Organization of Photosystem I Polypeptides'

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, James A. 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 complexes with 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; Gol- beck, 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, FA and FB, each a [4Fe- 451 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 acknowl- edge 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.

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 recon- stitution 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-2- fold 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 characterized the 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 cyanobacterial mutants 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- -

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1058 Xu et al. Plant Physiol. Vol. 106, 1994

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 stabi- lizing association of PsaD and PsaL with the PSI core.

MATERIALS A N D METHODS

Preparation of Photosynthetic Membranes and PSI Complexes

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, respec- tively. 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.

Treatment of PSI Complexes with Na1 and Thermolysin

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-1 O0 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.

Cross-Linking of PSI Subunits

Purified wild-type PSI complexes at a concentration of 150 Fg Chl/mL were-treated with 15 m~ glutaraldehyde (Sigma) in the presence of 10 mM Mops-HC1 (pH 7.0) and 0.05%

Triton X-100 for 30 min on ice except as otherwise indicated. Glutaraldehyde was stored at -2OOC prior to use. The cross- linking 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

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 (Amer- sham). Following preparative Tricine-urea-SDS-PAGE, gel strips containing PsaE, PsaF, and PsaL of PSI from Synecho- cystis 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.

RESULTS

Subunit Composition of PsaD-less PSI

We purified PSI complexes from the wild-type and PsaD- less 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 wild- type 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 cyanobacterial strain have drastically reduced ability to form trimers (Chitnis artd Chitnis, 1993). This suggested an easier loss of PsaL from the PsaD- less 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

Organization of PSI 1059

anti-PsaB anti-PsaD anti-PsaF anti-PsaEFigure 1. Western blotting of purified PSI complexes in the wild-type (WT) and ADC4 strains. Subunits of PSI complexes containing10 ng of Chl were separated by Tricine-urea-SDS-PACE, blotted toImmobilon-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 poly-peptide that matched the position of the PsaL polypeptide inthe wild-type but not in the PsaL-less PSI complexes (Fig.2A, lanes 3 and 4). Western blot analysis of purified PsaD-less PSI complexes revealed a drastically reduced level ofPsaL in PSI of the ADC4 strain (Fig. 2B). To estimate theapproximate level of PsaL in PsaD-less PSI, we comparedimmunoreactivity of PsaL in the ADC4 PSI to that in a 20%amount (on Chl basis) of wild-type PSI. PsaD-less PSI ofADC4 strain contained far less than 20% of the wild-typelevel of PsaL (Fig. 2B). In contrast, the membranes of thewild-type and ADC4 strains contained similar levels of PsaL(Fig. 2C). These data suggest that an interaction betweenPsaD and PsaL is crucial for maintaining the association ofPsaL with the PSI core during PSI purification.

Removal of PsaD by Nal in PsaL-less PSI

To investigate further the interaction between PsaD andPsaL, we examined the association of PsaD in the PsaL-lessPSI complex. The membranes of the wild-type and PsaL-lessstrains contain similar levels of PsaD (Chitnis et al., 1993).The purified wild-type and PsaL-less PSI complexes also hadsimilar levels of PsaD, as estimated by Coomassie blue stain-ing (Fig. 3). The availability of defined PSI complexes and animproved Tricine-urea-SDS-PAGE system provided a suita-ble system in which to study the organization of PSI usingsusceptibility to protease cleavage and to chaotropic extrac-tion as the criteria to analyze subunit interactions. In thewild-type PSI complex, there was a differential sensitivityamong PSI subunits to thermolysin (Fig. 3). Incubation ofwild-type PSI with thermolysin resulted in a progressivecleavage of the PsaA-PsaB polypeptides. PsaD, PsaL, and

PsaC were largely resistant to proteolytic cleavage. It wasdifficult to estimate the sensitivity of PsaF to cleavage bythermolysin because an unidentified proteolytic degradationproduct comigrated with the intact PsaF (Xu et al., 1994b).PsaA-PsaB in the PsaL-less PSI complexes had increasedsusceptibility to thermolysin cleavage, possibly because ofthe increased exposure of these subunits caused by the ab-sence of PsaL. In contrast, the other subunits in the mutantcomplexes showed similar susceptibility to the protease as inthe wild type (Fig. 3). The level of PsaD in the PsaL-less aswell as wild-type PSI declined slightly and to a similar extent.In contrast, PsaD is more susceptible to thermolysin cleavagein the PsaE-less PSI (Xu et al., 1994a). Therefore, PsaLdomains exposed on the n-side of the membranes do notshield PsaD from proteases as much as does PsaE.

When the wild-type PSI complexes were exposed to in-creasing concentrations of Nal, PsaC, PsaD, and PsaE werereleased from the PSI complexes in a concentration-depend-ent fashion (Fig. 4). Complete removal of these peripheralcomponents can also be achieved upon treatment of PSI with

PsaA-B-

Figure 2. Western blot analysis of PsaL in purified PSI and photo-synthetic membranes. A, Polypeptides of purified wild-type (lanes1 and 3) and ALC7-3 (lanes 2 and 4) PSI complexes equivalent to10 Mg of Chl were separated by Tricine-urea-SDS-PAGE and trans-ferred to Immobilon-P membranes. The proteins were visualizedby Coomassie blue staining (lanes 1 and 2) or probed using an anti-PsaL antibody (lanes 3 and 4). The antigen-antibody reaction wasvisualized by using a horseradish peroxidase-conjugated secondaryantibody and an enhanced chemiluminescence detection system.B, The proteins from purified PSI complexes of wild type (10 Mg ofChl in lane 1), ADC4 (10 Mg of Chl in lane 2), and wild type (2 Mgof Chl in lane 3) were resolved using Tricine-urea-SDS-PACE andprobed with an anti-PsaL antibody. Immunodetection was per-formed as described in A. C, The proteins from photosyntheticmembranes of the wild type (lane 1) and ADC4 (lane 2) containing10 Mg of Chl per lane were fractionated using Tricine-urea-SDS-PACE and probed with an anti-PsaL antibody. Immunodetectionwas performed as described in A.


Wild type ALC7-3

0 5 2 0 4 0 6 0 0 5 2 0 4 0 6 0 Time (min)

PsaA-

PuD-Pi«F-PuL-

P«E_PwC

Figure 3. Digestion of wild-type and PsaL-less PSI by thermolysin.The wild-type and PsaL-less PSI complexes were incubated withthermolysin for 0, 5, 20, 40, and 60 min. The thermolysin-cleavedPSI complexes equivalent to 10 ̂ g of Chl per lane were solubilized,and proteins were separated by Tricine-urea-SDS-PAGE. Proteinswere 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 hydropho-bic 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 wasexposed to 2 M Nal, all PsaD was removed from the PSI core.In contrast, only approximately 50% of PsaD in the wild-type PSI was removed by the same concentration of Nal (Fig.4). Complete removal of PsaD in the wild-type PSI could beachieved only after incubation with 3 M Nal (Fig. 4). PsaE inPsaL-less PSI was also more susceptible for removal by Nal,most likely because of the easier loss of PsaD. Incubationwith Nal more readily removes PsaE from the PsaD-lessmembranes (Cohen et al., 1993). PsaC was equally suscepti-ble to removal by Nal in the wild-type and PsaL-less PSIcomplexes. PsaF and several low-mass polypeptides, such asPsaK, PsaJ, and PsaM in the PsaL-less PSI were resistant tochaotropic extraction with up to 3 M Nal. The easier removalof PsaD from the PsaL-less mutant may result from a weakerassociation 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 andenhanced susceptibility of PsaD to removal by a chaotropein the PsaL-less PSI suggest that an interaction between PsaDand PsaL is important to their stable assembly within PSI.These results imply, but do not demonstrate, direct physicalcontacts between these two proteins. Therefore, we examinedthe physical proximity of these subunits in PSI complexes bychemical cross-linking. The zero-length cross-linker 1-ethyl-

3(3-dimethylaminopropyl)-carbodiimide has been effectivelyused to cross-link native electron donors or acceptor proteinsto PSI subunits (Zanetti and Merari, 1987; Wynn and Malkin,1988; Wynn et al., 1989). It, however, caused no significantcross-linking among PSI subunits at concentrations of lessthan 100 mM (data not shown).

Here we used glutaraldehyde, a bifunctional cross-linkingreagent, that reacts principally with amino groups of lysylresidues (Mclntosh, 1992). Because higher concentrations ofglutaraldehyde may have unspecific cross-linking effects, wefirst optimized conditions that result in intermolecular cross-linking within a complex without forming intercomplex cross-links or excessive protein aggregates. When the wild-type PSIwas treated with increasing concentrations of glutaraldehydeat 4°C, increasing amounts of two major cross-linked prod-ucts with apparent molecular masses of 29 and 25 kD wereformed (Fig. 5A). At a higher glutaraldehyde concentration(25 mM), we observed high-mass protein aggregates and anoverall decrease in the relative amounts of PsaD, PsaF, PsaL,PsaE, and PsaK. In contrast, the low-mass subunits, such asPsal, PsaJ, and PsaM, resisted cross-linking. Interestingly,accumulation of the 25-kD species increased, whereas thatof the 29-kD species decreased, when the glutaraldehydeconcentration increased from 1 to 25 mM. Glutaraldehydetreatment at 24°C resulted in an enhanced accumulation ofcross-linked products; this is expected from the elevated ratesof the cross-linking reactions at higher temperatures (Kiehmand Ji, 1977).

To determine the contribution of intracomplex cross-link-ing relative to intercomplex cross-linking at 4°C, we treatedthe wild-type PSI complexes at 50, 150, and 250 /*g Chl/mLwith 15 mM glutaraldehyde (Fig. 5B). Analyses of cross-linkedPSI samples by Tricine-urea-SDS-PAGE showed no apparent

Wild type ALC7-3

0 1 0 1 MNal1

PsaA-B —

PsaDPsaFPsaL

PsaE-PsaC-PsaK —

PsalPsaJ

PsaM-

Figure 4. Accessibility of subunits in PSI complexes to removal byNal. The PSI complexes were exposed to 0, 1, 2, or 3 M Nal for 30min on ice, followed by desalting and removal of salt-extractedproteins in a Centricon-100. The proteins from PSI complexescontaining 4 /ig of Chl per lane were denatured and separated byTricine-urea-SDS-PACE. The polypeptides were visualized by silverstaining.


Temperature 4'C 25T

BISO 50 100 250 ngChl/mL

mM glutaraldehyde 0 1 5 25 1

PsaA-PsaB- |

29kD-25 kD-

PsaD-PsaF-PsaL-

PsaEPsaCPsaK

Psal

PsaM

0 15 15 15 mM glutaraldehyde

-29kD25 kD

Figure 5. Cross-linking of subunits in PSI complexes. A, The wild-type PSI complexes were exposed to 0, 1, 5, and 25mM glutaraldehyde for 30 min on ice or at 25°C, followed by termination of cross-linking reaction with addition of Clyand filtration in a Centricon-100. The proteins from PSI complexes containing 5 /xg of Chl per lane were denatured andseparated by Tricine-urea-SDS-PAGE. The polypeptides were visualized by silver staining. B, The wild-type PSI complexeswere adjusted to 50, 100, 150, or 250 Mg/mL concentration and then treated with 15 mM glutaraldehyde for 30 min onice. After termination of the cross-linking reaction with addition of Cly and filtration in a Centricon-100, the proteinsfrom PSI complexes containing 10 jig of Chl per lane were denatured and separated by Tricine-urea-SDS-PACE. Thepolypeptides were visualized by Coomassie blue staining.

CB Staining anti-PsaD anti-PsaL

mM glutaraldehyde 0 15 0 IS 0 15

PsaA-PsaB -

29 kD25 kD

PsaK

Figure 6. Identification of the 29-kD cross-linked species by west-ern blotting. The wild-type PSI complexes were exposed to 0 or 15mM glutaraldehyde for 30 min on ice, followed by termination ofthe cross-linking reaction and Centricon-100 ultrafiltration. Theproteins from PSI complexes containing 10 n%oi Chl per lane weredenatured and separated by Tricine-urea-SDS-PACE. The polypep-tides were transferred to Immobilon-P membranes and visualizedby Coomassie blue (CB) staining or probed with anti-PsaD and anti-PsaL antibodies. The blot was first probed with anti-PsaD antibody.Subsequently, the membrane was stripped of the bound antibodiesin 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-linkedPsaD and PsaL species.

difference in the amounts and number of the cross-linkedproducts (Fig. 5B). Cross-linking within a PSI complex is notexpected to depend on the PSI concentration, whereas cross-linking between two mobile PSI complexes would increasewith higher concentrations of PSI during the glutaraldehydetreatment (Kiehm and Ji, 1977). The data in Figure 5B sug-gested that the 25- and 29-kD products resulted from cross-linking between proteins within a PSI complex.

To test whether the interaction between PsaD and PsaLleads to the formation of a cross-linked product betweenthem, we used western blotting to detect the presence ofthese polypeptides in the cross-linked proteins (Fig. 6). Whenthe PSI complexes were treated with 15 mM glutaraldehyde,29- and 25-kD cross-linked species were formed (Fig. 6). Theanti-PsaD antibodies recognized both 25- and 29-kD speciesas well as the non-cross-linked PsaD. When the blot wasreprobed, an anti-PsaL antibody recognized the 29-kD butnot the 25-kD cross-linked product (Fig. 6). Thus, the 29-kDspecies was composed of intramolecular cross-linking be-tween PsaD (16 kD) and PsaL (14 kD). The 29-kD PsaD-PsaL cross-linked product could also be detected by westernblotting using anti-PsaD and anti-PsaL antibodies when thewild-type photosynthetic membranes were treated with 15mM glutaraldehyde (data not shown). The 25-kD species wasalso recognized by antibodies against PsaE and PsaC, indi-cating cross-linking between PsaD and PsaE or between PsaDand PsaC (data not shown).

DISCUSSION

Cyanobacterial PSI contains at least 11 polypeptides (Fig.5). Interactions among these polypeptides are not completelyunderstood. Biochemical studies have shown interactions


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 Suc- gradient 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 predom- inantly 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). Glutaralde- hyde may cross-link one of these residues to PsaD.

The absence of PsaD or PsaL does not affect the steady- state level of the other subunit in the photosynthetic mem- bianes 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 N- terminal 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

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 anti- PsaL polyclonal antibody and Vaishali P. Chitnis for expert technical assistance.

Received June 13, 1994; accepted July 28, 1994. Copyright Clearance Center: 0032-0889/94/106/1057,’07.

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