 Zilber AL, Malkin R (1992) Organization and topology of photosystem I subunits. Plant Physiol99 901-911 Downloaded from on August 2, 2017 - Published by www.plantphysiol.org Copyright © 1994 American Society of Plant Biologists. All rights reserved.