conjugated to hemocyanin (Henry et al., 1992). For amino ... N-terminal se- quences were determined on an Applied Biosystems (Foster. City, CA) 477A ...
Plant Physiol. (1994) 106: 1057-1063
Organization of Photosystem I Polypeptides' A Structural Interaction between the PsaD and PsaL Subunits Qiang Xu, Trent S. Armbrust, JamesA. Cuikema, and Parag R. Chitnis*
Division of Biology, Kansas State University, Manhattan, Kansas 66506-4901
l h e wild-type, PsaD-less, and PsaL-less strains of the cyanobacterium Synechocystis sp. PCC 6803 were used to study subunit interactions in photosystem I (PSI). When the membranes of a PsaD-less strain were solubilized with Triton X-100 and PSI was purified using ion-exchange chromatography and sucrose-gradient ultracentrifugation, the PsaL subunit was substantially removed from the core of PSI, whereas other subunits, such as PsaE and PsaF, were quantitatively retained during purification. When the wild-type PSI was exposed to increasing concentrations of Nal, the PsaE, PsaD, and PsaC subunits were gradually removed, whereas PsaF, PsaL, PsaK, and PsaJ resisted removal by up to 3 M Nal. l h e absence of PsaL enhanced the accessibility of PsaD to removal by Nal. Treatment of the wild-type PSI complexeswith glutaraldehyde at 4'C resulted in a 29-kD cross-linked product between PsaD and PsaL. The formation of such cross-linked species was independent of PSI concentrations, suggesting an intracomplex cross-linking between PsaD and PsaL. Taken together, these results demonstrate a structural interaction between PsaD and PsaL that plays a role in their association with the PSI core.
PSI in cyanobacteria and chloroplasts is a multisubunit membrane-protein complex that catalyzes electron transfer from reduced plastocyanin (or Cyt c6) to oxidized Fd (or flavodoxin) (Chitnis and Nelson, 1991; Bryant, 1992; Golbeck, 1993). The PsaA and PsaB subunits of PSI form a heterodimeric core that harbors approximately 100 antenna Chl a molecules, the primary electron donor, P700, and a chain of electron acceptors, Ao, A,, and Fx. The PsaC subunit binds the terminal electron acceptors, FAand FB, each a [4Fe451 cluster. PsaD provides an essential Fd-docking site on the reducing side of PSI (Zanetti and Merati, 1987; Wynn et al., 1989; Xu et al., 1994a) and is also required for in vitro assembly of PsaC and PsaE into the PSI complex (Li et al., 1991b; Chitnis and Nelson, 1992). PsaE may be involved in Fd reduction (Sonoike et al., 1993; Strotmann and Weber,
' This work was supported in part by grants from the National Science Foundation (MCB 9202751 and MCB 9405325 to P.R.C.), the US. Department of Agriculture-National Research Initiative Competitive Grants Program (USDA-NRICGP) (92-37306-7661 to P.R.C. and 93-37306-9147 to J.A.G.),and the National Aeronautics and Space Administration (NAGW 2328 to J.A.G:).We also acknowledge an equipment grant from the USDA-NRICGP (93-3731 1-9456 to P.R.C.).This is contribution 94-486-J from the Kansas Agricultural Experiment Station. * Corresponding author; fax 1-913-532-6653. 1057
1993; Lelong et al., 1994; Xu et al., 1994a) and cyclic electron flow around PSI (Yu et al., 1993). PsaL is required for the formation of PSI trimers (Chitnis and Chitnis, 1993). PsaF is exposed to the p-side (luminal) of the photosynthetic membranes (Wynn and Malkin, 1988; Hippler et al., 1989) but is not necessary for cyt c6 docking (xu et al., 1994b). Other subunits, such as PsaJ, PsaK, PsaI, and PsaM, are conserved from cyanobacteria to higher plants (Ikeuchi et al., 1991, 1993), but their roles are yet to be identified. The exact organization of the 11 or more polypeptides, approximately 100 antenna Chls, and electron transfer centers in the 340-kD PSI complex will be best understood from x-ray diffraction studies of PSI crystals. The PSI complex from Synechococcus elongatus has been crystallized, and a model for the three-dimensional structure at 6 A resolution has been proposed (Krauss et al., 1993). The electron density could be fitted to include the three [4Fe-4S] clusters Fx, FA, and FB, 28 (Y helices of proteins, and 45 Chl a molecules. Our understanding of the organization of PSI is also based on biochemical studies. In higher plants, specific domains of the PsaD, PsaE, and PsaL subunits are exposed to proteases (Zilber and Malkin, 1992). Cross-linking and in vitro reconstitution experiments have revealed that PsaD, PsaE, and PsaC are exposed on the n-side (stromal in chloroplasts and cytoplasmic in cyanobacteria), with a considerable part of PsaC buried under PsaD and PsaE (Oh-oka et al., 1989; Li et al., 1991b; Chitnis and Nelson, 1992). PsaC is positioned near the center of each monomeric PSI on a local pseudo-2fold axis of symmetry (Krauss et al., 1993; Kruip et al., 1993) Clearly, the detailed interactions among other PSI subunits remain largely unexplored. The cyanobacterium Synechocystis sp. PCC 6803 provides an attractive system to study the organization and function of PSI. We have cloned and characterizedthe genes that code for PsaD, PsaE, PsaF, PsaJ, PsaL, and PsaI subunits of PSI from Synechocystis sp. PCC 6803 and have subsequently generated mutants in which these genes have been interrupted or deleted (Chitnis et al., 1989a, 1989b, 1991, 1993; Xu et al., 1994b).This approach has allowed us to assess the functions of these subunits in vivo. At the same time, the availability of these cyanobacterialmutants provides a unique system to investigate protein interactions in PSI. In the present study, we isolated photosynthetic membranes and PSI complexes from the wild-type strain and the mutants that lack PsaD or PsaL and investigated their PSI organization. We also used chemical cross-linking - to examine the near-
Xu et al.
neighborhood relationship between PsaD and PsaL. These studies reveal a structural interaction between the PsaD and PsaL subunits of PSI. Such interaction plays a role in stabilizing association of PsaD and PsaL with the PSI core. MATERIALS A N D M E T H O D S Preparation of Photosynthetic Membranes and PSI Complexes
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Triton X-100 for 30 min on ice except as otherwise indicated. Glutaraldehyde was stored at -2OOC prior to use. The crosslinking reactions were quenched by the addition of Gly to a final concentration of 100 mM for 15 min. Subsequently, the samples were diluted with an excess of 10 mnn Mops-HC1 (pH 7.0) and filtered using Centricon-100 ultrafiltration. Analytical Gel Electrophoresis, Western Blotting, and Peptide Sequencing
A Glc-tolerant strain of Synechocystis sp. PCC 6803 was used as the wild type (Williams, 1988). The ADC4 (Cohen et al., 1993; Xu et al., 1994a) and ALC7-3 (Chitnis et al., 1993) strains were used as PsaD-less and PsaL-less strains, respectively. In these mutant stains, psaD or psaL is replaced or interrupted by a gene for chloramphenicol resistance. The ALC7-3 strain has completely functional PSI (Chitnis et al., 1993; Xu et al., 1994a), whereas the PsaD-less PSI is unable to reduce NADP' via Fd (Xu et al., 1994a). Cyanobacterial cultures were grown in BG-11 with or without Glc (5 m) and a selective antibiotic (30 pg chloramphenicol/mL) under a light intensity of 21 pmol m-' s-'. Cells were harvested at the late exponential phase of growth and were resuspended in 0.4 M SUC,10 mM NaCI, 200 PM PMSF, 5 mM benzamidine, and 10 mM Mops-HC1 (pH 7.0). Photosynthetic membranes were isolated using a bead beater (Chitnis and Chitnis, 1993). To isolate PSI, the membranes were solubilized with Triton X- 100, followed by DEAE-cellulose chromatography and Suc-gradient ultracentifugation (Reilly et al., 1988). The complexes purified by this procedure are suitable for analysis of PSI electron transport using native electron donors and acceptors (Xu et al., 1994a, 1994b). Chl concentrations were determined in 80% (v/v) acetone (Amon, 1949). The isolated membranes or PSI complexes were stored at -2OOC until needed.
PSI complexes and photosynthetic membranes were solubilized with 1% SDS and 10 mM 2-mercaptoethanol, and proteins were resolved by modified Tricine-urea -SDS-PAGE (Xu et al., 1994b). After electrophoresis, gels were stained with Coomassie blue or silver nitrate. Altemativdy, proteins were transferred to the polyvinylidine difluoride membranes (Immobilon-P; Millipore, Bedford, MA). Immunodetection was performed using enhanced chemilumineswnce (Amersham). Following preparative Tricine-urea-SDS-PAGE, gel strips containing PsaE, PsaF, and PsaL of PSI from Synechocystis sp. PCC 6803 were excised and used to immunize rabbits for production of antibodies against PsaII, PsaF, and PsaL. The antibody against PsaD was a kind gift of Dr. John H. Golbeck (University of Nebraska, Lincoln, NIi). The antibody against PsaB was raised using a C-terminal peptide conjugated to hemocyanin (Henry et al., 1992). For amino acid sequencing, polypeptides were blotted to Irnmobilon-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.
Treatment of PSI Complexes with Na1 and Thermolysin
Subunit Composition of PsaD-less PSI
To study the association of peripheral subunits with the PSI core, the PSI preparations were adjusted to 150 pg Chl/ mL and incubated with O, 1, 2, or 3 M Na1 for 30 min on ice. The samples were diluted with an excess amount of 10 m~ Mops-HC1 (pH 7.0), containing 0.05% Triton X-100, and desalted by ultrafiltration through a Centricon-1O0 apparatus (Amicon, Beverly, MA). This ultrafiltration also separates the PSI complex from the proteins released during incubation with NaI. To minimize experimental variables during the remova1 of proteins by NaI, the wild-type and mutant PSI complexes were treated identically during incubation with Na1 and subsequent desalting. For protease accessibility studies, PSI complexes isolated from wild-type and the ALC7-3 mutant strain (150 pg Chl/mL) were incubated with thermolysin (Sigma) at a concentration of 20 Pg protease/mg Chl in the presence of 5 mM CaC&at 37OC for 5, 20, 40, and 60 min. The reactions were terminated with 20 m~ EDTA.
We purified PSI complexes from the wild-type and PsaDless strains and compared their composition by westem blotting (Fig. 1).When normalized on an equal Chl basis, PsaB, PsaF, and PsaE were present at similar levels in the wildtype and PsaD-less PSI complexes. As expected, PsaD was absent in the ADC4 strain. When the PSI subunits were resolved by a Tricine-urea-SDS-PAGE and visudized with silver nitrate, we observed similar levels of the remaining small subunits in the wild-type and the PsaD-less PSI complexes (data not shown). Thus, analyses of subunit composition of PsaD-less PSI showed that the PSI coinplex was assembled in the absence of PsaD and could be isolated using the same procedure that was used for purification of the wild-type PSI. PsaL is essential for the formation of PSI timeis. The PSI complexes from a PsaD-less cyanobacterialstrain have drastically reduced ability to form trimers (Chitnis artd Chitnis, 1993). This suggested an easier loss of PsaL from the PsaDless membranes. To examine the relative amount of PsaL in the PsaD-less PSI, we generated a polyclonal antibody against PsaL. Tricine-urea-SDS-PAGE clearly resolved PsaL and other subunits of the wild-type PSI (Fig. 2A, lane 1).As expected, the PsaL subunit was absent in the PS[ from the
Cross-Linking of PSI Subunits
Purified wild-type PSI complexes at a concentration of 150 Chl/mL were-treated with 15 m~ glutaraldehyde (Sigma) in the presence of 10 mM Mops-HC1 (pH 7.0) and 0.05%
Organization of PSI
anti-PsaB anti-PsaD anti-PsaF anti-PsaE Figure 1. Western blotting of purified PSI complexes in the wildtype (WT) and ADC4 strains. Subunits of PSI complexes containing 10 ng of Chl were separated by Tricine-urea-SDS-PACE, blotted to Immobilon-P membranes, and probed with antibodies against PsaB, PsaD, PsaF, and PsaE.
ALC7-3 strain that lacked functional psal (Fig. 2A, lane 2). The anti-PsaL antibody specifically recognized a single polypeptide that matched the position of the PsaL polypeptide in the wild-type but not in the PsaL-less PSI complexes (Fig. 2A, lanes 3 and 4). Western blot analysis of purified PsaDless PSI complexes revealed a drastically reduced level of PsaL in PSI of the ADC4 strain (Fig. 2B). To estimate the approximate level of PsaL in PsaD-less PSI, we compared immunoreactivity of PsaL in the ADC4 PSI to that in a 20% amount (on Chl basis) of wild-type PSI. PsaD-less PSI of ADC4 strain contained far less than 20% of the wild-type level of PsaL (Fig. 2B). In contrast, the membranes of the wild-type and ADC4 strains contained similar levels of PsaL (Fig. 2C). These data suggest that an interaction between PsaD and PsaL is crucial for maintaining the association of PsaL with the PSI core during PSI purification. Removal of PsaD by Nal in PsaL-less PSI
To investigate further the interaction between PsaD and PsaL, we examined the association of PsaD in the PsaL-less PSI complex. The membranes of the wild-type and PsaL-less strains contain similar levels of PsaD (Chitnis et al., 1993). The purified wild-type and PsaL-less PSI complexes also had similar levels of PsaD, as estimated by Coomassie blue staining (Fig. 3). The availability of defined PSI complexes and an improved Tricine-urea-SDS-PAGE system provided a suitable system in which to study the organization of PSI using susceptibility to protease cleavage and to chaotropic extraction as the criteria to analyze subunit interactions. In the wild-type PSI complex, there was a differential sensitivity among PSI subunits to thermolysin (Fig. 3). Incubation of wild-type PSI with thermolysin resulted in a progressive cleavage of the PsaA-PsaB polypeptides. PsaD, PsaL, and
PsaC were largely resistant to proteolytic cleavage. It was difficult to estimate the sensitivity of PsaF to cleavage by thermolysin because an unidentified proteolytic degradation product comigrated with the intact PsaF (Xu et al., 1994b). PsaA-PsaB in the PsaL-less PSI complexes had increased susceptibility to thermolysin cleavage, possibly because of the increased exposure of these subunits caused by the absence of PsaL. In contrast, the other subunits in the mutant complexes showed similar susceptibility to the protease as in the wild type (Fig. 3). The level of PsaD in the PsaL-less as well as wild-type PSI declined slightly and to a similar extent. In contrast, PsaD is more susceptible to thermolysin cleavage in the PsaE-less PSI (Xu et al., 1994a). Therefore, PsaL domains exposed on the n-side of the membranes do not shield PsaD from proteases as much as does PsaE. When the wild-type PSI complexes were exposed to increasing concentrations of Nal, PsaC, PsaD, and PsaE were released from the PSI complexes in a concentration-dependent fashion (Fig. 4). Complete removal of these peripheral components can also be achieved upon treatment of PSI with
Figure 2. Western blot analysis of PsaL in purified PSI and photosynthetic membranes. A, Polypeptides of purified wild-type (lanes 1 and 3) and ALC7-3 (lanes 2 and 4) PSI complexes equivalent to 10 Mg of Chl were separated by Tricine-urea-SDS-PAGE and transferred to Immobilon-P membranes. The proteins were visualized by Coomassie blue staining (lanes 1 and 2) or probed using an antiPsaL antibody (lanes 3 and 4). The antigen-antibody reaction was visualized by using a horseradish peroxidase-conjugated secondary antibody and an enhanced chemiluminescence detection system. B, The proteins from purified PSI complexes of wild type (10 Mg of Chl in lane 1), ADC4 (10 Mg of Chl in lane 2), and wild type (2 Mg of Chl in lane 3) were resolved using Tricine-urea-SDS-PACE and probed with an anti-PsaL antibody. Immunodetection was performed as described in A. C, The proteins from photosynthetic membranes of the wild type (lane 1) and ADC4 (lane 2) containing 10 Mg of Chl per lane were fractionated using Tricine-urea-SDSPACE and probed with an anti-PsaL antibody. Immunodetection was performed as described in A.
Xu et al.
Wild type 0 5 2 0 4 0 6 0 0
ALC7-3 5 2 0 4 0 6 0
Figure 3. Digestion of wild-type and PsaL-less PSI by thermolysin. The wild-type and PsaL-less PSI complexes were incubated with thermolysin for 0, 5, 20, 40, and 60 min. The thermolysin-cleaved PSI complexes equivalent to 10 ^g of Chl per lane were solubilized, and proteins were separated by Tricine-urea-SDS-PAGE. Proteins were visualized with Coomassie blue.
6.8 M urea (Li et al., 1991a). In contrast, PsaF, PsaL, PsaK, PsaJ, and PsaM resisted removal from PSI by up to 3 M Nal. The amino acid sequences of these subunits have hydrophobic regions that are typical of transmembrane proteins (Scheller et al., 1989; Chitnis et al., 1993; Kjaerulff et al., 1993; Muhlenhoff et al., 1993). When the PsaL-less PSI was exposed to 2 M Nal, all PsaD was removed from the PSI core. In contrast, only approximately 50% of PsaD in the wildtype PSI was removed by the same concentration of Nal (Fig. 4). Complete removal of PsaD in the wild-type PSI could be achieved only after incubation with 3 M Nal (Fig. 4). PsaE in PsaL-less PSI was also more susceptible for removal by Nal, most likely because of the easier loss of PsaD. Incubation with Nal more readily removes PsaE from the PsaD-less membranes (Cohen et al., 1993). PsaC was equally susceptible to removal by Nal in the wild-type and PsaL-less PSI complexes. PsaF and several low-mass polypeptides, such as PsaK, PsaJ, and PsaM in the PsaL-less PSI were resistant to chaotropic extraction with up to 3 M Nal. The easier removal of PsaD from the PsaL-less mutant may result from a weaker association of PsaD with the PSI core in the absence of PsaL. Cross-Linking of PsaD and PsaL Subunits
The decreased level of PsaL in the PsaD-less PSI and enhanced susceptibility of PsaD to removal by a chaotrope in the PsaL-less PSI suggest that an interaction between PsaD and PsaL is important to their stable assembly within PSI. These results imply, but do not demonstrate, direct physical contacts between these two proteins. Therefore, we examined the physical proximity of these subunits in PSI complexes by chemical cross-linking. The zero-length cross-linker 1-ethyl-
Plant Physiol. Vol. 106, 1994
3(3-dimethylaminopropyl)-carbodiimide has been effectively used to cross-link native electron donors or acceptor proteins to PSI subunits (Zanetti and Merari, 1987; Wynn and Malkin, 1988; Wynn et al., 1989). It, however, caused no significant cross-linking among PSI subunits at concentrations of less than 100 mM (data not shown). Here we used glutaraldehyde, a bifunctional cross-linking reagent, that reacts principally with amino groups of lysyl residues (Mclntosh, 1992). Because higher concentrations of glutaraldehyde may have unspecific cross-linking effects, we first optimized conditions that result in intermolecular crosslinking within a complex without forming intercomplex crosslinks or excessive protein aggregates. When the wild-type PSI was treated with increasing concentrations of glutaraldehyde at 4°C, increasing amounts of two major cross-linked products with apparent molecular masses of 29 and 25 kD were formed (Fig. 5A). At a higher glutaraldehyde concentration (25 mM), we observed high-mass protein aggregates and an overall decrease in the relative amounts of PsaD, PsaF, PsaL, PsaE, and PsaK. In contrast, the low-mass subunits, such as Psal, PsaJ, and PsaM, resisted cross-linking. Interestingly, accumulation of the 25-kD species increased, whereas that of the 29-kD species decreased, when the glutaraldehyde concentration increased from 1 to 25 mM. Glutaraldehyde treatment at 24°C resulted in an enhanced accumulation of cross-linked products; this is expected from the elevated rates of the cross-linking reactions at higher temperatures (Kiehm and Ji, 1977). To determine the contribution of intracomplex cross-linking relative to intercomplex cross-linking at 4°C, we treated the wild-type PSI complexes at 50, 150, and 250 /*g Chl/mL with 15 mM glutaraldehyde (Fig. 5B). Analyses of cross-linked PSI samples by Tricine-urea-SDS-PAGE showed no apparent
PsaD PsaF PsaL PsaEPsaCPsaK — Psal PsaJ PsaMFigure 4. Accessibility of subunits in PSI complexes to removal by Nal. The PSI complexes were exposed to 0, 1, 2, or 3 M Nal for 30 min on ice, followed by desalting and removal of salt-extracted proteins in a Centricon-100. The proteins from PSI complexes containing 4 /ig of Chl per lane were denatured and separated by Tricine-urea-SDS-PACE. The polypeptides were visualized by silver staining.
Organization of PSI
mM glutaraldehyde 0
100 250 ngChl/mL
PsaA-PsaB- | 29kD-
PsaE PsaC PsaK Psal PsaM
Figure 5. Cross-linking of subunits in PSI complexes. A, The wild-type PSI complexes were exposed to 0, 1, 5, and 25 mM glutaraldehyde for 30 min on ice or at 25°C, followed by termination of cross-linking reaction with addition of Cly and filtration in a Centricon-100. The proteins from PSI complexes containing 5 /xg of Chl per lane were denatured and separated by Tricine-urea-SDS-PAGE. The polypeptides were visualized by silver staining. B, The wild-type PSI complexes were adjusted to 50, 100, 150, or 250 Mg/mL concentration and then treated with 15 mM glutaraldehyde for 30 min on ice. After termination of the cross-linking reaction with addition of Cly and filtration in a Centricon-100, the proteins from PSI complexes containing 10 jig of Chl per lane were denatured and separated by Tricine-urea-SDS-PACE. The polypeptides were visualized by Coomassie blue staining.
CB Staining anti-PsaD
PsaA-PsaB 29 kD 25 kD
Figure 6. Identification of the 29-kD cross-linked species by western blotting. The wild-type PSI complexes were exposed to 0 or 15 mM glutaraldehyde for 30 min on ice, followed by termination of the cross-linking reaction and Centricon-100 ultrafiltration. The proteins from PSI complexes containing 10 n%oi Chl per lane were denatured and separated by Tricine-urea-SDS-PACE. The polypeptides were transferred to Immobilon-P membranes and visualized by Coomassie blue (CB) staining or probed with anti-PsaD and antiPsaL antibodies. The blot was first probed with anti-PsaD antibody. Subsequently, the membrane was stripped of the bound antibodies in 100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCI (pH 6.7) at 50°C for 30 min. The blot was reprobed with anti-PsaL antibody. The blots were overlaid for proper identification of cross-linked PsaD and PsaL species.
difference in the amounts and number of the cross-linked products (Fig. 5B). Cross-linking within a PSI complex is not expected to depend on the PSI concentration, whereas crosslinking between two mobile PSI complexes would increase with higher concentrations of PSI during the glutaraldehyde treatment (Kiehm and Ji, 1977). The data in Figure 5B suggested that the 25- and 29-kD products resulted from crosslinking between proteins within a PSI complex. To test whether the interaction between PsaD and PsaL leads to the formation of a cross-linked product between them, we used western blotting to detect the presence of these polypeptides in the cross-linked proteins (Fig. 6). When the PSI complexes were treated with 15 mM glutaraldehyde, 29- and 25-kD cross-linked species were formed (Fig. 6). The anti-PsaD antibodies recognized both 25- and 29-kD species as well as the non-cross-linked PsaD. When the blot was reprobed, an anti-PsaL antibody recognized the 29-kD but not the 25-kD cross-linked product (Fig. 6). Thus, the 29-kD species was composed of intramolecular cross-linking between PsaD (16 kD) and PsaL (14 kD). The 29-kD PsaDPsaL cross-linked product could also be detected by western blotting using anti-PsaD and anti-PsaL antibodies when the wild-type photosynthetic membranes were treated with 15 mM glutaraldehyde (data not shown). The 25-kD species was also recognized by antibodies against PsaE and PsaC, indicating cross-linking between PsaD and PsaE or between PsaD and PsaC (data not shown). DISCUSSION Cyanobacterial PSI contains at least 11 polypeptides (Fig. 5). Interactions among these polypeptides are not completely understood. Biochemical studies have shown interactions
Xu et al.
among PsaD, PsaE, and PsaC proteins (Oh-oka et al., 1989). Characterization of subunit-deficient mutants have indicated interactions of PsaE with PsaF (Xu et al., 1994b). Here we present several lines of evidence that show a structural interaction between PsaD and PsaL in the organization of PSI. First, the absence of PsaD affected the association of PsaL with the PSI core (Fig. 2). The PsaD-less PSI complexes that had been purified by ion-exchange chromatography and Sucgradient ultracentrifugation contained a significantly reduced level of PsaL (Fig. 2) but maintained the wild-type level of other PSI subunits (Fig. 1).Thus, PsaD, which functions as a Fd-docking protein (Zanetti and Merati, 1987; Wynn et al., 1989), is also involved in the assembly or positioning of PsaL in the organization of PSI. Second, the absence of PsaL destabilizes the association of PsaD within the PSI complex as shown by enhanced susceptibility of PsaD to chaotropic extraction (Fig. 4). PsaL-less PSI is functional in vivo, in the membranes, and in isolated complexes (Chitnis et al., 1993; Xu et al., 1994a). Also, the absence of PsaL specifically increases susceptibility of PsaD to chaotropic removal. The absence of PsaE or PsaF-PsaJ does not influence the accessibility of PsaD to removal by Na1 (Xu et al., 1994a, 1994b). Therefore, the enhanced susceptibility of PsaD to chaotropic removal is specifically caused by the absence of PsaL and is less likely to be due to global changes in PsaL-less PSI structure. Third, PsaD and PsaL in the wild-type PSI complexes can be cross-linked under conditions in which predominantly intracomplex cross-linking occurred. The occurrence of cross-linking between PsaD and PsaL in the PSI complexes demonstrates a close association of PsaD and PsaL in organization of PSI. Initial attempts to identify the 29-kD species by N-terminal sequencing were unsuccessful. This may be due to a glutaraldehyde-dependent blockage of the N termini. Altematively, one or both N termini may be involved in cross-linking. Glutaraldehyde is a bifunctional cross-linking agent, reacting mostly with primary amines in lysyl residues (McIntosh, 1992). PsaL contains only two primary amines, the N terminus and Lys4’ (Chitnis et al., 1993). Glutaraldehyde may cross-link one of these residues to PsaD. The absence of PsaD or PsaL does not affect the steadystate level of the other subunit in the photosynthetic membianes of the subunit-specific mutant strains (Fig. 1; Chitnis et al., 1993). Therefore, the interaction between PsaD and PsaL may not be required for in vivo assembly of these subunits into PSI. However, their interaction may play a role in quatemary organization of PSI. Both PsaD and PsaL affect the ability of PSI to form trimers (Chitnis and Chitnis, 1993). Whereas PsaL is essential for the organization of monomers into trimeric PSI, the absence of PsaD leads to a low yield of trimers. PsaL has been proposed to form “the connecting domain” that joins individual PSI monomers to form a trimer (Chitnis and Chitnis, 1993). The structural interaction between PsaD and PsaL may be involved in integrating the connecting and the catalytic domains of PSI. Hydropathy analysis of PsaL suggests the presence of a hydrophilic Nterminal domain, followed by two potential transmembrane domains (Zilber and Malkin, 1992; Chitnis et al., 1993). A relatively large N-terminal domain of PsaL in stacked spinach thylakoids resists proteolysis and is predicted to be located on the stromal side (Zilber and Malkin, 1992). Therefore, the
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N-terminal domain of PsaL may interact with PsiiD and link the connecting domain to the catalytic domain in the PSI trimers. ACKNOWLEDGMENTS
We thank W.R. Odom for valuable help in the preparation of antiPsaL polyclonal antibody and Vaishali P. Chitnis for expert technical
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