©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Mutational Analysis of Photosystem I Polypeptides in the Cyanobacterium Synechocystis sp. PCC 6803
TARGETED INACTIVATION OF psaI REVEALS THE FUNCTION OF PsaI IN THE STRUCTURAL ORGANIZATION OF PsaL (*)

Qiang Xu , Dan Hoppe , Vaishali P. Chitnis , William R. Odom , James A. Guikema , Parag R. Chitnis (§)

From the (1)Division of Biology, Kansas State University, Manhattan, Kansas 66506-4901

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We cloned, characterized, and inactivated the psaI gene encoding a 4-kDa hydrophobic subunit of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803. The psaI gene is located 90 base pairs downstream from psaL, and is transcribed on 0.94- and 0.32-kilobase transcripts. To identify the function of PsaI, we generated a cyanobacterial strain in which psaI has been interrupted by a gene for chloramphenicol resistance. The wild-type and the mutant cells showed comparable rates of photoautotrophic growth at 25 °C. However, the mutant cells grew slower and contained less chlorophyll than the wild-type cells, when grown at 40 °C. The PsaI-less membranes from cells grown at either temperature showed a small decrease in NADP photoreduction rate when compared to the wild-type membranes. Inactivation of psaI led to an 80% decrease in the PsaL level in the photosynthetic membranes and to a complete loss of PsaL in the purified photosystem I preparations, but had little effect on the accumulation of other photosystem I subunits. Upon solubilization with nonionic detergents, photosystem I trimers could be obtained from the wild-type, but not from the PsaI-less membranes. The PsaI-less photosystem I monomers did not contain detectable levels of PsaL. Therefore, a structural interaction between PsaL and PsaI may stabilize the association of PsaL with the photosystem I core. PsaL in the wild-type and PsaI-less membranes showed equal resistance to removal by chaotropic agents. However, PsaL in the PsaI-less strain exhibited an increased susceptibility to proteolysis. From these data, we conclude that PsaI has a crucial role in aiding normal structural organization of PsaL within the photosystem I complex and the absence of PsaI alters PsaL organization, leading to a small, but physiologically significant, defect in photosystem I function.


INTRODUCTION

Photosystem (PS)()I from cyanobacteria and chloroplasts is a multisubunit membrane-protein complex that catalyzes electron transfer from reduced plastocyanin (or cytochrome c) to oxidized ferredoxin (or flavodoxin)(1, 2, 3, 4) . The PsaA and PsaB subunits of PS I form the heterodimeric core of the complex which harbors 100 antenna chlorophyll a, the primary electron donor (P700), and a chain of electron acceptors (A, A, and F). PsaC binds the terminal electron acceptors, F and F, which are two [4Fe-4S] centers. PsaD serves as a ferredoxin-docking site (5, 6, 7) and may also be required for in vitro assembly of PsaC and PsaE into the PS I complex(8, 9) . PsaE may also be involved in ferredoxin reduction (10-12) and cyclic electron flow around PS I(13) . PsaL is essential for the formation of PS I trimers(14, 15) . PsaF is exposed to the p-side (lumenal) of the photosynthetic membranes(16, 17) but is not necessary for cytochrome c docking (18). Other subunits, such as PsaJ, PsaK, PsaI, and PsaM, are conserved from cyanobacteria to higher plants(19, 20) , but their functions are not known.

The cyanobacterium Synechocystis sp. PCC 6803 provides an attractive system for studying the organization and function of PS I. Its PS I complex is structurally and functionally similar to that from higher plants, but its genome is simpler and can be more easily manipulated. Synechocystis sp. PCC 6803 can take up extraneous DNA and incorporate it into the genome by homologous recombination(21) , thus enabling one to mutate specific genes. We have embarked on a program to identify functions of the subunits of PS I through targeted mutagenesis of the genes encoding these subunits in Synechocystis sp. PCC 6803. Previously, we have cloned the genes that code for PsaD, PsaE, PsaF, PsaJ, and PsaL from Synechocystis sp. PCC 6803 and subsequently generated mutants in which these genes have been interrupted or deleted(14, 22, 23, 24, 25) . This approach has allowed us to demonstrate in vivo importance of these subunits in function and organization of PS I. In this paper, we describe molecular cloning, characterization, and targeted inactivation of psaI that codes for a 4-kDa hydrophobic protein of PS I. We investigated the role of PsaI in the function and structural organization of PS I using the PsaI-less strain.


EXPERIMENTAL PROCEDURES

Screening and Nucleotide Sequencing

Oligonucleotide probes corresponding to the amino-terminal residues of PsaI (19) were used to screen a genomic library of Synechocystis DNA in pBluescript II SK+/-. The oligonucleotides were phosphorylated using [-P]ATP by T4 polynucleotide kinase. Plasmid DNA was isolated from positive colonies by the alkaline lysis method and screened further by dot blots and Southern blots(26) . Both strands of a 0.7-kb Sau3A fragment containing the psaI gene were completely sequenced. Overlapping deletions in this fragment were generated by exonuclease III digestion (27) and their nucleotide sequences determined by the dideoxy termination method(28) . Nucleotide sequences were aligned and analyzed using GeneWorks software (Intelligenetics, Mountain View, CA).

Southern and Northern Analysis

Southern blotting was performed using 1 µg of Synechocystis genomic DNA for each restriction digestion. For Northern analysis, total cellular RNA was isolated from Synechocystis sp. PCC 6803, 10 µg of RNA was resolved by electrophoresis on a formaldehyde-containing agarose gel and then transferred to Magnacharge nylon membranes (Fisher Biotech). A 200-bp fragment containing the complete psaI was amplified by polymerase chain reaction, labeled with [-P]dCTP by random primer labeling, and used as a probe in Southern and Northern analyses. Hybridization analyses were performed using a rapid hybridization mixture (Amersham Corp.).

Targeted Mutagenesis of psaI

A mutant strain lacking psaI was generated by transforming the wild-type strain of Synechocystis sp. PCC 6803 with DNA of a clone (pK81C) in which psaI was interrupted by a gene conferring resistance to chloramphenicol (see ``Results'' for more details). Transformation was carried out according to previously described methods(21) . The transformants resistant to chloramphenicol were segregated for a few generations by a combination of single colony selection and growth in a liquid BG11 medium(14) . Interruption of psaI in the mutant strain AIC9 was confirmed by Southern analysis.

Characterization of Photosynthesis in the PsaI-less Strain

The wild-type and mutant AIC9 strain with inactive psaI were grown in BG-11 medium (21) with or without 5 mM glucose and 30 µg ml chloramphenicol under a light intensity of 21 µmol m s. Chlorophyll content in whole cells was determined according to Ref. 21. For high temperature growth experiments, the culture flasks were incubated in a 40 °C water bath and the cultures were constantly aerated by bubbling air through the medium. The growth of bacterial cultures was monitored by measuring the absorbance of the cultures at 730 nm. Rates of oxygen evolution or uptake were determined according to Ref. 14.

Chlorophyll to P700 ratios in the photosynthetic membranes were determined based on the oxidized-minus-reduced difference spectrum of P700(29) . Spectra of chlorophyll fluorescence emission at 77 K were measured with isolated photosynthetic membranes and PS I trimers. The samples were adjusted to 5 µg of chlorophyll (ml) in 60% (v/v) glycerol to minimize crystal fracturing and quickly cooled to 77 K in liquid N. The photosynthetic membranes and PS I trimers were positioned in a 430-nm excitation beam, and the fluorescence spectra at wavelengths 600-850 nm were monitored with a SPEX Fluorolog (SPEX Industries, Edison, NJ) equipped with a Dewar assembly. The rate of ferredoxin-mediated NADP photoreduction was measured using a modified Shimadzu spectrophotometer according to Ref. 12, except that both ferredoxin and cytochrome c used in the reaction mixture were obtained from Synechocystis sp. PCC 6803.

Isolation of Photosynthetic Membranes and PS I Complexes

Photosynthetic membranes from the wild-type and mutant cells were isolated and suspended in 0.4 M sucrose, 10 mM NaCl, 10 mM MOPS-HCl (pH 7.0)(14) . Chlorophyll concentrations were determined in 80% (v/v) acetone(30) . To isolate Triton X-100-solubilized PS I complexes, the membranes were solubilized with Triton X-100, and PS I was purified by DEAE-cellulose chromatography and sucrose-gradient ultracentrifugation(18) . To isolate PS I monomers and trimers, the photosynthetic membranes were solubilized with n-dodecyl -D-maltoside and fractionated by sucrose-gradient ultracentrifugation(15) . The PS I complexes purified by these procedures are suitable for analysis of PS I electron transport using native electron donors and acceptors(18) .

Cross-linking, Chaotropic Extraction, and Protease Treatment of PS I Subunits

Photosynthetic membranes in the wild-type and PsaI-less strains were adjusted to 150 µg of chlorophyll (ml) and were treated with 10 mM glutaraldehyde (Sigma) for 30 min on ice. Glutaraldehyde was stored at -20 °C prior to use. The cross-linking reactions were quenched by the addition of glycine to a final concentration of 10 mM for 15 min. Subsequently, the samples were diluted with an excess of 10 mM MOPS-HCl (pH 7.0) and pelleted. The membrane samples were analyzed by Western blotting. For chaotropic extraction, photosynthetic membranes of the wild-type and AIC9 strains were adjusted to 200 µg of chlorophyll (ml) and exposed to 0, 1, 2, and 3 M NaI for 30 min on ice. Subsequently, the samples were diluted with an excess of 10 mM MOPS-HCl (pH 7.0) and pelleted. The pellets were resuspended, washed once, and analyzed by Western blotting. For protease treatments, the wild-type and AIC9 membranes were incubated with chymotrypsin (Sigma) (1 mg of protease/mg of chlorophyll) at 37 °C for 0, 5, 20, and 40 min. The reactions were terminated by addition of phenylmethylsulfonyl fluoride (10 µM final concentration). PS I subunits in the protease-treated membranes were analyzed using Western blotting.

Overexpression of PsaI Protein and Production of Anti-PsaI Antibody

The psaI gene was amplified using the PC264 and PC267 primers that added EcoRI and EcoRV restriction sites at the beginning and end of the psaI open reading frame, respectively. The amplified fragment was digested with EcoRI and EcoRV restriction endonucleases and ligated into pGEX-KG vector that had been digested with EcoRI and EcoICRI enzymes(31) . The resultant clone contained psaI fused in-frame to the end of a gene that codes for glutathione S-transferase. The fused gene was expressed in Escherichiacoli, and inclusion bodies containing the fusion protein were isolated(31) . After solubilization of the inclusion bodies with SDS, the glutathione S-transferase-PsaI fusion protein was separated using preparative Tricine/urea/SDS-PAGE, gel strip was excised and used to immunize rabbits. Postimmune specificity and titer were determined by Western blotting.

Analytical Gel Electrophoresis and Western Blotting

Polypeptide composition of PS I was analyzed by modified Tricine/urea/SDS-PAGE(12) . After electrophoresis, gels were stained with Coomassie Brilliant Blue or silver nitrate. Alternatively, proteins were transferred to polyvinylidene difluoride (Immobilon-P) membranes (Millipore). Western blotting was performed using enhanced chemiluminescence reagents (Amersham). Polyclonal antibodies against Anacystisnidulans PsaA-PsaB were raised in rabbit(32) . Antibodies against PsaC and PsaD were from Dr. John H. Golbeck, University of Nebraska, Lincoln. Antibodies against PsaF, PsaL, and PsaE were generated against the respective proteins from Synechocystis sp. PCC 6803(25, 42) .


RESULTS

Molecular Cloning and Sequencing of psaI

We synthesized an oligonucleotide probe corresponding to the NH-terminal sequence of PsaI(19) . A genomic library was screened using a P-end-labeled probe and positive clones were isolated. Southern analysis of the pK81 plasmid indicated that the psaI gene resided in an 0.7-kb Sau3A fragment. In Southern blot analysis of genomic DNA, psaI-specific probe hybridized to a single band in all restriction digests (data not shown), indicating that psaI is present as a single copy in the genome of Synechocystis sp. PCC 6803.

The nucleotide sequence of the region within the 0.7-kb fragment containing the psaI gene revealed an open reading frame comprising psaI gene (Fig. 1). It is 120 bp long and encodes a protein of 40 amino acid residues. The amino acid sequence determined by chemical protein sequencing is found beginning at residue 1 of PsaI protein(19) . Therefore, the protein encoded by psaI is not post-translationally processed. In contrast, the product of psaI from another cyanobacterium, Anabaena variabilis ATCC 29413, contains an NH-terminal presequence(33) . Examination of the nucleotide sequence also revealed the presence of an open reading frame that started 183 bp downstream from psaI and continued beyond the cloned fragment. The protein encoded by this open reading frame was homologous to MurG protein of E. coli that functions as UDP-N-acetylglucosamine:N-acetylmuramyl-(pentapeptide)pyrophosphoryl-undecaprenol N-acetylglucosamine transferase involved in the membrane steps of peptidoglycan synthesis(34, 35) . Another open reading frame that codes for PsaL of PS I was found 90 bp upstream from psaI(14) .


Figure 1: Genomic region containing psaL and psaI of Synechocystis sp. PCC 6803. Restriction map of the region in the genome of Synechocystis sp. PCC 6803 that contains psaL and psaI is shown on the upper line. The lower line represents the Synechocystis sp. PCC 6803 DNA cloned in pK81 plasmid. Arrows indicate size and direction of open reading frames. Numbers indicate size of the DNA fragments (bp). The open reading frame corresponding to murG extends beyond the sequenced region shown in this figure.



Hydropathy analysis of the deduced amino acid sequence of PsaI indicated the presence of a 26-amino acid long hydrophobic domain flanked by hydrophilic amino and carboxyl termini (Fig. 2A). Comparison of PsaI from various sources showed that PsaI of Synechocystis has considerable homology to PsaI proteins from Synechococcus elongatus (65% identity)(36), A. variabilis (35% identity) (20), Marchatis polymorpha (50% identity)(37) , Aegilops crassa (33% identity) (GenBank/EMBL accession number X62118), Hordeum vulgaris (33% identity) (38), Nicotiana tabaccum (35% identity)(39) , and Oryza sativa (35% identity) (40) (Fig. 2B). The length and primary structure of the hydrophobic domain are highly conserved among PsaI proteins from cyanobacteria and higher plants.


Figure 2: Analysis of the deduced amino acid sequence of PsaI from Synechocystis sp. PCC 6803. A, hydropathy profile of the deduced amino acid sequence for PsaI was analyzed by the GeneWorks program using the Eisenberg algorithm (51) with a window size of 21 amino acids (solid line) or using Kyte-Doolittle algorithm (56) using a window size of 11 amino acids (broken line). B, comparison of deduced amino acid sequences of PsaI from various sources was performed using the GeneWorks program. Conserved hydrophobic domain is shaded. The presequence of the polypeptide encoded by psaI from A. variabilis is not shown. Sources for sequences: Synechocystis sp. PCC 6803 (this work), S. elongatus (36), A. variabilis (20), M. polymorpha (37), A. crassa (GenBank/EMBL with accession number X62118), H. vulgaris (38), N. tabaccum (39), and O. sativa (40).



Targeted Mutagenesis of psaI

Plasmid pK81 was treated with NcoI that has a unique site in psaI (Fig. 1), the DNA ends were made blunt using the Klenow fragment of DNA polymerase I, and a gene for chloramphenicol resistance was ligated to yield plasmid pK81C. In this plasmid, psaI is interrupted by the gene for chloramphenicol acetyltransferase which was isolated from plasmid pUC4C (14) after digestion with restriction endonuclease SmaI. The orientation of the resistance cassette with respect to psaI was determined to ensure that it would be transcribed in the same direction as psaL. The plasmid pK81C was used to transform the wild-type cells of Synechocystis sp. PCC 6803, and chloramphenicol-resistant transformants were selected and segregated to generate the AIC9 mutant strain. Fig. 3A shows the Southern blot analysis of genomic DNA from the wild-type and AIC9 strains. The genomic DNAs were digested completely with EcoRI or HindIII, transferred to nylon membranes, and hybridized with the [-P]dCTP-labeled DNA fragments containing the psaI gene. When digested with EcoRI, the probe for the PsaI-coding region hybridized with a 18-kb fragment in wild-type DNA but fragments of 15 and 3.2 kb were seen in the mutant DNA (Fig. 3A). Introduction of an additional EcoRI site in the mutant chromosome was expected since the gene for chloramphenicol acetyltransferase contains an EcoRI site. In HindIII-digested DNAs, the probe recognized a 3.0-kb fragment in wild-type DNA but a 4.4-kb fragment in the mutant DNA, as expected due to insertion of the 1.4-kb DNA cassette containing the gene for chloramphenicol resistance. Therefore the AIC9 strain contains only the interrupted psaI gene.


Figure 3: Characterization of the AIC9 strain of Synechocystis sp. PCC 6803. A, Southern blot of genomic DNA from the wild-type (WT) and AIC9 mutant strains. Genomic DNA was completely digested with EcoRI or HindIII and then electrophoresed on 0.75% agarose gel, transferred to a Magnacharge nylon membrane, and probed with [-P]dCTP-labeled probes specific for the psaI gene. B, Northern blot analysis of psaI from Synechocystis sp. PCC 6803. 10 µg of total RNA from the wild-type and AIC9 strains was isolated and subjected to electrophoresis in 1.2% agarose gel containing formaldehyde, transferred to a Magnacharge nylon membrane, and hybridized with a probe specific to psaI. In the wild type, the sizes of psaI transcripts were 0.94 and 0.32 kb, whereas in the AIC9 strain the smaller species was 0.26 kb. Sizes were calculated based on the migration of radiolabeled 0.16-1.77-kb RNA ladder (Life Technologies, Inc.) using [-P]ATP and T4 polynucleotide kinase after dephosphorylation. C, Western blot analysis of PsaI in the wild-type (WT) and AIC9 mutant strains. Polypeptides of photosynthetic membranes equivalent to 10 µg of chlorophyll were separated by Tricine/urea/SDS-PAGE and transferred to Immobilon-P membranes. The proteins were probed with an anti-PsaI antibody. The antigen-antibody reaction was visualized using a horseradish peroxidase-conjugated secondary antibody and enhanced chemiluminescence. D, low molecular weight polypeptides of PS I from the wild-type (WT) and AIC9 mutant strains. The PS I complexes containing 5 µg of chlorophyll from the wild-type and AIC9 strains were solubilized. The subunits were separated by Tricine/urea/SDS-PAGE and visualized by silver staining.



Total RNA was isolated from the wild-type and AIC9 mutant strains and used for Northern analysis of psaI transcripts. Northern hybridization of a psaI probe to total RNA from Synechocystis sp. PCC 6803 revealed two distinct RNA species in the wild type (Fig. 3B). It seems that the psaI gene was transcribed on two distinct transcripts of 0.94 and 0.32 kb. The possibility that the smaller transcript was derived from processing of the larger transcript is less likely, because when psaL is interrupted in the ALC7-3 strain, the 0.32-kb transcript is present in normal amounts (data not shown). When the same Magnacharge nylon membrane was stripped of the psaI probe and hybridized with a probe for psaL, a RNA species of 0.94 kb was recognized (data not shown). Similarly, a DNA fragment could be amplified from total RNA of the wild-type strain by reverse transcription-coupled polymerase chain reaction using a primer in the PsaL-coding region and a primer in the psaI gene (data not shown). Computer-assisted search of DNA sequences failed to identify any potential prokaryotic transcription termination sequences in the intergenic region between psaL and psaI. In contrast, the DNA sequences in the 3`-flanking region of psaI may form step-loop structures that are typical of prokaryotic transcriptional terminators. Therefore, the larger transcript is a bicistronic RNA for psaL and psaI. The smaller transcript may be derived from only the psaI gene (Fig. 3B). Northern hybridization of a psaI probe to total RNA in the AIC9 strain revealed only one RNA species (Fig. 3B). This species was smaller in size when compared with 0.32-kb psaI transcript in the wild type. This was expected as insertion of the chloramphenicol-resistance gene in psaI would introduce a transcriptional terminator.

We generated a polyclonal antibody against PsaI using a glutathione S-transferase-PsaI fusion protein that was expressed in E. coli. Western blotting of the photosynthetic membrane proteins showed that the anti-PsaI antibodies specifically recognized a protein species that matched the position of PsaI (Fig. 3C). The PsaI subunit was absent in the AIC9 mutant strain. We also purified PS I complexes from the wild-type and AIC9 mutant strains and separated their polypeptides by Tricine/urea/SDS-PAGE to confirm the absence of PsaI in PS I. Electrophoresis resolved several low mass subunits of PS I, including PsaE, PsaC, PsaK, PsaI, PsaJ, and PsaM from the wild-type PS I (Fig. 3D). As expected from Southern, Northern, and Western analyses, the PS I preparation from the AIC9 mutant strain specifically lacked the 4-kDa PsaI subunit. Taken together, the AIC9 mutant strain had an interrupted psaI and lacked the PsaI subunit.

Characterization of Photosynthesis in the PsaI-less Strain

compares the photosynthetic characteristics of the wild-type and AIC9 strains. When the mutant and wild-type strains were grown at 25 °C under photoautotrophic conditions, the doubling time of the AIC9 strain was similar to that of the wild-type strain (). Furthermore, the mutant strain could normally grow under photoheterotrophic conditions in the presence of 3-(3,4-dichlorophenyl)-1-1-dimethylurea and glucose, indicating that the cyclic electron flow around PS I is normal in the absence of PsaI. Chlorophyll per cell remained similar in the mutant and wild-type cells. When grown at 40 °C, the wild-type and mutant cells had 26- and 39.9-h doubling times, respectively. A similar decrease in growth rate was also observed in the PsaL-less strain (36.3-h doubling time). At higher temperature, the total cellular chlorophyll decreased in the mutant cells (3.7 µg of chlorophyll/OD at 730 nm), but not in the wild-type cells (4.3 µg of chlorophyll/OD at 730 nm).

The light-dependent oxygen evolution or uptake by whole cells were used as indicators of photosynthetic electron transfer. Both the wild-type and AIC9 cells showed about the same rate of overall photosynthetic activity (). As expected, PS II activity remained unchanged in the mutant. To measure PS I activity (as the rate of oxygen uptake) in the intact cells, PS II activity was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and then ascorbate and diaminodurene were used to donate electrons to PS I. The rates of oxygen uptake due to electron transport through PS I were similar in the wild-type and mutant cells.

The mutant photosynthetic membranes contained about the same numbers of chlorophylls per P700 as the wild-type, indicating that the absence of PsaI does not affect the number of active PS I reaction centers in the membranes. The chlorophyll fluorescence emission spectra of isolated photosynthetic membranes were examined at 77 K following excitation at 430 nm. 77 K fluorescence emission spectrum of Synechocystis PS I exhibits a single peak with a maximum at 725 nm(41) . The wild-type and AIC9 mutant strains showed identical fluorescence emission maxima originating from PS I, suggesting that absence of PsaI did not cause significant changes in the organization of pigments. NADP photoreduction activity of PS I was determined using reduced cytochrome c from Synechocystis sp. PCC 6803 as the electron donor, oxidized ferredoxin from Synechocystis sp. PCC 6803 as the electron acceptor, and spinach ferredoxin:NADP oxidoreductase to catalyze NADPH production. The reductase activity of PS I in PsaI-less mutant membranes was 15% less than that of the wild-type PS I. PsaL-less membranes also show a similar decrease in NADP photoreduction(12) . The PsaI- or PsaL-less membranes from cultures grown at 40 °C also showed a 15% decrease in the ferredoxin-mediated NADP photoreduction rates. This minor effect on reductase activity of PSI could be an indirect result of changes in the abundance or organization of other crucial proteins such as PsaD.

Accumulation of PS I Subunits in Membranes of PsaI-less Strain

Photosynthetic membranes were isolated from the wild-type and AIC9 mutant cells. Western blotting was used to detect the presence of different subunits of PS I in the membranes (Fig. 4). PsaA-B, PsaC, PsaD, PsaF, and PsaE were present in approximately the same amounts in the wild-type and mutant membranes, indicating that the lack of PsaI did not affect the assembly of these proteins into membranes. In contrast, the level of PsaL was drastically reduced in the PsaI-less membranes (Fig. 4A). To estimate the relative level of PsaL in the membranes, immunoquantification was performed using different amounts of wild-type membranes (Fig. 4B). The absence of PsaI resulted in approximately 80% reduction in the steady state level of PsaL. Reduced PsaL levels in the AIC9 photosynthetic membranes could be due to a decreased level of psaL transcripts, defects in the assembly of PsaL, and/or enhanced turnover of PsaL.


Figure 4: Immunodetection of PS I subunits in photosynthetic membranes of the wild-type and AIC9 mutant strains of Synechocystis sp. PCC 6803. A, proteins from photosynthetic membranes containing 5 µg of chlorophyll were solubilized, separated by Tricine/urea/SDS-PAGE, and then transferred to Immobilon-P membrane. The blot was immunodecorated with antibodies raised against PsaA-PsaB, PsaC, PsaD, PsaE, PsaF, and PsaL. The antibody-antigen reaction was recognized as in Fig. 3C. B, quantification of PsaL in the photosynthetic membranes of the AIC9 mutant strain. The proteins from the photosynthetic membranes containing 5 µg of chlorophyll in the wild-type (WT) and AIC9 mutant strains were solubilized, separated, and blotted as described above. Amounts of PsaL in the AIC9 strain were immunoquantified based on the immunoreactivity of wild-type photosynthetic membranes containing 1, 2, and 3 µg of chlorophyll.



Transcript Level of PsaL in the AIC9 Mutant Strain

A significant decrease in the PsaL level in the PsaI-less membranes (Fig. 4) prompted examination of transcript levels of psaL. Northern analysis of total RNA from the wild-type and AIC9 strains using the psaL probe revealed a single RNA species in the wild type (Fig. 5). Strain AIC9 contained a single distinct RNA species that hybridized with the psaL probe. There was about 5% psaL RNA in AIC9 strain compared to that in the wild type. Thus, insertional inactivation of psaI that is located downstream from psaL drastically reduced steady state RNA levels for psaL, thereby decreasing the amount of PsaL in the mutant membranes.


Figure 5: Northern blot analysis of psaL from Synechocystis sp. PCC 6803. 10 µg of total RNA from the wild-type and AIC9 strains was subjected to electrophoresis in 1.2% agarose gel containing formaldehyde, transferred to a Magnacharge nylon membrane, and hybridized with a probe specific to psaL.



Composition of PS I from AIC9 Strain

To examine the role of PsaI in the assembly of the complex, PS I was purified from the photosynthetic membranes of the wild-type and AIC9 mutant strains by solubilizing membranes with Triton X-100. Subsequently, the polypeptides of purified PS I were separated by Tricine/urea/SDS-PAGE (Fig. 6). The PS I preparation from the AIC9 mutant strain specifically lacked the PsaI subunit (Fig. 3, C and D) and PsaL (Fig. 6), but maintained wild-type levels of PsaA-B, PsaD, PsaF, PsaE, PsaC, and PsaK (Fig. 6). Apparently, the absence of PsaI specifically causes loss of PsaL, and not of other subunits, during purification of PS I. Since PsaL was present in the membranes, albeit in reduced amounts, these results suggest that absence of PsaI destabilizes association of PsaL with the core of PS I.


Figure 6: Polypeptide composition of PS I complexes isolated from the wild-type and AIC9 mutant strains. Proteins in the wild-type (WT) and AIC9 PS I complexes containing 15 µg of chlorophyll were separated by Tricine/urea/SDS-PAGE. The polypeptides were visualized by Coomassie Brilliant Blue.



Isolation of PS I Trimers

Since the absence of PsaI specifically affects the presence of PsaL in isolated PS I, we examined function and organization of PsaL in the PsaI-less mutant strain. PsaL is required for the formation of PS I trimers(15) . When the wild-type photosynthetic membranes were solubilized using n-dodecyl -D-maltoside, followed by sucrose gradient ultracentrifugation, PS I could be resolved into two fractions (Fig. 7A). The heavier fraction represented the PS I trimers while the lighter fraction contained PS II and PS I monomers (15). Interestingly, when the photosynthetic membranes from the AIC9 strain were solubilized and fractionated under the conditions that resulted in resolution of PS I trimers and monomers in the wild type, PS I trimer fraction was completely absent although tightly migrating PS I monomers were formed (Fig. 7A). This observation suggested that PsaI may play a role in trimerization of PSI, either through a direct involvement in the formation of PS I trimers or by altering the organization of PsaL. We determined the level of PsaL in the monomeric fraction from the wild-type and AIC9 strains by Western blotting (Fig. 7B). There were equal amounts of PsaA-PsaB in the monomeric fractions from the wild-type and AIC9 mutant strains. In contrast, PsaL could be detected only in the monomeric fraction from the wild-type strain, but not in PS I monomers from the AIC9 strain (Fig. 7B). The absence of PsaE, PsaF, or PsaJ does not affect the association of PsaL with PS I core(12) . These results indicated that the absence of PsaI affects PS I trimerization by causing loss of PsaL during detergent solubilization.


Figure 7: Fractionation of PS I monomers, trimers, and immunodetection of PsaL in the wild-type and AIC9 mutant strains. A, fractionating of PS I trimers and monomers in the wild-type (WT) and AIC9 mutant strains. Trimers and monomers of PS I were fractionated using sucrose-gradient ultracentrifugation, when the photosynthetic membranes in wild-type and AIC9 mutant strains were solubilized with n-dodecyl -D-maltoside. B, proteins in the PS I monomers of the wild-type (WT) and AIC9 mutant strains containing 5 µg of chlorophyll were separated by Tricine/urea/SDS-PAGE, blotted on the Immobilon-P membranes, and probed with antibodies against PsaA-PsaB or PsaL.



Cross-linking between PsaL and PsaD in the PsaI-less Membranes

PsaL was completely lost during isolation of PsaI-less PSI complexes (Fig. 7). These results imply altered interactions of PsaL with other PSI subunits in PsaI-less membranes. We have previously reported a structural interaction between PsaD and PsaL(42) . Ability to cross-link PsaD and PsaL can be used as an indicator of their close proximity. Thus, we performed cross-linking experiments using the wild-type and PsaI-less photosynthetic membranes. When the wild-type photosynthetic membranes were treated with 10 mM glutaraldehyde at 4 °C, two major cross-linked products with apparent molecular masses of 29 and 25 kDa were formed (Fig. 8). The 29-kDa species was recognized by both anti-PsaD and anti-PsaL antibodies. The PsaD-PsaL cross-linked product was also observed after treatment of PsaI-less membranes with glutaraldehyde (Fig. 8). The cross-linked species between PsaD and PsaL was absent as expected when the PsaL-less photosynthetic membranes from the ALC7-3 strain (14) were used to perform the above cross-linking experiment (data not shown). The 25-kDa species was recognized by antibodies against PsaD, PsaE, and PsaC, indicating cross-linking between PsaD and PsaE, or PsaD and PsaC (data not shown). The formation of cross-linked species between PsaD and PsaL in the AIC9 strain suggested that the positioning of the glutaraldehyde-reactive residues that are involved in cross-linking between PsaL and PsaD was not greatly altered in the absence of PsaI.


Figure 8: Cross-linking of PS I subunits. The photosynthetic membranes in the wild-type and AIC9 mutant stains were exposed to 0 or 10 mM glutaraldehyde for 30 min on ice, followed by termination of cross-linking reaction. The proteins in the photosynthetic membranes containing 10 µg of chlorophyll were denatured and separated by Tricine/urea/SDS-PAGE. The polypeptides were transferred to Immobilon-P membranes. 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-HCl (pH 6.7) at 50 °C for 30 min. The blot was reprobed with anti-PsaL antibody. The antigen-antibody reaction was visualized as described in the legend to Fig. 3C. Apparent molecular masses were determined from the migration of prestained markers (Life Technologies, Inc.).



Removal of PS I Polypeptides from PsaI-less Membranes by NaI or Proteases

PsaL is an integral membrane protein that may contain two or three transmembrane helices(14, 43) . Thus, it is expected that completely assembled PsaL would resist chaotropic extraction. When wild-type membranes were treated with up to 3 M NaI, PsaL resisted removal from the membranes (Fig. 9A). PsaL in the membranes of the AIC9 strain was equally resistant to chaotropic extraction (Fig. 9A), demonstrating that PsaL is inserted in the PsaI-less membranes. In contrast, there was an enhanced loss of PsaD from the AIC9 membranes after treatment with NaI. This could be attributed to a decreased level of PsaL in the photosynthetic membranes. We have reported that the absence of PsaL leads to an enhanced loss of PsaD to chaotropic extraction in the ALC7-3 strain(42) . Further analysis of PsaL organization in PS I was performed by examining susceptibility of PsaL to proteases. When the photosynthetic membranes were treated with chymotrypsin, PsaL in the wild-type membranes resisted proteolytic cleavage (Fig. 9B). In contrast, chymotrypsin could digest PsaL in PsaI-less membranes. PsaD was equally accessible to chymotrypsin in both wild-type and AIC9 strains. In addition, the cleavage patterns of PsaD were remarkably similar in the wild-type and mutant strains. PsaA-PsaB subunits were also similarly resistant to chymotrypsin cleavage in both wild-type and AIC9 strains (Fig. 9B). These data, together with unchanged chlorophyll fluorescence emission at 77 K (), suggested that the absence of PsaI does not cause a gross change in the PS I organization, but specifically affected the accessibility of PsaL to proteases.


Figure 9: Accessibility of PS I subunits to removal by NaI or to proteolysis. A, SDS-PAGE analyses of NaI-treated photosynthetic membranes isolated from the wild-type and AIC9 strains. The photosynthetic membranes were exposed to various concentrations of NaI for 30 min on ice, followed by desalting. The proteins in the photosynthetic membranes containing 5 µg of chlorophyll were separated by Tricine/urea/SDS-PAGE, electroblotted to Immobilon-P membranes, and probed with antibodies against PsaL or PsaD. The antigen-antibody reaction was visualized as in Fig. 3C. B, digestion of wild-type and AIC9 photosynthetic membranes by chymotrypsin. The wild-type and AIC9 photosynthetic membranes were incubated with chymotrypsin at a concentration of 1 mg of protease (mg chlorophyll) for 0, 5, 20, and 40 min. The protease-treated photosynthetic membranes equivalent to 5 µg of chlorophyll per lane were solubilized and proteins were separated by Tricine/urea/SDS-PAGE. The proteins were blotted to Immobilon-P membranes and probed with anti-PsaL antibody. Subsequently, the membrane was stripped of the bound antibodies as described in the legend to Fig. 8 and reprobed with anti-PsaD or anti-PsaA-B antibodies. The antigen-antibody reaction was visualized as in Fig. 3C.




DISCUSSION

The composition, structure, and function of the PS I reaction centers from cyanobacteria and higher plants are remarkably conserved. The psaD(44) , psaE(23) , psaF(24) , psaA-B(45) , psaC(46, 47) , psaJ(18, 25) , and psaL(14) genes encoding subunits of PS I from Synechocystis sp. PCC 6803 have been isolated and characterized. Like these genes, psaI encodes a polypeptide that is similar to the homologous counterparts from higher plants (Fig. 2). PsaI subunits contain a central hydrophobic domain flanked by hydrophilic amino- and carboxyl termini (Fig. 2; Ref. 38). The central hydrophobic domain potentially could span the membrane. PsaI in intact spinach thylakoids is not accessible to proteolytic digestion(43) . Furthermore, PsaI is resistant to removal by chaotropic agents(25, 38, 42) . Therefore, PsaI is probably an integral membrane protein containing one transmembrane helix. The carboxyl-terminal hydrophilic domain of PsaI in barley contains several positively charged residues (38). If the ``positive inside rule'' for predicting topology of membrane proteins (48) applies to PsaI of barley, the carboxyl-terminal domain of this protein may face the n-side (stromal or cytoplasmic side) of the photosynthetic membranes. While PsaI in cyanobacteria and higher plants have similar hydropathy profiles, the carboxyl termini of cyanobacterial and liverwort PsaI contain several negatively charged residues (Fig. 2). Thus, the orientation of PsaI with respect to membranes cannot be predicted based on the distribution of its charged amino acids. The significance of the differences in overall charges in the carboxyl termini of PsaI from cyanobacterial and higher plants is not understood. PsaI proteins do not contain obvious consensus sequences for binding prosthetic groups.

Biochemical methods and generation of cyanobacterial mutant strains lacking specific proteins have been successfully used to study functions of PS I proteins(4) . We generated a cyanobacterial mutant strain lacking PsaI. The mutant strain AIC9 has an interrupted psaI gene (Fig. 3), and lacks PsaI in its membranes and in purified PS I complexes (Fig. 3). The absence of PsaI had a significant effect on the organization of PsaL in the membranes. In cyanobacteria, PsaL makes the connecting domain of the PS I complex that links the catalytic domains to make a PS I trimer(15, 49, 50) . PsaL in the PsaI-less membranes was resistant to chaotropic extraction (Fig. 9A). Therefore, PsaL can integrate into membranes without PsaI. However, our results demonstrate that PsaL in the PsaI-less membranes has an altered conformation that differs in its interaction with the PS I core. First, the PsaI-less PS I complexes that had been purified by DEAE-cellulose chromatography and sucrose-gradient ultracentrifugation completely lacked PsaL (Fig. 6). The PsaD-less PS I complexes purified by the same method also contained a significantly reduced level of PsaL(42) . However, PsaD and PsaL could be cross-linked in the PsaI-less membranes (Fig. 8). Also, PsaI-less PS I complexes maintained wild-type levels of PsaD and other subunits (Fig. 6). Thus, the effect of PsaI on association of PsaL with PS I core is not mediated by PsaD or other PS I subunits. Therefore, the absence of PsaI caused easier loss of PsaL from the PS I core during purification which requires detergent solubilization of membranes (Fig. 6). Second, PS I trimers could not be obtained when the PsaI-less membranes were solubilized with n-dodecyl -D-maltoside (Fig. 7A). PsaL is required for the formation of PS I trimers(15) . It is likely that the structural interactions between PsaL and PsaI are crucial in maintaining the trimeric organization of PS I. Third, the absence of PsaI specifically affected the accessibility of PsaL, but not of PsaD or PsaA-PsaB, to proteases (Fig. 9B). Although PsaL of Synechocystis sp. PCC 6803 has 21 potential chymotrypsin cleavage sites(14) , it is resistant to proteolytic cleavage in the wild-type photosynthetic membranes, perhaps due to shielding, steric hindrance, or other conformational factors. Although the positioning of PsaL relative to PsaD in the AIC9 strain is not significantly altered (Fig. 8), the absence of PsaI may expose the cleavage sites in PsaL to proteases. In conclusion, the absence of PsaI alters interaction of PsaL with the PS I core and causes conformational changes in parts of PsaL. These results also indicate that PsaI, along with PsaL(15) , is present in the ``connecting domain'' that links PS I monomers to form trimers(49) .

The role of PsaI in the organization of PsaL, as indicated by our results, may involve structural interactions between the hydrophobic or extramembraneous domains. Although PsaL and PsaI are both integral membrane proteins(14, 43) , the average hydrophobicity value calculated using the Eisenberg algorithm (51) in the hydrophobic domain of Synechocystis PsaI is much higher than those of potential transmembrane helices of PsaL. It is likely that the highly hydrophobic region of PsaI may stabilize the less hydrophobic transmembrane domain(s) of PsaL, thus preventing PsaL from detergent extraction. The role of PsaI in maintaining normal organization of PsaL is highly specific. PsaI, PsaJ, and PsaM of PS I have similar features in their primary sequences. Yet PsaJ and PsaM cannot substitute function of PsaI in maintaining organization of PsaL. Similarly the absence of other transmembrane proteins such as PsaF and PsaJ does not affect organization of PsaL(18, 25) . Therefore, PsaL and PsaI may interact in a highly specific and probably direct manner. Specificity in transmembrane associations relies mainly upon a detailed sterochemical fit between helices and upon the protein outside the lipid bilayer (52). PsaL-PsaI interactions may include both components. The transmembrane domain of PsaI contains three conserved prolines which may give this domain a peculiar conformation and make its interaction with PsaL highly specific. The function of PsaI in assisting proper assembly of PsaL may be common to other similar proteins found in the heteromultimeric membrane-protein complexes. PS I(4) , PS II(53) , cytochrome b/f complex(54) , and cytochrome c oxidase (55) contain hydrophobic polypeptides of 4 kDa. These proteins have one putative transmembrane helix flanked by short hydrophilic, charged domains. Similar to PsaI, these polypeptides may assist in the correct organization of other subunits by stabilizing transmembrane helices in the lipid bilayer or by anchoring peripheral proteins.

The decreased levels and altered organization of PsaL in the PsaI-less mutant makes it difficult to assess the direct role of PsaI in the electron transfer and light-trapping functions of PS I. The absence of PsaI had small effects on PS I function. The normal rates of electron transfer to methyl viologen suggested that the electron transfer within PS I was not altered in the PsaI-less cells, but the electron transfer from PS I to ferredoxin was marginally decreased as indicated by the ferredoxin-mediated NADP photoreduction rates (). Similar differences were observed in the PsaL-less strain, which contains the wild-type levels of PsaI. Therefore, we believe that the major function of PsaI is to aid normal organization of PsaL. Under laboratory growth conditions, the defects caused by the absence of PsaI were not crucial enough to cause major physiological alterations. However, the small decrease in chlorophyll contents and NADP photoreduction rates, that were observed in the cells grown at high temperature, might have resulted in a vital disadvantage that disabled growth of the mutant cells under stress conditions. Therefore the presence of PsaI offers a small but physiologically significant increase in the activity of PS I. This may provide an overall evolutionary advantage that led to the occurrence of this highly conserved polypeptide in the PS I complexes from cyanobacteria and chloroplasts.

  
Table: Photosynthetic characteristics the wild-type and AIC9 mutant strains

Rates of oxygen evolution or uptake were determined using cells that were actively growing at 25 °C. Chlorophyll to P700 ratios and 77 chlorophyll fluorescence spectra were performed using thylakoid membranes from cells grown at 25 °C.



FOOTNOTES

*
This work was supported in part by National Science Foundation Grants MCB 9202751 and MCB 9405325 (to P. R. C.), United States Department of Agriculture-National Research Initiative Competitive Grants Program Equipment Grants 93-37311-9456 (to P. R. C.) and 93-37306-9147 (to J. A. G.), and NASA Grant NAGW 2328 (to J. A. G.). This is contribution 95-484-J from the Kansas Agricultural Experiment Station. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) L24773.

§
To whom correspondence should be addressed. Tel.: 913-532-6303; Fax: 913-532-6653; E-mail: chitnis@ksu.ksu.edu.

The abbreviations used are: PS, photosystem; bp, base pair(s); MOPS, 4-morpholinepropanesulfonic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; PAGE, polyacrylamide gel electrophoresis; kb, kilobase(s).


ACKNOWLEDGEMENTS

We acknowledge Trent S. Armbrust and Jeffrey D. Westberg for valuable help in the preparation of anti-PsaI polyclonal antibody. We also thank David Rintoul for advice regarding use of the SPEX Fluorolog spectrometer.


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