From the
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
Photosystem (PS)
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.
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
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
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.
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
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
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
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
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.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
(
)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.
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.
) 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) .
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.
-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.
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).
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.
-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) .
/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.
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
/EMBL Data Bank with accession number(s)
L24773.
, N., Hinrichs, W., Witt, I., Fromme, P., Pritzkow, W., Dauter, Z., Betzel, C., Wilson, K. S., Witt, H. T., and Saenger, W. (1993) Nature361, 326-331
[CrossRef]
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.