Recent studies of the mutants that were blocked in State 1 revealed that thylakoid protein kinase Stt7 from green alga Chlamydomonas reinhardtii and its higher plant orthologue STN7 are
Trang 1photosystem I of Chlamydomonas reinhardtii under State 2 conditions
Joanna Kargul1, Maria V Turkina2, Jon Nield1, Sam Benson1, Alexander V Vener2
and James Barber1
1 Wolfson Laboratories, Division of Molecular Biosciences, Imperial College London, UK
2 Division of Cell Biology, Linko¨ping University, Sweden
Excitation of the membrane-bound protein complexes
photosystem I (PSI) and II (PSII) by light must be
optimized to ensure the highest efficiency of
photosyn-thetic electron transport Redistribution of excitation
energy between both photosystems as an immediate
and dynamic response to changing illumination
condi-tions occurs during the process termed ‘State
transi-tions’, where State 1 is induced by excess PSI light and
State 2 by excess PSII light [1] State 1 to State 2
transition occurs in response to the reduction of the
plastoquinone pool, triggering the activation of
thyla-koid-bound kinases which in turn phosphorylate the
mobile light-harvesting complex II (LHCII) antenna
[2–5] The phosphorylated LHCII is proposed to
transfer physically from PSII to PSI to balance energy
distribution between, and optimize the rate of electron transfer through, the two photosystems or induce cyclic electron flow around PSI [6–9] Conversely, in PSI-favouring light, oxidation of plastoquinone occurs, leading to deactivation of LHCII-specific kinases and dephosphorylation of mobile LHCII by redox-inde-pendent phosphatases As a consequence, LHCII deta-ches from PSI and functionally couples to PSII (State
2 to State 1 transition) Recent studies of the mutants that were blocked in State 1 revealed that thylakoid protein kinase Stt7 from green alga Chlamydomonas reinhardtii and its higher plant orthologue STN7 are required for phosphorylation of several LHCII poly-peptides [4,5], thus providing further evidence that pro-tein phosphorylation is essential for State transitions
Keywords
Chlamydomonas; CP29; photosynthesis;
protein phosphorylation; State transitions
Correspondence
J Barber, Division of Molecular Biosciences,
Imperial College London, South Kensington
Campus, London SW7 2AZ, UK
Fax: +44 20 7594 5267
Tel: +44 20 7594 5266
E-mail: j.barber@imperial.ac.uk
(Received 17 June 2005, revised 29 July
2005, accepted 2 August 2005)
doi:10.1111/j.1742-4658.2005.04894.x
The State 1 to State 2 transition in the photosynthetic membranes of plants and green algae involves the functional coupling of phosphorylated light-harvesting complexes of photosystem II (LHCII) to photosystem I (PSI)
We present evidence suggesting that in Chlamydomonas reinhardtii this coupling may be aided by a hyper-phosphorylated form of the LHCII-like CP29 protein (Lhcbm4) MS analysis of CP29 showed that Thr6, Thr16 and Thr32, and Ser102 are phosphorylated in State 2, whereas in State 1-exposed cells only phosphorylation of Thr6 and Thr32 could be detected The LHCI–PSI supercomplex isolated from the alga in State 2 was found
to contain strongly associated CP29 in phosphorylated form Electron microscopy suggests that the binding site for this highly phosphorylated CP29 is close to the PsaH protein It is therefore postulated that redox-dependent multiple phosphorylation of CP29 in green algae is an integral part of the State transition process in which the structural changes of CP29, induced by reversible phosphorylation, determine the affinity of LHCII for either of the two photosystems
Abbreviations
Chl, chlorophyll; DDM, b-dodecyl maltoside; EM, electron microscopy; IMAC, immobilized metal affinity chromatography; LHCII, light-harvesting complex II; PSI, photosystem I; PSII, photosystem II; S1 and S2, State 1 and State 2.
Trang 2Although it is well established that State transitions
are driven by the redox control of phosphorylation⁄
dephosphorylation of a mobile pool of LHCII and
that this mobile antenna system shuttles between PSI
and PSII [6–9], little is known about the structural
changes involved Lunde et al [11] showed that the
PsaH protein of PSI was important in establishing
State 2 and suggested that it could be the docking site
for phosphorylated LHCII This idea was recently
reinforced by the 4.4 A˚ X-ray structure of higher plant
PSI [12], in which the PsaH protein was shown to be
located at an exposed hydrophobic surface of PSI and
to bind a chlorophyll (Chl) molecule which may aid
energy transfer from phosphorylated LHCII to the PSI
complex The X-ray structure, however, lacked density
indicative of binding of LHCII in this region Indeed,
to date, there is no direct structural evidence of how
phosphorylated LHCII binds to PSI, although recent
cross-linking and antisense studies have provided some
evidence for binding of the LHCII antenna within the
PsaH⁄ I ⁄ O region of the PSI core in State 2 conditions
[13,14]
In this study, we set out to characterize the physical
association of phosphorylated LHCII to PSI in State 2
using biochemical analyses, electron microscopy and
single particle image averaging of LHCI–PSI
super-complexes isolated from the green alga C reinhardtii
Compared with the LHCI–PSI supercomplex isolated
from cells in State 1, we found an additional protein
density in the isolated State 2 LHCI–PSI supercomplex
in the vicinity of the PsaH protein region This extra
density seems to be due to the presence of a 35 kDa
phosphoprotein which was shown by MS analyses to
be the minor LHCII-like subunit, CP29 MS also
revealed that CP29 in thylakoids isolated from algal
cells, exposed to either State 2 or State 1 conditions,
underwent multiple differential phosphorylation events
Therefore, our data indicate involvement of CP29
phosphorylation in State transitions and suggest that
hyperphosphorylated CP29 may provide a functional
link between a mobile LHCII antenna and the PSI
core in State 2
Results
Biochemical characterization of State 1 and
State 2 LHCI–PSI supercomplexes
It is well established that when Chlamydomonas is
sub-jected to anaerobic conditions in the dark, the cells
convert from State 1 to State 2 due to over-reduction
of the redox pool linking PSI and PSII [10,15] Using
this procedure we were able to establish that this
conversion occurs by monitoring their low-temperature Chl emission spectrum and comparing it with that of Chlamydomonas cells in State 1 induced by normal aerobic dark conditions As shown in Fig 1, in State 2 the yield of fluorescence from PSI (peaking at
715 nm), which is a measure of its absorption cross-section, was significantly higher than that from PSI of State 1 cells, based on normalization with the fluores-cence from PSII (peaking at 685 nm) This result confirmed the increase of functional light-harvesting antenna in PSI during State 1 to State 2 transition Using previously optimized sucrose gradient frac-tionation of thylakoid membranes partially depleted from PSII and solubilized with 0.9% b-dodecyl malto-side (DDM) [16], we isolated LHCI–PSI complexes from State 1 (S1) and State 2 (S2)-induced Chlamydo-monas cells Three Chl-containing fractions were obtained with the densest fractions corresponding to the LHCI–PSI supercomplexes (S1–F3 and S2–F3 frac-tions) [16] The protein profiles of S1 and S2 thyla-koids (Thy) and also of the S1⁄ S2–F3 sucrose-gradient factions (Fig 2A) were essentially identical (as judged
by Coomassie Brilliant Blue staining) and similar to the S1 profiles reported previously [16] Western blot-ting and spectroscopic analyses of S1–F3 and S2–F3 fractions confirmed the presence of PSI core subunits and the functionally coupled LHCI antenna which
Fig 1 State transitions in C reinhardtii We measured 77 K fluor-escence emission spectra measured from the psbD-His cells, induced to State 1 (solid) or State 2 (dotted) Note the resultant rel-ative change in fluorescence of PSI (715 nm) and PSII (685 nm) owing to relative changes in the absorption cross-section of each photosystem Spectra were obtained from dark-adapted aerated cells (State 1) or from cells preadapted to anaerobic conditions in the dark (State 2).
Trang 3form the outer light-harvesting proteins of PSI (data
not shown) [16] These LHCI proteins are not
phos-phorylated in State 1 and indeed, there is no reported
evidence that they become phosphorylated in State 2
[17–19] Nevertheless, as shown in Fig 2B, significantly
increased phosphorylation of several proteins was
detected within intact S2 thylakoid membranes by
western blotting with antiphosphothreonine serum In
S1 thylakoids, only the 10 kDa phosphoprotein was
clearly detected under the conditions used, whereas in
S2 thylakoids additional phosphorylated proteins were
clearly distinguished in the range of 29–35 kDa
(Fig 2B) The latter proteins correspond to
phosphor-ylated CP29, CP26 and unresolved major LHCII
antenna polypeptides undergoing phosphorylation in
S2 thylakoids [4,10] In the case of the S2–F3 fraction,
a single phosphorylated protein of 35 kDa was
spe-cifically detected (arrowed in Fig 2B), which was not
present in the S1–F3 fraction Although
antithreonine serum readily interacted with the
phospho-LHCII solubilized from S2 thylakoid membranes, it
was less effective at detecting phospho-CP29 either in
the thylakoid membrane or in S2 LHCI–PSI
super-complex fractions (Fig 2B)
To identify the 35 kDa phosphoprotein in the S2–F3
fraction, the protein band was excised from the
poly-acrylamide gel and digested with trypsin The peptides
were extracted after the procedure of tryptic in-gel
digestion and subjected to tandem MS
Collision-induced fragmentation of peptide ions revealed
sequences of four peptides ranging in length from 12
to 27 amino acids (Table 1, peptides 1–4) The blast
database search [20] identified that all the subsequent peptides originate from the minor LHCII-like subunit CP29 The positions of the sequenced peptides in the sequence of the mature CP29 are indicated in Table 1 These data also confirmed recent findings [21] that the putative transit peptide of the nuclear-encoded CP29
in Chlamydomonas is not removed but processed by methionine excision and acetylation (peptide 1 in Table 1) However, we were unable to detect any phos-phopeptides from CP29, which could be explained by the frequently observed loss of the phosphorylated peptides during the in-gel digestion procedure and the following peptide extraction, as well as the suppressed ionization of the phosphorylated peptides in the pres-ence of nonphosphorylated ones [22] Importantly, no peptides corresponding to CP29 were detected in the S1–F3 sample subjected to identical tandem MS analy-sis, even though all the proteins present in the region
of 25–40 kDa were analysed by in-gel digestion fol-lowed by MS characterization
In order to investigate the status of CP29 phos-phorylation in the algal cells exposed to State 2 condi-tions, we subjected isolated thylakoid membranes to proteolytic ‘shaving’ and enriched the phosphopeptides
by immobilized metal affinity chromatography (IMAC) using the procedure described previously [21] Sequen-cing of the phosphopeptides obtained by nanospray quadrupole time-of-flight MS revealed four distinct phosphorylated peptides from the CP29 protein (Table 1, peptides 5–8)
Identification and mapping of the three previously unknown phosphorylation sites in CP29 was achieved
Fig 2 Protein composition and phosphorylation of thylakoids and LHCI–PSI obtained from State 1 and State 2 C reinhardtii cells (A) Protein profiles of thylakoids (Thy) and LHCI–PSI (F3) complexes obtained from psbD-His State 1- (S1) and State 2 (S2)-induced cells (B) Phosphory-lation of thylakoids and LHCI–PSI complexes isolated from psbD-His cells Proteins were separated on SDS ⁄ PAGE at 5 lg of Chl per lane Detection of phosphoproteins was performed with antiphosphothreonine serum as described previously [16] Protein size markers are indica-ted on the left The 35 and 10 kDa phosphobands are marked with arrows in (B) The 35 kDa protein was identified as CP29 by MS Posi-tions of other proteins were identified by western blotting as in Kargul et al [16].
Trang 4by collision-induced dissociation of the corresponding
peptide ions and examination of the resultant spectra
for the presence of the signals produced by ‘neutral
loss’ of phosphoric acid, which are characteristic
of phosphorylated peptides [22–24] Analysis of the
spectra for the presence of N- and C-terminal frag-ments that contain phosphate and show neutral loss of phosphoric acid allowed unambiguous localization of the exact phosphorylation sites The first (Fig 3A) and second (Fig 3B) peptides contained three threonine residues each, but only the third N-terminal threonine
Table 1 The sequences of tryptic peptides from CP29 revealed by
tandem MS A single-letter amino acid code is used; Ac- designates
N-terminal acetylation; the lower case ‘t’ and ‘s’ specify
phosphor-ylated threonine and serine residues, correspondingly Positions of
the peptides in the sequence of the mature CP29 are indicated by
corresponding amino acid numbers The sequences of the peptides
1–4 were obtained after in-gel digestion of the putative
phospho-protein from the State 2 LHCI–PSI supercomplex preparation The
sequences of the phosphorylated peptides 5–8 were obtained after
phosphopeptide enrichment from State 2 thylakoid membranes
(see Experimental procedures).
No Peptide sequence
Amino acid numbers
m/z
200 400 600 800
A
A G t T A T K P A P K
b 2 3 4 5 7 9
10 9 8 7 6 5 4 3 2 1 y
10 20
40
519.7
y9*
y9* 2+
y2
b4*
b3
y8 b9*
y7 y6
b7*
y4
b5*
b2 y1
b3*
y3
y5
C
N N K G s V E A I V Q A T P D E V S S E N R
13 12 11 10 9 8 6 5 4 2 y
m/z
20 40 60
200 600 1000 1400
832.1
y13
y12 y11
y9
b10
y10 b9
b8 y8 y9 2+
y6 y5
a5 a5*
y4
y2 a5* 2+
B
20 40
80
60
m/z
V A t S T G T R
7 6 5 4 3 2 1 y
443.7
y3 y4
y6* 2+
y7 b3*
y1
y6 y7*
y6*
b7*
y5 y5-H20
b6*
394.7
y2-H20
b2 a2
Fig 3 MS sequencing of three phosphorylated peptides from CP29
in C reinhardtii cells exposed to State 2 conditions The b
(N-terminal) and y (C-terminal) fragment ions are labelled and the
peptide sequences shown The lower case t and s in the sequences
designate phosphorylated Thr and Ser residues, respectively The
sites of phosphorylation were localized according to the pattern of
the fragment ions that do not contain phosphate and complimentary
ions containing phosphate and satellite signals with the neutral loss
of phosphoric acid (b and y ions marked with the asterisk) (A)
Frag-mentation spectrum of the doubly protonated peptide ion with
m ⁄ z ¼ 568.7 The pronounced doubly charged ion indicated at
m ⁄ z ¼ 519.7 corresponds the neutral loss of phosphoric acid from
the parent ion (568.7 · 2 ) 519.7 · 2 ¼ 98, which is the mass of
H 3 PO 4 ) Thr3 in the peptide is phosphorylated: see, particularly, b3
ion with the phosphate, b3* after the neutral loss of H3PO4 and
complementary y8 ion without phosphate (B) Fragmentation
spec-trum of the doubly protonated peptide ion with m ⁄ z ¼ 443.7
(indica-ted) The ion originated after the neutral loss of phosphoric acid is
indicated at m ⁄ z ¼ 394.7 Thr3 in the peptide is phosphorylated:
see, particularly, y5 ion without phosphate and y6 with the
phosphate plus b3* ion after the neutral loss of H 3 PO 4 (C)
Frag-mentation spectrum of the triply protonated phosphopeptide ion
with m ⁄ z ¼ 832.1 and corresponding ‘neutral loss’ signal at m ⁄ z ¼
799.4 (832.1 · 3 ) 799.4 · 3 ¼ 98) The peptide is phosphorylated
at Ser5: see y11 to y13 fragments without phosphate and b8 to b10
ions with the phosphate This pattern of fragment ions can only
originate from the peptide in which Ser5 is phosphorylated.
Trang 5in each of these peptides was found to be
phosphoryl-ated These residues correspond to positions 16 and 32
in the sequence of the mature CP29 (Table 1) The
third peptide contained one threonine and three serine
residues (Fig 3C) However, the fragmentation
spec-trum (Fig 3C) revealed that only the serine
corres-ponding to position 102 in the amino acid sequence of
CP29 was phosphorylated All three newly identified
phosphorylation sites are located in the long
N-termi-nus of CP29 exposed to the stromal side of thylakoid
membranes These findings (Table 1, Fig 3) are unique
because there is no other report of any thylakoid
pro-tein undergoing quadruple phosphorylation
To determine the extent of CP29 phosphorylation in
State 1 we performed similar MS analyses of thylakoid
membranes isolated from algal cells exposed to State 1
conditions This study identified only two
phosphoryl-ated peptides derived from CP29, which corresponded
to phosphorylation of Thr6 and Thr32 (Table 1) The
level of both phosphopeptide ions was significantly
lower than in samples from the same amount of
thyla-koids in State 2, probably accounting for the lack of
detection of phospho-CP29 in State 1 thylakoids by
antiphosphothreonine blotting (Fig 2B) However, MS
measurements that do not include labelling with stable
isotopes are generally not quantitative and the exact levels of CP29 phosphorylation at positions 6 and 32
in State 1 and 2 conditions will be addressed in a sep-arate study We did not find any phosphorylation of CP29 at residues 16 and 102 in State 1 thylakoid membranes and therefore, we conclude that phos-phorylation of these residues is specific to the State 2 condition
Single particle image averaging of State 1 and State 2 LHCI–PSI supercomplexes
Both S1–F3 and S2–F3 sucrose density gradient frac-tions were analysed by electron microscopy of negat-ively stained particles followed by single-particle averaging In the S1–F3 fraction, the population of the most structurally intact particles (3881 particles) cor-responded to LHCI–PSI supercomplexes described pre-viously for State 1 [16] In the S2–F3 fraction, a novel population of larger LHCI–PSI supercomplexes (1675 particles) was identified Top-view projection maps of the LHCI–PSI supercomplex isolated from State 1 and State 2 are compared in Fig 4 The former (Fig 4A) has maximum dimensions of 190· 170 A˚ (excluding detergent shell), whereas the State 2 supercomplex
Fig 4 Top-view projections of S1 and S2
LHCI–PSI supercomplexes of C reinhardtii,
as viewed from their stromal sides (A)
Pro-jection of State 1 LHCI–PSI, derived from an
analysis of negatively stained particles by
electron microscopy (B) Projection of State
2 LHCI–PSI (C,D) Outline (black) of the pea
three-dimensional X-ray model 1qzv.pdb [12]
emphasizing the monomeric PSI core and
the four LHCI subunits overlaid onto
projec-tions of State 1 and State 2 LHCI–PSI,
respectively Scale bar ¼ 50 A˚.
Trang 6(Fig 4B) is larger with maximum dimensions of
190· 205 A˚ This size difference is due to additional
protein density (Fig 4B) To gain further insight into
the organization of Chlamydomonas LHCI–PSI
super-complexes, the outline of the X-ray map of the
recently published higher plant LHCI–PSI [12] was
overlaid onto the S1 and S2 LHCI–PSI projections
(Fig 4C,D, respectively) using the crescent-like
four-domain LHCI antenna as a visual reference for the
fit-ting In addition to the four Lhca subunits present
within the X-ray model of the higher plant LHCI–PSI
supercomplex, we were able to identify density which
could accommodate two further LHC subunits in the
S1 LHCI–PSI supercomplexes (Figs 4C and 5)
Import-antly, in the S2 LHCI–PSI particles, the additional
density compared with that of higher plant PSI, was
larger than for State 1 particles, and the extra density
corresponded to that expected for an additional LHC
subunit (Fig 5) As can be seen in Fig 5, all the extra
density was observed in the region adjacent to PsaH
(highlighted in white in Fig 5)
Discussion
The recent X-ray structure of the higher plant LHCI– PSI supercomplex revealed several unique features of the organization of the LHCI antennae and its bind-ing to the PSI core First, the number of Lhca pro-teins constituting the higher plant LHCI appears to
be lower than previously estimated from biochemical and spectroscopic studies Four rather than eight Lhca subunits form the light-harvesting belt asymmet-rically located on the PsaG⁄ J ⁄ K side of the core domain [12] Second, the LHCI crescent is much more densely populated with Chl molecules than previously estimated, with 56 Chls bound within the peripheral LHCI antenna region and an additional 10 Chls pre-sent in the so-called ‘gap’ region, which are involved
in energy transfer from the antenna to the reaction centre [12]
The crystal structure of the higher plant LHCI–PSI supercomplex prompted us to extend the modelling of the Chlamydomonas homologue visualized by electron microscopy [16] We propose that in Chlamydomonas, the four major Lhca subunits of LHCI form a crescent positioned asymmetrically on the PsaG⁄ J ⁄ K side of the core complex similar to the higher plant LHCI antenna However, it is well established that the Chlamydomonas LHCI antenna complex comprises a larger number of Lhca proteins than in higher plants [25–27] Therefore, as argued previously [16], the addi-tional density detected in the Chlamydomonas LHCI– PSI supercomplex particles from State 1 cells is likely
to accommodate extra LHCI antenna subunits which are also retained in the supercomplex isolated from cells placed in State 2 According to modelling using the X-ray structure of higher plant PSI [12], we con-clude that S1 and S2 LHCI–PSI supercomplexes of Chlamydomonas contain six Lhca subunits (Fig 5, red)
The LHCI–PSI supercomplex, isolated from Chlamy-domonas cells in State 2 and present in the S2–F3 fraction of the sucrose density gradient, contained a single phosphoprotein with an apparent molecular mass of 35 kDa (Fig 2B) Subsequent analyses by tandem MS identified this protein as CP29 whose well-established function is to aid the binding of LHCII to the PSII reaction centre core complex [28,29] We there-fore suggest that the additional density observed in the S2 LHCI–PSI supercomplex in the vicinity of PsaH is indeed phosphorylated CP29, modelled in blue in Fig 5 according to the X-ray structure of the LHCII protein [30] In order to further test the hypothesis that CP29 plays a role in the binding of phospho-LHCII to facilitate the State 1 to State 2 transition, we
Fig 5 Detailed modelling of the projection map for the LHCI–PSI
supercomplex isolated from C reinhardtii cells placed in State 2.
Modelling is based on higher plant coordinates 1qzv.pdb [12] with
PSI core (green), LHCI antenna (red), PsaJ (yellow), PsaK
(magenta), PsaG (purple), PsaI (orange), PsaL (cyan) and PsaH
(white) The additional density observed in State 2 LHCI–PSI
super-complex which is able to accommodate an additional LHC subunit
is coloured blue and is suggested to be phospho-CP29 (see text).
Chlorophylls are shown in yellow, but were excluded from the
addi-tional density attributed to the LHCI and CP29 subunits The
deter-gent shell surrounding the particles in the hydrophobic membrane
plane sits within any stain present and this shell is assigned here
as an 15 A˚ wide outer contour (yellow) Scale bar ¼ 50 A˚.
Trang 7conducted studies on a mutant of Chlamydomonas
gen-erated by dsRNA antisense technology having an
unde-tectable level of CP29 (A Kanno and J Minagawa,
unpublished observations) We found that although
this mutant was highly unstable with regards to CP29
suppression (experiments are currently being conducted
to stabilize inhibition of CP29 expression; A Kanno
and J Minagawa, unpublished observations), a
prelim-inary mutant line depleted of CP29 did not contain the
35 kDa phosphoprotein in the purified State 2 LHCI–
PSI complex even though thylakoid membranes from
which it was isolated contained several
phosphopro-teins including major LHCII (J Kargul, J Nield,
S Benson, A Kanno, M Turkina, A Vener, J
Mina-gawa & J Barber, unpublished observations)
Conco-mitant with this finding, electron microscopy and
single-particle analysis showed that density attributed
to phospho-CP29 was absent in the LHCI–PSI particles
isolated from the State 2-induced CP29 mutant cells
(J Kargul, J Nield, S Benson, A Kanno, M Turkina,
A Vener, J Minagawa and J Barber, unpublished
observations)
Although it is known that CP29 can undergo
revers-ible N-terminal phosphorylation [21,31,32], it has not
previously been shown to bind to PSI or be implicated
with State transitions CP29 in Chlamydomonas is
unique because it is the only nuclear-encoded
thyla-koid protein in which the transit chloroplast-targeting
peptide is not removed but processed by excision of
the N-terminal methionine, followed by acetylation
and phosphorylation of Thr6 [21] It has been
proposed that it is the functional importance of this
phosphorylation site which leads to retention of the
transit peptide in the mature protein [21] Importantly,
our MS analyses identified three novel phosphorylation
sites, in addition to Thr6, within the N-terminal domain of CP29 in Chlamydomonas exposed to State 2 conditions We also found that phosphorylation of these sites is dynamically regulated by redox conditions
in the photosynthetic membranes Phosphorylation of CP29 in State 2 is more pronounced and two of the newly found modification sites are exclusively phos-phorylated only under conditions associated with the State 1 to State 2 transition It is feasible that under State 2 conditions, these additional phosphorylations perturb the electrostatic properties of CP29 and trigger
a conformational change leading to dissociation of this protein from PSII and its subsequent attachment to PSI
In conclusion, our results suggest that phospho-CP29, possibly in a multiphosphorylated form, strongly associates with PSI in State 2, adjacent to the PsaH protein The absence of the mobile pool of LHCII in our State 2 LHCI–PSI supercomplex prepar-ation is likely to be a consequence of its weak interac-tion with PSI compared with phospho-CP29, and its displacement following DDM treatment [13] Our data suggest that the functional role of the phospho-CP29 bound to LHCI–PSI is to act as a docking site for the mobile phospho-LHCII, as depicted in Fig 6 The extent of LHCII binding to LHCI–PSI will depend on the degree of excitation imbalance between PSI and PSII Therefore, in Chlamydomonas, it seems that CP29 may functionally couple LHCII to PSI as well as
to PSII, with the former occurring under State 2 con-ditions Previously, we estimated that the LHCI–PSI supercomplex in State 1 binds about 214 Chls [16] and
if CP29 binds 14 Chls, as does each monomer of LHCII [30], then CP29 alone would increase the absorption cross section of the State 2 LHCI–PSI
Fig 6 Diagrammatic representation of how
phospho-CP29 could tightly associate with
PSI in State 2 and therefore facilitate the
binding of mobile LHCII in order to regulate
the absorption cross-section of PSI.
Trang 8supercomplex by 7% compared with its State 1
counterpart This increase in antenna size would be
enhanced by the functional association of
phospho-LHCII, which in the case of Chlamydomonas can be
very extensive compared with higher plants [4–6]
Whe-ther hyperphosphorylation of CP29 occurs in higher
plants and whether this phosphoprotein associates with
PSI in State 2 has yet to be determined Importantly,
one phosphorylation site identified in this study
exclu-sively in State 2 thylakoid membranes (Thr16) is fully
conserved between higher plant (Arabidopsis and
maize) CP29 and its Chlamydomonas counterpart
Experimental procedures
Culturing and State transitions
C reinhardtii psbD-His cells [33] were grown to mid-log
phos-phate medium as described previously [16] The cells were
placed in either State 1 by aerobic dark incubation for 2 h
or in State 2 by anaerobic dark incubation (bubbling with
nitrogen) for 20 min in the presence of 40 mm NaF to
inhi-bit phosphatase activity, as described previously [15] The
ability of the cells to carry out State transitions was
checked by monitoring room fluorescence yield changes in
response to illumination by light preferentially absorbed by
PSII, light 2 (Balzer BG18 filter, Milan, Italy) or light
pref-erentially absorbed by PSI, light 1 (Schott RG695 filter,
Mainz, Germany) The room temperature fluorescence
emission was monitored at > 650 nm using a Waltz
chloro-phyll fluorimeter (PAM-101; Effeltrich, Germany) State
transitions were also monitored by recording chlorophyll
fluorescence spectra at 77 K using a Perkin–Elmer LS50
luminescence spectrophotometer (Beaconsfield, UK) with
an excitation wavelength of 435 nm
Biochemical isolation and characterization
Using a procedure reported previously [16], thylakoid
mem-branes were isolated from cells that had been placed in
procedures [15] In the case of cells in State 2, 40 mm NaF
was present in order to prevent dephosphorylation of
phos-phoproteins LHCI–PSI supercomplexes were isolated from
DDM followed by sucrose density gradient centrifugation
as detailed in Kargul et al [16] This procedure produced
three Chl-containing bands, F1–F3, where F3 consists of
the LHCI–PSI supercomplex as shown previously [16]
immunoblotting with antiphosphothreonine serum (Zymed
Laboratories Inc., South San Francisco, CA, USA) [16]
and by tandem MS (see below)
Mass spectroscopy For protein identification, the procedures of in-gel digestion and peptide extraction were made as described previously [22] Phosphorylated peptides were obtained after treatment
of the isolated thylakoids by trypsin, conversion of the released peptides to methyl esters by methanolic HCl and following enrichment of the phosphopeptides by IMAC as described earlier [21] Electrospray ionization tandem MS was performed on a hybrid spectrometer Q-STAR Pulsar I (Applied Biosystems, Foster City, CA, USA) equipped with
a nano-electrospray ion source (MDS Protana, Odense, Denmark) Collision-induced dissociation of selected pre-cursor ions was performed with manual control of collision energy during spectrum acquisition
Electron microscopy and densitometry
imaged using a Philips CM100 electron microscope (FEI Company, Eindhoven, the Netherlands) at a calibrated
dis-cernible drift or astigmatism, were digitized using a Leafscan
45 densitometer at a step size of 10 lm and transferred to a networked cluster of Linux-based PC workstations
Image processing The densitometry resulted in a sampling frequency of 1.97 A˚ per pixel on the specimen scale All subsequent pro-cessing was performed using the imagic-v software environ-ment [34,35] The first minima of the micrographs’ power spectra were measured to be in the range of 20.5–21.8 A˚
No correction was made for the contrast transfer function Datasets consisting of 10 933 and 5195 particle images for State 1 LHCI–PSI (S1–F3) and State 2 LHCI–PSI (S2–F3) samples, respectively, were compiled by interactively select-ing all possible sselect-ingle particles from the micrographs Mul-tivariate statistical analyses and reference-free alignments identified a number of subpopulations within each dataset [34,35] Each of these subpopulations was extracted from the total data set and treated de novo, gaining initial two-dimensional class averages and then iterative refinement fol-lowed in order to obtain improved class averages Standard molecular modelling programs were used to visualize the protein data bank file, 1qzv.pdb, of the higher plant LHCI– PSI structure [12] The views obtained were subsequently overlaid onto the improved two-dimensional class averages
by visual inspection
Acknowledgements
We thank Jun Minagawa (Hokkaido University, Japan) for donating the Chlamydomonas psbD-His
Trang 9strain and preliminary CP29 mutant line For financial
support, JB acknowledges the Biotechnology and
Bio-logical Sciences Research Council (BBSRC) AV is
grateful for support by grants from the Swedish
Research Council for Environment, Agriculture and
Space Planning (Formas), Nordiskt Kontaktorgan fo¨r
Jordbruksforskning (NKJ) and Graduate Research
School in Genomics and Bioinformatics (FGB) JN is
a Royal Society University Research Fellow
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