Download The Primary Structure of a 4.0-kDa Photosystem I Polypeptide

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OF BIOLOGICAL
CHEMISTRY
0 1989 by The Americsn Society for B i ~ ~ e ~and
i Molentlar
s t ~
Biology, Inc.
Val, 264, No. 31, I s ~ u of
e November 5, pp. 18402-18406 1989
Printat in
tlS.A.
The Primary Structureof a 4.0-kDa Photosystem I Polypeptide
Encoded by the Chloroplastpsujr Gene*
(Received for publication, July 14, 1989)
Henrik Vibe SchellerS,Jens Sigurd OkkelsS, Peter Bordier
HaljS, Ib SvendsenQ, Peter Roepstorffl,
and Birger LindbergM~llerS
From the $Department of Pkmt Phys~olog~,
Royal V e t e r and
~ ~Agr~u~tural
~
Uni~ersity~
40 T ~ r v a ~ s e ~
DK--1871
~ej,
Frederiksherg C, the §Department of Chemistry, Carlsberg Laboratory, 10 Gamle Carlsherg Vej, DK-2500 Valhy, and the
v e j , Odense M,Denmark
%Departmentof Mo~cularBiology, Odense University, 55~ a m ~ ~ DK-5230
Partial amino acid sequences have been determinedcyanobacteria, algae, and plants. This suggests that thePS I
for a 4.0-kDa photosystem I polypeptide from barley. complex has the same structure in all species. One copy of
A comparisonwiththesequenceofthechloroplast
each subunit is present per reaction center (11-13).
genome of Nicotiana tabacum and Marchantia polyIn a previous paper f13), we have reported the presence of
morpha identified the polypeptide as chloroplast-en- two hitherto unnoticed subunits with apparent molecular
psal and masses of 4 and 1.5 kDa in barley PS I preparations. In the
coded. We designate the corresponding gene
the polypeptide PSI-I. The barley
chloroplastpsalgene present paper, the amino acid sequence forthe 1.5-kDa polywas sequenced. The gene encodesa polypeptide of 36 peptide and the localization and nucleotide sequence of the
amino acid residues with a deduced molecular
mass of corresponding gene on the barley chloropIast genome is re4008 Da,The 4.0-kDa polypeptide is N-terminally
blocked witha formyl-methionine residue. Plasma de-ported. The polypeptide has an actual molecular mass of 4.0
in a ~ e e m e n t
sorption mass spectrometry established that the poly- kDa and is referred to as PSI-I in the fo~~owing
with
the
nomenclature
proposed
by
Schantz
and
Bogorad
peptide is not post-translationally processed except for
the PS I p o l ~ e p t i d e s
possible conversion of a methionine residue into me- (14). The nomenclature system used for
thionine sulfone. The hydrophobic 4.0-kDa polypep- in barley i s shown in Table 1.
tide is predicted to have one membrane-spanning aMATERIALS ANDMETHODS
helix and is homologous to transmembranehelix E of
Isolation and Amino Acid Sequencing of the PSI-I Polypeptidethe D2 reaction center polypeptide
of photosystem11.
PS 1 particles were prepared from barley (Hordeum uulgare L,cv.
Sval0fs Bonus), and the PSI-I polypeptide was isolated from PS I
particles as described previously (1, 13).
Enzymatic cleavage of the PSI-I polypeptide with pepsin was
Photosystem I (PS I)’ in plants and cyanobacteriacatalyzes
the photochemical transfer of electrons from plastocyanin to carried out by dissolving the lyophilized PSI-I polypeptide (5 nmol)
in 100 p l of 0.2% acetic acid (adjusted to pH 2.0 with HCl) and
ferredoxin. Pigments and theelectron acceptors P700, Ao, AI, incubating with porcine pepsin (0.4 pg) at 37 “C for 20 min. The
and X are thought to be bound to two polypeptides with
reaction was stopped by quickly freezing the sample. The mixture of
apparent molecufar massesof 82-83 kDa and encoded by the fragments was lyophilized, dissolved in a small volume of0.1%
chloroplastpsaA and psaB genes. The heterodimeric pigment- trifluoroacetic acid, and subjected to reverse-phase high performance
protein complex is known
as CPI. The two remainingelectron liquid chromatography on a C, column (4.6 X 250 mm, Vydac, The
acceptors of PS I, iron-sulfur centers A and B, are carried by Separations Group, Hesperia, CA). Elution was carried out with a
linear gradient from 0 to 54% acetonitrile in the presence of0.1%
a 9-kDa p o ~ ~ e p t i which
d e is encoded by the chloroplast gene trifluoroacetic acid (90 min, flow rate 1 mllmin, eluate monitored a t
psaC ( I f .PS I electron transport has been the subject of two 215 nm). Amino acid sequencing of the isolated PSI-I polypeptide
recent reviews (2, 3). Apart from these three subunits, PS I and of the isolated fragments was carried out aspreviously described
core preparations contain a number of polypeptides in the 8- (1).Prior to sequencing of the uncleaved polypeptide, the N-terminal
20-kDa region.The exact numberhas not been fully clarified, blocking group was partially removed by overnight incubation in 70%
but complete amino acidsequences corresponding to five formic acid at 40 “C.
Plasma Desorption Mass Spectrometry-For molecular mass deterdifferent nuclear-encodedsubunits have beenreported (4-10). mination, the lyophilized polypeptide was dissolved in 0.1% trifluoThe amino acid sequence of the three chloroplast and five roacetic acid, adsorbed to a nitroceltuIose-covered aluminum foil,
nuclear-encoded subunits are all highly conserved between washed with 0.1% trifluoroacetic acid, and mounted in the plasma
desorption spectrometer (16). The plasma desorption mass spectrom*This work was supported in part by grants from the Danish eter used was a Bio-Ion Bin 10K instrument (Bio-Ion Nordic AB,
Governmentaf Program for Biotechnology Research, the Danish Ag- Uppsala, Sweden) which is basically similar to the instrument dericultural Research Council, the Danish Natural Science Research scribed by Sundqvist et al. (17).
Cloning and Nucleotide Sequencing-Based on the determined
Council, the Danish Technical Science Research Council, Dansk
Inves~ringsfond,Thomas B. Thriges Foundation, the Carlsherg amino acid sequences, open reading frames coding for homologous
Foundation, the Tuborg Foundation, Stiftelsen Hofmansgave, and polypeptides were identified in the chloroplast genomes of tobacco
Legatstiftelsen Pedersholm. The costs of publication of this article (18) and Marchantia polymorpha (19). A 13.5-kb PstI clone (pHvwere defrayed in part by the payment of page charges. This article C186) covering the corresponding region in the barley chloroplast
must therefore be hereby marked “aduertisement” in accordance with genome (Fig. 1 A ) was obtained from Dr. C. Poulsen, Dept. of Molecular Biology and PlantPhysiology, University of Arhus (20). Plasmid
18 U.S.C. Section 1734 solelyto indicate this fact.
The nucleotide sequence(s)reported in this paper
has been submitted DNA was prepared according to Birnboim (21). The 13.5-kb insert of
to the GenBankmf/EMBL Data Bank with accession numherCs) pHvC186 was subcloned in the pTZ18/19 plasmids (22) as outlined
in Fig. 1B. Single-stranded DNA was obtained from the pTZ18/19
505104.
The abbreviations used are: PS I, photosystem I; PS 11, photosys- piasmick using helper phage M13K07 (23). DNA sequencing was
carried out by the method of Sanger et al. (24) using C Y - [ ~ ~ P ] ~ A T P
tem II; CP1, chlorophyll a-protein 1; kb, kilobase pairs.
18402
APolypeptide
4.0-kDa
of Photosystem
I
18403
TABLEI
Nomenclature system for the polypeptides of PS I in barley
Polypeptides of PS I preparationsfrom barley using the nomenclature proposed by Schantz and Bogorad (14) (A) and thenomenclature
of Bengis and Nelson (45) (B) are listed. The localization of the
corresponding genes in the chloroplast (C) or nuclear (N) genomes is
indicated. Apparent molecular masses are based on electrophoretic
mobilities in sodium dodecyl sulfate-polyacrylamide gels (1, 13). Calculated molecular masses are based on amino acid or nucleotide
sequencing data (4,10, 15).
Polypeptide
subunit
Molecular
mass
Gene
(A)
(B)
Calculated
Apparent
kDa
(C)
(C)
p ~ a A(C)I
psaE
psaC (C)
PsaD (N)
PsaE (N)
PsaF (N)
PsaC (N)V
(N)
PsaH
psaI
PSI-A
PSI-B
I
PSI-C VIb VII,
PSI-D
I1
PSI-E
IV
PSI-F
I11
PSI-G
PSI-H VIa VI,
82
82
9
18
16
15b
ND
9.5
1.5
14
4
ND"
ND
8.8
ND
10.8
ND
ND
10.2
4.0
ND
ND
ND, not determined in barley.
H. V. Scheller, B. Andersen, and B. L. Mprller, unpublished data.
p s a ~ psa A
400 bp
rbcL
P Hc
E
H
(Amersham International, Buckinghamshire, UK) and a Sequenase
kit (United States Biochemical Corp., Cleveland, OH).
Additional Procedures-Hydropathy plots and secondary structure
predictions were carried out with the Sequanal program package (A.
R. Crofts, Biotechnology Center, University of Illinois).
The protein database provided by the National Biomedical Research Foundation was used to search for homologous polypeptides.
The standard mutation matrix was used to evaluate the found homologies.
RESULTS
The 13.5-kb fragment of the barley chloroplast genome
cloned in pHvC186 starts 164 bases into the coding region of
rbcL, the gene for the large subunit of ribulose-bisphosphate
carboxylase (25). Restriction mapping of the 13.5-kb fragment
showed that the gene encoding the PSI-I polypeptide begins
3.0 kb downstream of the start codon for the rbcL gene (Fig.
1). The corresponding distance is 4488 base pairs in tobacco
(18) and 2838 base pairs in M . polymorph (19). We designate
the gene for the PSI-I polypeptide psaZ. Downstream to psal
is an intergenic region followed by an open reading frame
which is highly homologous toan open reading frame,
ORF184, located inasimilar
position on the chloroplast
genomes of tobacco (18) and M. polymorph (19). The open
reading frame was partially sequenced in barley, and the
deduced amino acid sequence for the first 130 amino acid
residues is 86% and 62% identical with the corresponding
sequence in tobacco and M. polymorpha, respectively. The
high level of conservation of ORF184 indicates that it is an
actively transcribed gene although the product has not been
identified. The nucleotide sequence and deduced amino acid
sequences for the PSI-I polypeptide and ORF184 in barley
are shown in Fig. 2.
The isolated PSI-I polypeptide was N-terminally blocked
preventing Edman degradation. The blocking group could be
partially removed by incubation with formic acid and subsequent amino acid sequencing provided the first 15 residues of
the N-terminal sequence (except Phe-10 which was not identified). An additional 12 amino acid residues were determined
by digestion of the PSI-I polypeptide with pepsin at pH 2.0
and sequencing of the isolated fragments (Fig. 2). The remaining 10 amino acid residues were deduced from the nu-
.
B
S
H
44
H
S
S
E
AI
EEH
4
l
"
FIG. 1. Restriction map of the barley chloroplast genome.
A , a PstI restriction map of the barley chloroplast genome (20)
showing the position of psal, ORF184, and some other genes (20, 26,
27). psbA andpsbD encode D l and D2 of PS 11, respectively, whereas
psbC encodes the 47-kDa polypeptide of PS 11. The inverted repeat
regions ( I R ) are indicated. E , restriction map of a part of the 13.5-kb
PstI fragment containingpsal andORF184. The sequencing strategy
and different sets of subclones are indicated. Only the subclone
containing the psaIgene was sequenced on both strands. HincII and
SspI sites have only been determined for the central HindIII-EcoRI
fragment. P, PstI; E, EcoRI; H, HindIII; Hc, HincII; S, SspI.
cleotide sequence of the corresponding gene. Cleavage with
pepsin is rather unspecific and may result in very small
fragments or even single amino acids. However, larger fragments areobtained by limiting the incubation period. Pepsin
digestion may be the method of choice for very hydrophobic
polypeptides since enzymatic cleavage with trypsin and chymotrypsin was unsuccessful, presumably due to the low solubility of the hydrophobic polypeptide at pH8.0.
The molecular mass of the PSI-I polypeptide was determined to be 4066 f 2 Da by plasma desorption mass spectrometry (Fig. 3). The psuZ gene encodes for a polypeptide
with a calculated molecular mass of 4008 Da leaving 58 f 2
Da unaccounted for in the amino acid sequence. The Nterminal blocking group is presumably a formyl group on the
methionine residue which would account for 28 Da. The PSII polypeptide contains 2 methionine residues. Amino acid
analysis of the isolated polypeptide showed that about 50%
of the methionine had been converted to methionine sulfone.
Only trace amounts of methionine sulfoxide were found. The
presence of methionine sulfone together with the N-terminal
formyl group accounts for the 58 k 2 Da difference between
the measured and calculated molecular mass of the PSI-I
polypeptide.
The PSI-Ipolypeptide migrates with an apparentmolecular
mass of 1.5 kDa on sodium dodecyl sulfate-polyacrylamide
gels (13). Large differences between apparentand actual
molecular mass have also been reported for other thylakoid
polypeptides like the 10.8-kDa PSI-E polypeptide of barley
(4). The discrepancy between the apparent andactual molec-
A 4.0-kDa Polypeptide of Photosystem I
18404
-35
-10
1 AATATTCCTTATAA~~~~~TACTTAATTATATCATAAGAATC~~~~~AmTTCGACTAGATAG~TAGT~
SD
p
Met Thr ASP Leu Asn Leu Pro Ser Ile Phe Val Pro Leu Val Gly
ACGGAT TTA AAC TTA CCT TCT ATT TTC GTG CCT TTA GTA GGC
81 GAATTFACACACCTATTCC ATG
Leu Val Phe Pro Ala Ile Ala Met Thr Ser Leu Phe Leu T r Val Gln L s L s Lys Ile
AAG ATT
1151 TTA GTA TTT CCG GCA ATT GCA ATG ACT TCT ITA TTT CTT T ~ T
GTG CAA
ALAL
1
2
J
FIG.2. Nucleotide sequence of the
psal gene and amino acid sequence
of the PSI-I polypeptide. The amino
acid sequences confirmed by peptide sequencing are underlined. The open reading frame starting at position 540 is homologous to ORF184 in tobacco (18)and
M.polyrnorpha (19).The slash at position 388 indicates an Ssp1 site used for
subcloning where no overlap was established. Therefore, it cannot be excluded
that a small stretch of nucleotide sequence is missing. Putative -35 and -10
promoterregions and Shine-Dalgarno
sequence (SD)are indicated with dots.
Val *
205 GTC TAG TATmTTAGTGTAAGTAATATAATATGGTAGGGTATG~ACmTTCTACACACAC~CTACACAC~
282 TGAAAAACGGCTATGGATGCAAGATATAGGCTACGAGCATACGAGCAT~TGCATGAATA~CAGAGC~TATAGCGAG~
361 T T T A ~ A T ~ M T T G A A T C A A C G A A T / A T T ‘ I T G A A T T ~ T A ~ G T C A A T G T A T C T A A C C T A T T A ~ C A C A G G A G T A C T
439 A G T T G C T G A A G G C G A T T T C A G A A T ~ G T A A A G G C ~ T T A T P T A ~ T T A T T C T C T ~ ~ C A A T C G A C C G C
Met Asn Trp Arg Ser Glu H i s Ile Trp Val Glu Leu Leu Lys
518 TGCTGGATPTAGTATATCTAAT ATG AAT TGG CGA TCA GAA CAC ATA
TGG GTA GAACTT CTA AAA
I
Gly Ser Arg Lys Arg Ser Asn Phe Phe Trp Ala Cys Ile Leu Phe Leu Gly Ser Leu Gly
582 /GGT TCT CGA AAA AGG AGT AAT TPT TTC TGGGCC TGT ATT CTT TIT CTA GGT TCA CTA GGA
1
a
Phe Leu Leu Val Gly Thr Ser Ser Tyr Leu Gly Lys Asn Ile Ile Ser Ile Leu Pro Ser 4
642 TTC TTA TTG GTT GGG ACT TCC AGT TAT CTT GGT AAG AAT ATT ATA TCT ATA CTT CCA TCT
Gln Glu Ile Leu Phe Phe Pro Gln Gly Val Val MetSer Phe Tyr Gly Ile Ala Gly Leu
702 CAA GAA ATT CTT TlT TTTCCG CAGGGG GTC GTG ATGTCT TTC TAC GGA ATC GCA GGC CTA
Phe Ile Ser Ser Tyr Leu Trp Cys Thr Ile Leu Trp Asn Val Gly Ser Gly Tyr Asp Arg
762 TTC ATT AGC TCC TAC CTG TGG TGT ACT ATT TTG TGGAAT GTA GGT AGT GGT TAT GAC CGA
Phe Asp Arg Lys Glu Gly
8 2 2 TTC GAT AGA AAA GAG GGA
Ile ValCys Ile Phe Arg Trp Gly Phe Pro Gly Ile Lys Arg
ATAGTT TGC ATT TIT CGT TGG GGA lTC CCT GGA ATAAAA CGT
Arg Val Phe Leu Arg Phe Leu Met Arg Asp Ile Gln Ser Ile Arg Ile
CGG GAT ATC CAA TCA ATT AGA ATT C 930
882 CGC GTC TTC CTT CGA TTC ClT ATG
++
MH
2
MH
+
FIG.3.Plasma desorption mass
spectrometry of the PSI-I polypeptide. Peaks of 4065.2and 2034.6correspond to the single- and double-charged
molecular ions. Correcting for the protons in the ions, an averagemolecular
mass of 4066 Da is calculated. The accuracy is estimated to +2 Da.
ular mass of the PSI-Ipolypeptide cannot be due to extensive
post-translational modification since such a modification
would have been detected by the mass spectrometric analysis.
The PSI-I subunit is very hydrophobic with a polarity index
(28) of 0.28 which is lower than the indices for any of the
eight other PS I subunits with known primary structure. The
hydropathy profile of the PSI-I polypeptide from barley indicates the presence of a central hydrophobic region flanked
by hydrophilic N- and C-terminals (Fig. 4). The hydropathy
plots for the psal gene products in tobacco and M. polymorph
are very similar except for the more hydrophobic N-terminal
region in M. polymorpha (not shown). Using the parameters
of Rao and Argos (29) for prediction of secondary structure
of membrane proteins, the central hydrophobic region was
predicted to form an a-helix with 23 residues. This helix may
span thethylakoid membrane. The PSI-Isubunit in barley is
89%and 64% homologous to thecorresponding gene products
in tobacco (18)and M. polyrnorpha (19), respectively. Comparison of the amino acid sequence in the threespecies (Fig.
5) shows that the central
hydrophobic region with the putative
a-helix conformation is highly conserved. The N-terminal
region is also conserved, whereas the C-terminal region is
strikingly different in M. polymorph in comparison with the
two higher plant species. According to the “positive inside
rule” of von Heijne and Gavel (30), the C-terminal of the PSII subunit from barley and tobacco wouldbe expected to
protrude into the stroma. This assumption is in agreement
with the observation by Michel et al. (31) that formyl-methionine residues which are not post-translationally removed
indicate a membrane-buried or translocated N-terminal. The
PSI-I polypeptide from M. polymorph has a negatively
charged C-terminal region, but the N-terminal region is hydrophobic and therefore the polypeptide from M. polymorph
is also predicted to have the C-terminal protruding into the
stroma.
A 4.0-kDa P ~ l y p e p t of
~ eP ~ ~ t ~ s y sI ~ e r n
18405
after removal of the detergent by repeated washing with 0.1%
trifluoroacetic acid after adsorption of the proteins to the
The psaI gene in barley encodes a 4.0-kDa PS I polypeptide nitrocellulose layer.
denoted PSI-I. The gene is positioned near the rbcL gene at
PSI-I is very hy~ophobicand tightly bound to CP1, the
a position similar to theposition in tobacco and M. potymor- heterodimer of the 82-83-kDa polypeptides PSI-A and PSIpha. Previously, three chloroplast genes,psaA,psaB, andpsaC B. Treatment of PS I with 3.2 M NaSCN completely dissohave been shown to encode PS I subunits. The position of ciates the 18-, lo&, and 9-kDa polypeptides (PSI-D, PSI-E,
psaA and psaB on the barley chloroplast genome has been and PSI-C, respectively) from CP1 and partlydissociates the
determined (26). The psaC gene has not been located on the 10.2-kDa polypeptide (PSI-H) from CP1 (l),whereas the 14barley chloroplast genome, but in other species it has been
kDa polypeptide, PSI-I,and the other 4-kDa polypeptide
located in the small single copy region (14, 18, 19, 32). The
remain associated with CP1 (13). P700, Ao, AI, and X have
gene psaI is therefore positioned far from the other known
all been reported to be bound to CP1(39-43). The treatments
genes which encode PS I components. Several subunits of
used to separate low molecular mass subunits from CP1 while
molecular mass below 5 kDa have been described in PS I1 and
in the cytochrome f/bs complex (33-38). Low molecular mass leaving the electron transfer components intact may not have
polypeptides derived by proteolytic degradation of higher been sufficiently harsh to dissociate the most hydrophobic
molecular mass subunits have also been reported (35). The low molecular mass polypeptides including PSI-I (39-43).
identification of the gene which encodesPSI-I eliminates the Evidence suggests that P700 is a chlorophyll dimer, A0 may
be a special chlorophyll molecule, and A, is thought to be a
possibility of PSI-I being a proteolytic degradation product.
pair
of quinones (2, 3, 42). X is a [4Fe-4S] iron-sulfur cluster
Plasma desorption mass spectrometry provides a conclusive
(13,
43). Four cysteine residues of the 82- and 83-kDa heterdetermination of the molecular mass of proteins andpeptides.
The molecular mass of PSI-I as determined by this method odimer are thought to provide the ligands for center X (40),
was higher than themolecular mass deduced fromthe nucleo- whereas it has notyet been possible to determine the binding
tide sequence. The difference in molecular mass is consistent sites for P700, A,, and AI. Contamination of CP1 with PSI-I
with the presence of a formyl group on the N-terminal me- would probably not have beendetected by the electrophoretic
thionine and the presence of methionine sulfone. It remains systems used in earlier studies (39-43). Therefore, it cannot
other 4-kDa polypeptide
to be shown whether the presence of methionine sulfone is be ruled out that PSI-I and the
caused by chemical oxidation during protein isolation or rep- participate in the binding of P700, Ao, or AI.
Search for proteins homologous to PSI-Iled to thediscovery
resents a specific post-translational modification in the native
polypeptide is homologous to helix E of the D2
PSI-I polypeptide. The mass spectrometric determination of that the PSI-I
the molecular mass of the isolated PSI-I polypeptide elimi- reaction center polypeptide of PS 11 (Fig. 6). Eleven residues
nates the possibility of further post-translational modifica- are identical corresponding to a homology of 31%. Eleven of
tion. The use of plasma desorption mass spectrometry for the the nonidentical amino acid residues represent conservative
determination of molecular masses of proteins haspreviously substitutions. In PS 11, a heterodimer of the polypeptides D l
been limited to soluble proteins, In thepresent study, we have and D2 is known to bind the electron transfer components (3,
found that good spectra of membrane proteins are obtained 44). D l and D2 are homologous to each other and to the L
and M subunits of the crystallized Rhdopseudomonas viridis
reaction center (31, 44). Each of the proteins has five transmembrane a-helices denoted A to E. Helices C and D are
intimately involved in the binding of the reaction center
chlorophyll, the accessory chlorophylls, and thepheophytins,
whereas the quinones QA and QB are bound to helix D and
t
the loop connecting helices D and E (44). A histidine residue
in helix E is involved in the binding of non-heme iron (44),
but this residue is not conserved in PSI-I. Helix E is oriented
with the N-terminal part toward the stroma which is opposite
of the predicted orientation for PSI-I. Apart from D2, the
search for homologous proteins showed several ubiquinoneNADH oxidoreductases to be homologous to PSI-I. The quinone-binding domain of the ubiquinone-NADH oxidoreduc+
++
tases has not been determined. Thus, theobserved homologies
provide no direct clue to thespecific function of PSI-I. NeverFIG. 4. Hydropathy plot of the PSI-I polypeptide from bar- theless, the homology between the PSI-Ipolypeptide and helix
ley. The hydropathy plot was calculated with an averaging window
of 7 amino acid residues and using membrane helix parameters E of D2 and the strong association of PSI-I to CP1makes it
according to Rao and Argos (29). The predicted (29) membrane- tempting to speculate that thehitherto overlooked P S I
spanning a-helix is indicated by the wavy line. Charged amino acid polypeptides with molecular masses below 5 kDa participate
residues are indicated.
in the binding of the electron transfer components of PS I. If
DISCUSSION
u _
I____
M-T-D-L-N-L-P-S-I-F-V-P-L-V-G-L-V-F-P-A-I-A-M-T-S-L-F-L-Y-V-Q-K-K-K-I-V
Barley:
1 1 : 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 l l l l : 1 1 1 1 . 1 1 1 : 1 1 1
Tobacco:
8-T-N-L-N-L-P-S-I-F-V-P-L-V-G-L-V-F-P-A-I-A-M-A-S-L-F-L-H-V-Q-K-N-K-I-V
Marchantia:
1 1 .
I l l l i l l l l l l : l l l i : I l I I I : . : : : : .
I :
M-T-A-S-Y-L-P-S-I-P-V-P-L-V-G-L-I-F-P-A-I-T-M-A-S-L-F-I-Y-I-E-Q-D-E-I-L
FIG. 5. Homology between PSI-I from different species. The amino acid sequence for PSI-I from tobacco
and M.p o l y ~ o r was
p ~ deduced from the nucleotide sequence of the chloroplast genomes (18,19). Wavy line,
putative membrane-spanning a-helix; vertical line, identical residues; double dot, conservative substitutions; single
dot, neutral substitutions with .
18406
A 4.O-kDuPolypeptide of P ~ t o s y s t 1e ~
D2
/K-R-W-L-B-F-F-M-L-F-V-P-V-T-G-L-W-M-S-A-I-G-V-V-G-L-A-L-N-L-R-A-Y-D-F-V/
PSI-I
ORP34
.
I : :
: I I I : . I I
. : l l : : . : l
I
: :
. : I
M-T-D-L-N-L-P-S-I-F-V-P-L-V-G-L-V-F-P-A-I-A-M-T-S-L-F-L-Y-V-Q-K-K-K-1-V
[ . . I
: : I :
: ] : : I
1 1
I t :
M-E-A-L-V-Y-T-F-L-L-V-S-T-L-G-I-I-F-F-A-I-F-F-R-E-P-P-K-V-P-T-K-K-N
.
. .
FIG. 6. Homology between PSI-I, D2, and a ~ y ~ t h e t34-residue
ic~
polypeptide from tobacco. The
amino acid sequence segment of D2 from barley was determined by Neumann (27). The amino acid sequence of
the 34-residue ORF34 product was deduced from the nucleotide sequence of the tobacco chloroplast genome (18).
Symbols: see legend to Fig. 5.
the PS I reaction center complex has a structure similar to
that of PS 11, an additional chloroplast-enc~edpolypeptide
homologous to PSI-I would be expected to be present in PS
I. Therefore, the tobacco and M. polymorph chloroplast
genomes (18,19) were searched for small open reading frames
encoding for products homologous to PSI-I. A hypothetical
polypeptide with 34 residues in tobacco and 35 residues in M.
polymorph was found to be weakly homologousto PSI-I with
a central hy~ophobicregion predicted to be membrane-spanning (Fig. 6). However, the hypothetical34/35-residue product is not particularly homologous to Dl. The barley PS I
preparations have a component of an apparent molecular
mass of 4 kDa in addition to PSI-I,but no amino acid sequence
information has been obtained for this component. Future
studies will showwhether the function of PSI-I and the other
4-kDa polypeptide of PS I is to participate inbinding of P700,
&, or AI of PS Iin a manner similar to thatof the membrane
helices of D l and D2 of PS 11.
Acknowledgments-Dr. C. Poulsen is thanked for kindly providing
the pHvC186 clone containing the psal gene and for providing additional information regarding the characteristics of this clone. Hanne
Linde Nielsen, Inga Olsen, Bodil Corneliussen, Pia Breddam, Lone
Serensen, and Lene Skou are thanked for skillful technical assistance.
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