Download ldentification of Surface-Exposed Domains on the Reducing Side of

Plant Physiol. (1994) 106: 617-624
ldentification of Surface-Exposed Domains on the Reducing
Side of Photosystem I'
Qiang Xu, James A. Cuikema, and Parag R. Chitnis*
Division of Biology, Kansas State University, Manhattan, Kansas 66506-4901
centers, A,, A,, and Fx. PsaC binds two [4Fe-4S] clusters, FA
and FB(Oh-oka et al., 1988). PsaD provides an essential Fddocking site on the reducing side of PSI (Zanetti and Merati,
1987; Zilber and Malkin, 1988; Lelong et al., 1994; Xu et al.,
1994a) and is also required for the stable assembly of PsaC
and PsaE into the PSI complex (Li et al., 1991; Chitnis and
Nelson, 1992). PsaE is required for Fd reduction (Rousseau
et al., 1993; Sonoike et al., 1993; Strotmann and Weber, 1993;
Xu et al., 1994a) and may be involved in cyclic electron flow
around PSI (Yu et al., 1993). PsaL is essential for the formation of PSI trimers (Chitnis and Chitnis, 1993). PsaF is
exposed to the p-side (lumenal) of the photosynthetic membranes and can be cross-linked with plastocyanin or Cyt c6
(Hippler et al., 1989; Wynn et al., 1989a, 1989b), but is not
essential for the photooxidation of Cyt c6 (xu et al., 199413).
Other subunits, such as PsaJ, PsaK, PsaI, and PsaM, are much
smaller ( c 5 . 5 kD) and are conserved from cyanobacteria to
higher plants, but their functions are not known (Ikeuchi et
al., 1993; Xu et al., 1994b).
The recent*x-raydiffraction analysis of cyanobacterial PSI
crystals at 6-A resolution (Krauss et al., 1993), in combination
with biochemical studies (Golbeck, 1993), provides a framework for understanding the overall architecture of PSI. PsaC,
PsaD, and PsaE are peripheral subunits, located on the nside (stromal in chloroplasts and cytoplasmic in cyanobacteria) of photosynthetic membranes, with PsaC positioned in
the center of each monomeric PSI on a local pseudo-2-fold
axis of symmetry (Krauss et al., 1993). Besides structural
studies, the subunit interactions in PSI are also deciphered
from biochemical experiments and characterization of PSI
mutants. For example, the availability of subunit-deficient
PSI complexes and improved Tricine-urea-SDS-PAGE has
revealed structural interactions among PsaE, PsaF, and/or
PsaJ (Xu et al., 1994b) or between PsaD and PsaL (Q. Xu,
P.R. Chitnis, unpublished data). These findings are corroborated by chemical cross-linking of these subunits (T.S. Armbrust, J.A. Guikema, unpublished data).
The detailed architecture of PSI is yet to be determined.
Protease accessibility studies have provided insights regarding organization and topology of PSI subunits (Zilber and
Malkin, 1992). In higher plant PSI, PsaA, PsaB, PsaD, PsaE,
PsaH, PsaK, and PsaL have stroma-exposed regions (Zilber
and Malkin, 1992). Mapping of the proteolytic fragments of
PsaD and PsaE indicates that N-terminal extensions of PsaD
and PsaE are accessible to proteolysis (Lagoutte and Vallon,
Photosystem I (PSI) i s a multisubunit enzyme that catalyzes the
light-driven oxidation of plastocyanin or cytochrome c6 and the
concomitant photoreduction of ferredoxin or flavodoxin. To identify the surface-exposed domains in PSI of the cyanobaderium
Synechocystis sp. PCC 6803, we mapped the regions in PsaE, PsaD,
and PsaF that are accessible to proteases and N-hydroxysuccinimidobiotin (NHS-biotin). Upon exposure of PSI complexes to a low
concentration of endoproteinase glutamic acid (CIu)-C, PsaE was
cleaved to 7.1- and 6.6-kD N-terminal fragments without significant cleavage of other subunits. C W and Clu6', located near the
C terminus of PsaE, were the most likely cleavage sites. At higher
protease concentrations, the PsaE fragments were further cleaved
and an N-terminal 9.8-kD PsaD fragment accumulated, demonstrating the accessibility of Clu residue(s) in the C-terminal domain
of PsaD to the protease. Besides these major, primary cleavage
products, severa1 secondary cleavage sites on PsaD, PsaE, and PsaF
were also identified. PsaF resisted proteolysis when PsaD and PsaE
were intact. Clu' and C I U ' ~
of~PsaF became susceptible to endoproteinase CIu-C upon extensive cleavage of PsaD and PsaE. Modification of PSI proteins with NHS-biotin and subsequent cleavage
by endoproteinase CIu-C or thermolysin showed that the intact
PsaE and PsaD, but not their major degradation produds lacking
C-terminal domains, were heavily biotinylated. Therefore, lysine74 at the C terminus of PsaE was accessible for biotinylation.
Similarly, lysine-107, or lysine-118, or both i n PsaD could be
modified by NHS-biotin.
PSI in cyanobacteria and chloroplasts is a Chl-binding
membrane-protein complex that catalyzes light-dependent
electron transfer from reduced plastocyanin or Cyt c6 to
oxidized Fd or flavodoxin (Chitnis and Nelson, 1991; Bryant,
1992; Golbeck, 1993). PSI contains at least 11 polypeptides
in cyanobacteria (Miihlenhoff et al., 1993) and three additional proteins, PsaG, PsaH, and PsaN, in higher plants
(Okkels et al., 1989, 1992; Knoetzel and Simpson, 1993).
PsaA and PsaB form a heterodimeric core that harbors the
primary electron donor, P700, and a chain of electron transfer
'
This work was supported in part by grants from the National
Science Foundation (MCB#9202751 to P.R.C.),the U.S. Department
of Agriculture-National Research Initiative Competitive Grants Program (USDA-NRICGP) (92-37306-7661 to P.R.C. and 93-373069147 to J.A.G.),and the National Aeronautics and Space Administration (NAGW 2328 to J.A.G.).We also achowledge an equipment
grant from the USDA-NRICGP (93-3731 1-9456 to P.R.C.).This is
contribution 94-538-J from the Kansas Agricultura1 Experiment
Station.
* Corresponding author; fax 1-913-532-6653.
Abbreviations: Glu-C, endoproteinase Glu-C; NHS-biotin,
N-hydroxysuccinimidobiotin.
61 7
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Xu et at.
61 8
1992; Zilber and Malkin, 1992). However, these N-terminal
extensions are absent in cyanobacterial PsaD and PsaE. Although the three-dimensional structure of cyanobacterial
PsaE in solution has recently been proposed from multidimensional NMR analysis (Falzone et al., 1994a, 1994b), data
regarding accessibility of cyanobacterial PsaE to proteases are
not available. Hence, the positioning of PsaE relative to the
photosynthetic membranes has not been determined. Clearly,
the interactions among subunits of PSI and surface topology
of the complex remain largely unknown.
Here we treated PSI complexes with Glu-C and mapped
the proteolytic peptides by N-terminal amino acid sequencing. We also labeled the PSI complexes with NHS-biotin and
mapped the biotinylation sites on PsaD and PsaE. Based on
these data, we identified the surface-exposed domains of
PsaD and PsaE in cyanobacterial PSI.
MATERIALS A N D METHODS
Materials
Prestained protein molecular mass standards were from
GIBCO-BRL. The centrifugal filtration apparatus (Centricon100 with 100-kD cutoff membrane) was from Amicon (Beverly, MA) and the polyvinylidine difluoride (Immobilon-P)
membrane was from Millipore (Bedford, MA). Avidin-peroxidase and NHS-biotin were from Sigma. Sequencing-grade
Glu-C (protease V8 from Staphylococcus aureus; EC 3.4.21.19)
was purchased from Boehringer Mannheim, and thermolysin
(protease type X from Bacillus thermoproteolyticus; EC
3.4.24.4) was from Sigma. The enhanced chemiluminescence
reagents for westem blotting were purchased from Amersham. Other chemicals were from Sigma or Fisher.
Plant Physiol. Vol. 106, 1994
The reaction was terminated with 20 m~ EDTA. After protease treatment, the association of cleavage products with the
PSI core could be examined by incubating the complexes
with Na1 (Xu et al., 1994a), followed by diluion with an
excess amount of 10 mM Mops-HC1 (pH 7.C9 containing
0.05% Triton X-100 and filtration with the Centricon-100.
This procedure desalts the PSI complexes and separates them
from the proteins released during incubation with NaI.
Modification of the PSI Subunits with NHS-Biotin
PSI complexes at 150 pg Chl/mL were incubated with 58
NHS-biotin, 10 mM Mops (pH 7.0), 0.05% ‘Triton X-100,
and 0.05% DMSO for 30 min at room temperature. The
reaction was quenched with 50 m~ ammonium bicarbonate
(pH 7.8). The labeled PSI subunits were separatcmd by Tricineurea-SDS-PAGE and electroblotted to Immobilon-P membranes. The blot was probed with an avidin-peroxidase
conjugate and developed with hydrogen peroxide and 4chloro-1-naphthol (Frankel and Bricker, 1992).
p~
Analytical Gel Electrophoresis and lmmunodelection
PSI complexes were solubilized with 1% SDS and 0.1% 2mercaptoethanol at room temperature for 1 h. Polypeptides
were resolved by Tricine-urea-SDS-PAGE (Xu et al., 1994a).
After electrophoresis, gels were stained with Coomassie blue
or silver nitrate. Altematively, proteins were tlectro-transferred to Immobilon-P membranes. Immunodetection was
performed using enhanced chemiluminescencereagents. The
antibody against PsaD of Synechococcus sp. PCC 7002 was a
gift from Dr. John H. Golbeck at the University of Nebraska.
Other antibodies were raised against Synechocystis sp. PCC
6803 proteins.
Isolation and Characterization of PSI Complexes
Synechocystis sp. PCC 6803 culture was grown in BG-11
under a light intensity of 21 pmol m-’ s-’ and harvested at
late exponential phase of growth. The cells were suspended
in 0.4 M SUC,10 m~ NaC1,200 m~ PMSF, 5 m~ benzamidine,
and 10 mM Mops-HC1 (pH 7.0), and photosynthetic membranes were isolated using a mini bead-beater (Xu et ai.,
1994b). The membranes were treated with 10 mg Triton/mg
Chl, and PSI complexes were purified by ion-exchange chromatography and Suc gradient ultracentrifugation (Xu et al.,
1994b). PSI isolated by this method can function as a lightdriven Cyt c6-Fd oxidoreductase (Xu et al., 1994b). Chl concentrations were determined in 80% (v/v) acetone (Amon,
1949).
Treatment of PSI Complexes with Proteases
Purified PSI was adjusted to 150 pg Chl/mL prior to
proteolytic cleavage. For Glu-C cleavage, PSI preparations
were incubated with O to 100 pg Glu-C/mg Chl in the
presence of 50 m~ ammonium bicarbonate (pH 7.8) at 15OC.
Under these conditions, Glu-C cleaves the peptide bond at
the carboxyl side of glutamyl residues (Houmard and Drapeau, 1972). The reaction was terminated with 10 m~ PMSF.
For cleavage by thermolysin, PSI was incubated with the
protease (20 pg/mg Chl) and 5 mM CaC& at 37OC for 40 min.
N-Terminal Amino Acid Sequencing
Peptides were blotted to Immobilon-P membranes, stained
with Coomassie blue containing 1% acetic acid for 2 min,
destained with 50% methanol, and rinsed extensively with
deionized water. N-terminal sequences were determined on
an Applied Biosystems (Foster City, CA) 477A sequencer at
the Biotechnology Core Facility of Kansas State University.
RESULTS
Accessibility of PSI Subunits to GIu-C
We treated PSI complexes with dfferent concentrations of
Glu-C and subsequently examined PSI subunits by SDSPAGE to estimate their relative accessibility to proteolysis
(Fig. 1).Upon exposure of PSI complexes to 1 p.g Glu-C/mg
Chl, most of the PsaE subunit was degraded without a
significant cleavage of other subunits and with s,imultaneous
appearance of 7.1- and 6.7-kD peptides, termed E1 and E11
(Fig. 1A). A polyclonal antibody against PsaE of Synechocystis
sp. PCC 6803 recognized both E1 and E11 fragments (data not
shown). At Glu-C concentrations of 5 pg/mg Chl or higher,
additional PSI subunits were degraded. Notably, a 9.8-kD
fragment (labeled DI) accumulated, with a concomitant decrease in the relative amount of PsaD. This fragment was
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Topology of PSI Subunits
^ Glu-C/mg Chl
0
1
5
20
50
100
Figure 1. Accessibility of PSI subunits to Clu-C. A, Purified PSI
complexes were treated with 0, 1, 5, 20, 50, and 100 fig Glu-C/mg
Chl at 15°C for 20 min. The polypeptides in PSI complexes containing 5 ng of Chl were separated by Tricine-urea-SDS-PACE and
visualized by silver staining. The subunits labeled in the figure have
been identified using immunodetection and N-terminal amino acid
sequencing (Xu et al., 1994b), and their molecular masses were
determined from migration of the following protein markers: insulin
(2.9 kD), bovine trypsin inhibitor (6.2 kD), lysozyme (14.3 kD), /3lactoglobulin (18.4 kD), carbonic anhydrase (29 kD), ovalbumin (43
kD), and BSA (68 kD). The PsaA-PsaB, PsaD, PsaF, PsaL, PsaE, PsaC,
PsaK, PsaJ, Psal, and PsaM of Synecriocyst/s sp. PCC 6803 migrated
on a Tricine-urea-SDS-polyacrylamide gel with apparent molecular
masses of 66, 18.2, 15.7, 14.2,8.9,8.0, 5.2, 3.4, 3.1, and 2.9 kD. B,
The PSI complexes were treated with 0 and 20 ^g Clu-C/mg Chl as
in A (lanes 0 and 20). An identical sample of protease-treated PSI
complexes was ultrafiltered using a Centricon-100. The retentate
was resuspended with 12% Sue, 0.05% Triton X-100, and 10 ITIM
Mops-HCI, pH 7.0 (lane 20*). The samples containing 10 ^g of Chl
were solubilized and proteins were separated by Tricine-urea-SDSPACE and visualized by staining the gel with Coomassie blue.
recognized by an anti-PsaD polyclonal antibody (data not
shown). PsaA-PsaB remained largely intact at Glu-C concentrations up to 20 ng/mg Chl. At higher protease concentrations, more fragments were observed; most of these were
detected only after silver staining. PsaL, PsaK, Psa], Psal, and
PsaM were resistant to Glu-C cleavage at all protease concentrations tested (Fig. 1A).
The results in Figure 1A would not distinguish between
proteolytic fragments that were released from PSI and those
that remained as peripheral PSI components. The El, EH, and
DI fragments were prominent species during cleavage of PSI
complexes with 20 ng Glu-C/mg Chl (Fig. IB). To determine
their relative association with the PSI core, the proteasetreated PSI complexes were filtered using the Centricon-100,
and retentates were analyzed by Tricine-urea-SDS-PAGE.
The El and EII cleavage products of PsaE were more tightly
associated with the PSI complexes than was the 9.8-kD DI
fragment of PsaD (Fig. IB, lane 20*). The stability of these
associations was further examined by incubation with 1 M
Nal prior to filtration. The El and EII fragments were relatively resistant to removal by Nal (data not shown).
When in solution, both PsaE (Rousseau et al., 1993) and
PsaD (data not shown) are completely cleaved by Glu-C.
Therefore, the data in Figure 1 demonstrated that specific
Glu residues of PsaD and PsaE are shielded from the protease
after their assembly into PSI complexes. These results
prompted identification of El, EII, and DI peptides by Nterminal amino acid sequencing (Table I). Both El and EII
fragments had N termini that matched the 2ALNRG terminus
of PsaE (Chitnis et al., 1989). Therefore Glu-C cleaved PsaE
at the Glu residues 1.8 and 2.3 kD from the C terminus to
generate the El and EII fragments. The N terminus of the DI
fragment had a sequence that matched the 2TELSG of PsaD
(Reilly et al., 1988) (Table I). Thus, a 6-kD C-terminal domain
of PsaD was cleaved by Glu-C. A 6-kD polypeptide was
observed on the polyacrylamide gel (Fig. 1A) but did not
accumulate to the same extent as the DI fragment. This
fragment may be more rapidly cleaved to smaller fragments.
These data showed that the Glu residues in the C-terminal
domains of PsaD and PsaE were accessible to Glu-C and thus
were exposed to the aqueous phase.
To identify secondary sites of Glu-C cleavage, we subjected
PSI complexes to extensive proteolysis using 100 jtg Glu-C/
mg Chl. Increased protease concentration yielded numerous
proteolytic fragments from PSI subunits, including FI, FII,
DII, Dili, DIV, DV, DVI, and EHI, with apparent molecular
masses of 13.3, 9.0, 6.4, 6.2, 4.4, 3.8, 3.5, and 2.5 kD,
respectively (Fig. 2). Although some of these fragments were
initially identified by western blotting (data not shown), we
determined amino acid sequences of their N termini for
accurate identification and mapping (Table H). These studies
revealed that all sequenced cleavage products were derived
from PsaD, PsaE, and PsaF. In contrast, PsaL and PsaK did
not decrease in intensity after extensive proteolysis, and none
of the peptides identified in this study (Tables I and II) were
derived from these subunits. This suggested that Glu residues
in PsaL and PsaK were not easily accessible to the protease.
The pattern of fragment appearance upon increasing proteolysis suggested a sequence of subunit accessibility to Glu-
Table I. N-terminal sequence analysis of prominent PSI fragments produced using Clu-C as described
in Figure /
Proteolytic
Fragment
Apparent
Mass"
El
Ell
DI
7.1
6.7
9.8
Sequencing
Cycle
Identification and Position of the
First Amino Acid
kD
ALNRC
ALNRC
TELSC
619
2
ALNRC of PsaE (Chitnis et al., 1989a)
ALNRC of PsaE (Chitnis et al., 1989a)
2
TELSC of PsaD (Reilly et al., 1988)
2
' Average of three independent estimates from the electrophoretic mobility.
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620
Xu et al.
Plant Physiol. Vol. 106, 1994
Hg Glu-C/mg Chl
0
^g Glu-C/mg Chl
100
0
1
5
20
50
100
PsaA-PsaB
Anti-PsaD
\nti-PsaF
EIII
Figure 2. Extensive cleavage of PSI complexes with Clu-C. The PSI
complexes (lane 0) were treated with 100 Mg Glu-C/mg Chl at 37°C
for 1 h (lane 100). The complexes containing 15 v% of Chl were
solubilized. The proteins were separated by Tricine-urea-SDS-PAGE
and electroblotted on Immobilon-P membranes. The membranes
were stained with Coomassie blue in 1% acetic acid.
C. For example, PsaE was readily cleaved at Glu-C concentrations that have minimal impact on PsaD (Fig. 1). When
levels of PsaD and PsaF were immunodetected after cleavage
of PSI with increasing concentrations of Glu-C (Fig. 3), PsaF
fragments were observed only after extensive degradation of
PsaA-PsaB, PsaD, and PsaE, which may expose Glu-C-sensitive sites on PsaF.
Figure 3. Immunodetection of PsaD and PsaF after Glu-C cleavage
of PSI. PSI was treated with 0, 1, 5, 20, 50, and 100 ^g Glu-C/mg
Chl as in Figure 1A. Proteins in the PSI complexes containing 5 ^g
of Chl were separated by Tricine-urea-SDS-PAGE, blotted on Immobilon-P membranes, and sequentially probed with antibodies
against PsaD (top) and PsaF (bottom).
Modification of PSI Subunits with NHS-Biotin
Analyses of the deduced amino acid sequences revealed
that PsaD and PsaE contain 12 and 4 lysyl residues, respectively (Reilly et al., 1988; Chitnis et al., 1989). NHS-biotin
reacts with the N terminus and the t-amino group of lysyl
residues (Bayer and Wilchek, 1990). Modification with NHSbiotin has been used to examine exposure of protein surfaces
to the aqueous phase (e.g. Frankel and Bricker, 1992). To
identify the surface-exposed lysyl residues in PSI polypeptides, we biotinylated the PSI complexes and detected the
protein modification using avidin peroxidase and a chromo-
Table II. N-terminal sequence analyses of the proteolytic fragments produced by extensive cleavage
of PSI complexes by Clu-C as described in Figure 2
Proteolytic
Apparent
Fragment
Mass"
Sequencing
Cycles
Identification and
Position of the First Amino Acid
kD
Fl
13.3
DDFANL
Dl
Fll"
9.8
9.0
TELSG
DDFAN
DM
6.4
6.2
4.4
3.8
3.5
2.5
KYAIT
VQYLH
GENLL
AQGTK
QCLAL
ALNRG
DIM
DIV
DV
DVI
Elllc
'DDFANL of mature PsaF (Chitnis and Nelson,
1991)
2
TELSG of PsaD (Reilly et al., 1988)
'DDFANL of mature PsaF (Chitnis and Nelson,
1991)
"KYATT of PsaD (Reilly et al., 1988)
'"VQYLH of PsaD (Reilly et al., 1988)
"GENLL of PsaD (Reilly et al., 1988)
""AQGTK of PsaD (Reilly et al., 1988)
"QCLAL of PsaD (Reilly et al., 1988)
2
ALNRG of PsaE (Chitnis et al., 1989a)
b
' Average of three independent estimates from the electrophoretic mobility.
To facilitate
proper identification of Fll, PSI complexes from a PsaE-less mutant strain (Chitnis et al., 1989a) were
c
digested under the same condition as for the wild-type PSI.
The fragment EIII migrated below
2.9-kD insulin marker and its apparent mass was calculated by extrapolation of the calibration curve.
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Copyright © 1994 American Society of Plant Biologists. All rights reserved.
621
Topology of PSI Subunits
DISCUSSION
PsaA-PsaB
PuDPsaFPsaL-
El
P«aK-
Figure 4. PsaE modification by NHS-biotin and cleavage with CluC. The biotinylated PSI complexes (lane 1) were cleaved with CluC at concentration of 2 /ig/mg Chl for 20 min to yield the PsaE
fragments lacking the C terminus (lane 2). The preparations equivalent to 10 Mg of Chl were solubilized and the proteins were
separated by Tricine-urea-SDS-PACE, electroblotted on Immobilon-P membranes, and stained with Coomassie blue. A replica blot
of lanes 1 and 2 was probed with avidin-peroxidase conjugate
(lanes 3 and 4). The immunoreaction was visualized using hydrogen
peroxide and 4-chloro-l-naphthol.
genie substrate for peroxidase. PsaA-PsaB, PsaD, PsaF, PsaL,
and PsaE showed significant amounts of biotin incorporation
(Fig. 4). When biotinylated PSI was subjected to GIu-C cleavage to generate El and EII fragments (Fig. 4, lanes 1 and 2),
most the biotin-avidin peroxidase-generated signal from PsaE
was lost upon its cleavage (Fig. 4, lane 4). There is a lysyl
residue (Lys74) at the C terminus of PsaE (Chitnis et al., 1989).
Based on the difference in biotinylation intensity of PsaE and
its proteolytic products El and EII, Lys74 is the most likely
NHS-biotin reactive site on PsaE.
The treatment of biotinylated PSI with higher concentration of Glu-C produced the DI fragment of PsaD (Fig. 5). A
comparison of the biotinylation-dependent signal and the
Coomassie blue staining intensity showed that most of the
biotinylation signal in the intact PsaD was not present in the
DI fragment (Fig. 5, lane 4). Four lysyl residues (Lys107, Lys118,
Lys132, and Lys136) in the C-terminal 6-kD domain of PsaD
(Reilly et al., 1988) are removed by Glu-C to generate the DI
fragment (Table III). Therefore, one or more of these residues
are exposed to the aqueous phase and can be biotinylated.
To identify modified lysyl residues in PsaD more accurately,
we treated the biotinylated PSI with thermolysin. A 13.8-kD
fragment was generated by thermolysin, albeit to a lesser
level than the Glu-C-generated fragments (Fig. 6). Thermolysin cleaves PsaD toward its C terminus to generate the
13.8-kD fragment containing Lys107 and Lys118 (Xu et al.,
1994a). This fragment was labeled with NHS-biotin (Fig. 6,
lane 4), indicating that Lys107, Lys118, or both could react to
NHS-biotin.
Chemical cross-linking, protease accessibility, and in vitro
reconstitution experiments have revealed that PsaD, PsaE,
and PsaC are exposed on the n-side of photosynthetic membranes and that a considerable part of PsaC is buried under
PsaD and PsaE (Oh-oka et al., 1989; Zilber and Malkin,
1992), PsaF is substantially exposed on the p-side of the
photosynthetic membranes (Wynn and Malkin, 1988; Hippler
et al., 1989; Wynn et al., 1989a, 1989b). Here we examined
the surface exposure of PsaD, PsaE, and PsaF in a topological
assessment of PSI. For this purpose we used proteolytic
cleavage and chemical modification experiments that probe
different reactive groups with different levels of accessibility
from the aqueous phase. Glu-C, a 34-kD protease, recognizes
peptide bonds that are C terminal to glutamyl residues, and
NHS-biotin, a smaller molecule, recognizes primary amines
in a protein.
The solution conformation of Synechococcus sp. PCC 7002
PsaE, proposed from NMR analysis, reveals that the protein
has an antiparallel five-stranded /3-sheet structure (Falzone
et al., 1994a, 1994b). Because of high sequence identity
among cyanobacterial PsaE, the structure of Synechococcus
sp. PCC 7002 PsaE provides a reliable model for Synechocystis
sp. PCC 6803 PsaE. There are five glutamyl residues at
positions of 15, 29, 63, 65, and 67 in PsaE of Synechocystis
sp. PCC 6803 (Chitnis et al., 1989). Among them, glutamyl
residues at positions 15, 63, 65, and 67 are conserved between
the PsaE subunits from the two cyanobacterial species. The
peptide mapping of the major PsaE fragments (Table I) and
1
PsaA-PsaB
Figure 5. PSI modification by NHS-biotin and cleavage with CluC. The biotinylated PSI complexes (lane 1) were cleaved with GluC (20 fig protease/mg Chl) for 20 min to yield the PsaD fragments
lacking the C terminus (lane 2). The preparations equivalent to 10
^g of Chl were solubilized and the proteins were separated by
Tricine-urea-SDS-PACE, electroblotted on Immobilon-P membranes, and stained with Coomassie blue. A replica blot of lanes 1
and 2 was probed with avidin-peroxidase conjugate (lanes 3 and
4). The immunoreaction was visualized using hydrogen peroxide
and 4-chloro-1-naphthol.
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Copyright © 1994 American Society of Plant Biologists. All rights reserved.
Xu et al.
622
Plant Physiol. Vcd. 106, 1994
Table 111. Predicted fragments of PsaE, PsaD, and PsaF after complete digestion with Clu-C
These analyses are based on t h e deduced amino acid sequences of psaE ((Chitniset al., 1989a), psaD (Reilly et al., 1988), and psaF (Chitnis
and Nelson, 1991).
Predicted CIu-C Fragment
PsaE fragments
'ALN RC DKVSlKRT' 5E
16SYWYG DVCTVASVZ9E
"KSG I LYPVIVRFDRVNY NC FSCSASGVNTNNFA63E
6 4 ~ 6 5 ~
6 6 ~ 6 7 ~
68LVQAAA74K
PsaD Fragments
'T3E
4LSCQPPKFGCSTGCLLSKAN RZ5E
26E
27KYAITWTSAS37E
38QVF4'E
42M
PTCGAAIMNs2E
53GE54
55NLLYLARK63E
MQCLALGTQLRTKFKPKlQDYKIYRVYPSG93E
94VQYLHPADCVFPlo6E
1 0 7 ~ ~ ~ 1 1 0 ~
111GR113E
114AQGTKTRRICNP127E
PVTlKFSGKAPY14"E
1
4
1
v
PsaF Fragments
DDFANLTPTSE"
1ZNPAYLAKSKNFLNTTNDPNSGKIRAE37
"RYASALCC PE47
48CYPH LlVDGRFTHACDFLlPSILFLYIAGW IGWVGRSYLIEB8
891RE91
92SKNPEq6
97MQEq9
'%I NVPLAlKKMLCCFLW PLAAVC E' 24
'25YTSCKLVMKDSE' 36
'371PTSPR142
predicted Glu-C fragments of PsaE (Table 111) indicate that
the primary Glu-C cleavage sites may be in a cluster of Glu
residues at positions 63, 65, and 67. Examination of the
structure of Synechococcus sp. PCC 7002 PsaE revealed that
the C-terminal peptide bonds of Glu"' and G1d6(corresponding to G l d 3 and G l d 7 in Synechocystis sp. PCC -6803 PsaE),
but not of G l d 4 (corresponding to G l d 5 in Synechocystis sp.
PCC 6803 PsaE), are accessible from the surface. Therefore,
GW3 and G l d 7 in Synechocystis sp. PCC 6803 PsaE are the
most likely sites of primary cleavage by Glu-C. The remaining
Glu residues in PsaE are not readily accessible to Glu-C and
therefore may be shielded by the other PSI subunits. The
loop of PsaE, located opposite the surface-exposed Gld3GhP7 region, varies in length depending on the source of
PsaE and may anchor PsaE in the PSI complex. Interestingly,
a 2.5-kD PsaE fragment with N termini of PsaE was accumulated when the PSI complexes were extensively degraded
by Glu-C. This implies that the Glu15, which is near the Nterminal domain of PsaE, may be relatively shielded from
Mass
Mass from the N
Terminus
kD
kD
1.585
1.531
3.690
0.261
0.260
0.699
1.585
3.116
6.806
7.067
7.327
8.026
0.248
2.199
O. 147
1.255
0.521
1.O90
0.204
1.118
3.541
0.248
2.447
2.594
3.849
4.370
5.460
5.664
6.782
10.323
1.470
0.488
0.360
1.554
1.435
0.117
11.793
12.281
12.641
14.195
15.630
15.747
1.210
2.861
1.O65
4.659
0.41 6
0.573
0.406
2.633
1.356
0.669
1.210
4.071
5.136
9.795
10.21 1
10.784
11.190
13.823
15.179
15.848
Proposed
Identification
i IDV
Dlll
}
FII
FI
aqueous phase. It is possible that proteolytic cleavage of the
C-terminal domain of PsaD exposed the G h z 9of PsaE.
Glutamyl residues in the C-terminal domain of PsaD are
the primary cleavage sites for Glu-C (Table I). The C-terminal
domain of PsaD is also accessible to thermolyfiin (Xu et al.,
1994a). Similarly, the proteolytically cleaved P5;aD fragment
with an N terminus identical to that of intact PsaD was found
after chaotrope-induced release of peripheral proteins from
purified PSI of Synechococcus sp. PCC 6301 (Li et al., 1991).
When PSI complexes were extensively cleaved with Glu-C,
Glu5' and G l d 3 were accessible. The peptide m,ipping of the
observed PsaD fragments (Tables I and 11) and fragments
predicted from the deduced amino acid sequerte (Table 111)
indicated that G1ulo6,Glu"", and Glu113were readily accessible to Glu-C. Interestingly, Lys'07 of PsaD in Synechocystis
sp. PCC 6803 can be cross-linked with Glu9,' of Fd by a
hydrophilic, zero-length cross-linker, N-ethyl-3-(3-dimethylaminopropy1)carbodiimide (Lelong et al., 1994).
The glutamyl residues in the C-terminal domain of PsaF
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Topology of PSI Subunits
1
2
3
PuA-PsaB
PsaD
PsaF
PsaL
PsaD
fragment
Figure 6. PSI modification by NHS-biotin and cleavage with thermolysin. The biotinylated PSI complexes (lane 1) were cleaved with
thermolysin (20 jig protease/mg Chl) for 20 min to yield the PsaD
fragments lacking the C terminus (lane 2). The preparations equivalent to 10 Mg of Chl were solubilized and the proteins were
separated by Tricine-urea-SDS-PACE, electroblotted on Immobilon-P membranes, and stained with Coomassie blue. A replica blot
of lanes 1 and 2 was probed with avidin-peroxidase conjugate
(lanes 3 and 4). The immunoreaction was visualized using hydrogen
peroxide and 4-chloro-1-naphthol.
were also accessible to Glu-C. The 13.3- and 9.0-kD PsaF
fragments with intact N termini gradually accumulated when
PsaE and PsaD had been cleaved by Glu-C. Based on the
apparent masses of these proteolytic fragments and deduced
amino acid sequence (Chitnis et al., 1991), Glu124 and GIu88
were the likely cleavage sites. A hydrophobic domain preceding Glu124 is 20 amino acids in length and may span a
membrane. Although chemical cross-linking and functional
reconstitution studies have demonstrated that PsaF is exposed
to the lumenal side (Hippler et al., 1989; Wynn et al., 1989a,
1989b), several lines of evidence suggest that PsaF is an
integral membrane protein. First, the amino acid sequences
of PsaF deduced from the gene sequences reveal two moderately hydrophobic regions in this protein (Chitnis et al.,
1991). Second, PsaF resists removal from wild-type PSI by
chaotropic agents (Xu et al., 1994a, 1994b). However, it could
be released from PSI core upon exposure to a high concentration of detergent (Bengis and Nelson, 1977). Third, a crosslinked product that is recognized by both anti-PsaE and antiPsaF antibodies can be obtained by treatment of PSI with
glutaraldehyde, showing that PsaE and PsaF are in close
proximity (T.S. Armbrust, J.A. Guikema, unpublished data).
PsaF is synthesized as a precursor protein with a transit
sequence typical of proteins targeted to the thylakoid lumen
(Chitnis et al., 1991), suggesting that PsaF is positioned in
the membranes with its N terminus on the lumenal side and
its C-terminal hydrophilic domain on the n-side of the membranes. Thus, it is possible that extensive cleavage of the nside components of PSI, most notably PsaE and PsaD, expose
623
the C-terminal domain of PsaF. Alternatively, the C-terminal
domain of PsaF may be shielded by PsaA-PsaB loops that
are degraded after extensive proteolysis.
There is an apparent difference in the sensitivity of PSI
subunits to proteolysis. PsaE was selectively cleaved by GluC before detectable degradation of PsaD, PsaC, or other PSI
subunits (Fig. 1). A similar sensitivity pattern of the n-side
components of PSI to extraction by Nal has been observed
(Xu et al., 1994a, 1994b). Upon treatment of PSI complexes
with Nal, a significant amount of PsaE was released before
removal of PsaD and PsaC. The absence of PsaE leads to an
enhanced cleavage of PsaD by thermolysin (Xu et al., 1994a).
Both PsaD and PsaE are required for efficient Fd-mediated
NADP+ photoreduction by PSI (Xu et al., 1994a). PsaD can
also be chemically cross-linked with Fd (Merati and Zanetti,
1987; Zilber and Malkin, 1988; Lelong et al., 1994). Thus,
PsaD and PsaE may partially overlap and form the docking
site for the electron acceptors of PSI. These functionally
important docking domains of PsaD and PsaE are expected
to be surface exposed. Hence, the domains of PsaD and PsaE
that were identified in this study may form the Fd-docking
region on the reducing side of PSI.
ACKNOWLEDGMENTS
We acknowledge John Tomich and Gary Radke for their valuable
advice about N-terminal amino acid sequencing. We thank Vaishali
P. Chitnis for expert technical assistance and William R. Odom for
critically reading the manuscript. We also thank Juliette T.J. Lecomte
for providing structural coordinates of PsaE before publication.
Received May 23, 1994; accepted July 1, 1994.
Copyright Clearance Center: 0032-0889/94/106/0617/08.
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Copyright © 1994 American Society of Plant Biologists. All rights reserved.