Linköping University Medical Dissertations
No. 1038
Functional proteomics of protein phosphorylation in
algal photosynthetic membranes
Maria Turkina
Linköping 2008
© Maria Turkina
Published articles have been reprinted with permission from copyright holder:
2006 © WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
2004 © Elsevier Inc.
2005 © Blackwell Publishing, Inc.
2006 © American Society for Biochemistry and Molecular Biology
Printed by LiU-Tryck, Linköping, Sweden, 2008
ISBN: 978-91-85523-02-3
ISSN 0345-0082
Abstract
Plants, green algae and cyanobacteria perform photosynthetic conversion of sunlight
into chemical energy in the permanently changing natural environment. For successful
survival and growth photosynthetic organisms have developed complex sensing and signaling
acclimation mechanisms. The environmentally dependent protein phosphorylation in
photosynthetic membranes is implied in the adaptive responses; however, the molecular
mechanisms of this regulation are still largely unknown. We used a mass spectrometry-based
approach to achieve a comprehensive mapping of the in vivo protein phosphorylation sites
within photosynthetic membranes from the green alga Chlamydomonas reinhardtii subjected
to distinct environmental conditions known to affect the photosynthetic machinery.
The state transitions process regulating the energy distribution between two
photosystems, involves the temporal functional coupling of phosphorylated light-harvesting
complexes II (LHCII) to photosystem I (PSI). During state transitions several of the thylakoid
proteins undergo redox-controlled phosphorylation-dephosphorylation cycles. This work
provided evidences suggesting that redox-dependent phosphorylation-induced structural
changes of the minor LHCII antenna protein CP29 determine the affinity of LHCII for either
of the two photosystems. In state 1 the doubly phosphorylated CP29 acts as a linker between
the photosystem II (PSII) core and the trimeric LHCII whereas in state 2 this quadruply
phosphorylated CP29 would migrate to PSI on the PsaH side and provide the docking of
LHCII trimers to the PSI complex. Moreover, this study revealed that exposure of
Chlamydomonas cells to high light stress caused hyperphosphorylation of CP29 at seven
distinct residues and suggested that high light-induced hyperphosphorylation of CP29 may
uncouple this protein together with LHCII from both photosystems to minimize the damaging
effects of excess light.
Reversible phosphorylation of the PSII reaction center proteins was shown to be
essential for the maintenance of active PSII under high light stress. Particularly
dephosphorylation of the light-damaged D1 protein, a central functional subunit of the PSII
reaction center, is required for its degradation and replacement. We found in the alga the
reversible D1 protein phosphorylation, which until our work, has been considered as plantspecific.
We also discovered specific induction of thylakoid protein phosphorylation during
adaptation of alga to limiting environmental CO2. One of the phosphorylated proteins has five
I
phosphorylation sites at both serine and treonine residues. The discovered specific low-CO2and redox-dependent protein phosphorylation may be an early adaptive and signalling
response of the green alga to limitation in inorganic carbon.
This work provides the first comprehensive insight into the network of environmentally
regulated protein phosphorylation in algal photosynthetic membranes.
II
Original publications
This thesis is based on the following papers, which will be referred to in the text by their
roman numerals:
I. Turkina M.V., Blanco-Rivero A., Vainonen J.P., Vener A.V., Villarejo A. (2006)
CO2 limitation induces specific redox-dependent protein phosphorylation in
Chlamydomonas reinhardtii. Proteomics 6:2693-2704.
II. Turkina M.V., Villarejo A., Vener A.V.
(2004) The transit peptide of CP29
thylakoid protein in Chlamydomonas reinhardtii is not removed but undergoes
acetylation and phosphorylation. FEBS Lett 564:104-108.
III. Kargul J., Turkina M.V., Nield J., Benson S., Vener A.V., Barber J. (2005) Lightharvesting complex II protein CP29 binds to photosystem I of Chlamydomonas
reinhardtii under State 2 conditions. FEBS J 272:4797-4806.
IV. Turkina M.V., Kargul J., Blanco-Rivero A., Villarejo A., Barber J., Vener A.V.
(2006) Environmentally modulated phosphoproteome of photosynthetic membranes in
the green alga Chlamydomonas reinhardtii. Mol Cell Proteomics 5:1412-1425.
III
Other publications
1. Turkina M.V., Vener A.V. (2007) Identification of phosphorylated proteins. Methods
Mol Biol 355:305-316.
2. Belogurov G.A., Malinen A.M., Turkina M.V., Jalonen U., Rytkonen K., Baykov
A.A., Lahti R. (2005) Membrane-bound pyrophosphatase of Thermotoga maritima
requires sodium for activity. Biochemistry 44:2088-2096.
3. Vainonen J.P., Aboulaich N., Turkina M.V., Stralfors P., Vener A.V. (2004) Nterminal processing and modifications of caveolin-1 in caveolae from human
adipocytes. Biochem Biophys Res Commun 320:480-486.
4. Belogurov G.A., Turkina M.V., Penttinen A., Huopalahti S., Baykov A.A., Lahti
R. (2002) H+-pyrophosphatase of Rhodospirillum rubrum. High yield expression in
Escherichia coli and identification of the Cys residues responsible for inactivation by
mersalyl. J Biol Chem 277:22209-22214.
5. Belogurov G.A., Fabrichniy I.P., Pohjanjoki P., Kasho V.N., Lehtihuhta E.,
Turkina M.V., Cooperman B.S., Goldman A., Baykov A.A., Lahti R. (2000)
Catalytically
important
ionizations
along
the
reaction
pathway
of
yeast
pyrophosphatase. Biochemistry 39:13931-13938.
6. Baykov A.A., Hyytia T., Turkina M.V., Efimova I.S., Kasho V.N., Goldman A.,
Cooperman B.S., Lahti R. (1999) Functional characterization of Escherichia coli
inorganic pyrophosphatase in zwitterionic buffers. Eur J Biochem 260:308-317.
7. Burobin A.V.,
Lomonova M.V.*, Skliankina V.A., Avaeva S.M. (1997)
Irreversible specific inhibition of E. coli inorganic pyrophosphatase with amines.
Bioorganic Chemistry (Moscow) 23:104-109.
* maiden name
IV
Abbreviations
ATP
adenosine-5´-triphosphate
CCM
CO2-concentrating mechanism
Ci
inorganic carbon
LHCI
light-harvesting complex I
light-harvesting complex II
LHCII
+
NADP
Nicotinamide adenine dinucleotide phosphate (oxidized)
NADPH Nicotinamide adenine dinucleotide phosphate (reduced)
PC
plastocyanin
PQ
plastoquinone
PQH2
plastoquinol
PSI
photosystem I
PSII
photosystem II
V
VI
Table of contents
Abstract....................................................................................................................................I
Original publications.........................................................................................................III
Abbreviations........................................................................................................................V
1. Introduction........................................................................................................................1
1.1. Photosynthetic energy conversion..........................................................................1
1.2. Role of protein phosphorylation in adaptive responses of the photosynthetic
apparatus.....................................................................................................................3
1.2.1. State transitions and phosphorylation of LHCII.................................................3
1.2.2. PSII turnover and phosphorylation under high light stress...............................6
1.2.3. Chlamydomonas reinhardtii as a model organism for the adaptive response
studies of the photosynthetic apparatus...............................................................7
1.2.4. Adaptation of alga to nutrient limitation: carbon concentrating mechanism....9
2. Present investigation...................................................................................................11
2.1. Aim of the research..................................................................................................11
2.2. Metodological aspects.............................................................................................11
2.2.1. Mapping of protein phosphorylation................................................................11
2.2.2. Vectorial proteomics approach........................................................................13
2.3. Exploring the phosphoproteome of photosynthetic membranes in
Chlamydomonas reinhardtii....................................................................................14
2.3.1. Specific protein phosphorylations induced at limited environmental CO2......14
2.3.2. Light-dependent protein phosphorylations......................................................16
2.3.3. Multiply phosphorylated light-harvesting phosphoprotein CP29 is an
integral part of the stress-response mechanism................................................18
3. Concluding remarks.....................................................................................................24
4. Acknowledgements......................................................................................................26
5. References......................................................................................................................28
VII
VIII
1. Introduction
1.1. Photosynthetic energy conversion
Oxygenic photosynthesis is a process of capture and conversion of sunlight into
chemical energy by photoautotrophic organisms. For the efficient energy conversion plants,
green algae and cyanobacteria have developed a complex molecular machinery. Most of the
reactions of photosynthesis occur in chloroplasts. Chloroplasts (see Figure 1) are plastid
organelles surrounded by two separate membranes: the outer chloroplast envelope and the
inner chloroplast envelop. Inside chloroplasts there is a third membrane system which is
called thylakoid and which forms a continuous three-dimensional network enclosing an
aqueous space called the lumen. The fluid compartment that surrounds the thylakoids is
known as the stroma (Nelson and Ben-Shem, 2004). Thylakoids are differentiated into two
distinct morphological domains: cylindrical stacked structures called grana and unstacked
membrane regions called stroma lamellae, which interconnect the grana (Figure 1) (Anderson
and Andersson, 1988; Mustardy and Garab, 2003). Algal thylakoids have a loose organization
with less amount of the stacked domains comparing to green plants (Aro and Ohad, 2003;
Steinback and Goodenough, 1975).
Thylakoid membrane provides light-dependent water oxidation, NADP+ reduction and
ATP formation. These reactions are catalyzed by four multi-subunit membrane-protein
complexes: photosystem I (PSI), photosystem II (PSII), the cytochrome b6f complex, and
ATP-synthase. Two photosystems are working in the photosynthetic membrane in parallel.
PSII absorbs light, oxidizes H2O to O2 and extracts electrons from water; the electrons are
then transported via electron transport chains in the thylakoid membrane to PSI. The electrons
from PSII are used for the reduction of plastoquinone (PQ) pool to plastoquinol (PQH2). The
cytochrome b6f protein complex then accepts electrons from PQH2. The cytochrome-b6f
complex mediates electron transport to PSI via plastocyanin (PC). PSI transfers the electrons
across the membrane and reduces NADP+ to form NADPH. NADPH is then used as reducing
power for the biosynthetic reactions. The proton-motive force generated by linear electron
flow from PSII to PSI powers ATP synthesis by F1F0-complex (Figure 2).
To synthesize ATP, photosynthesis provides an alternative route through which light
energy can be used to generate a proton gradient across the thylakoid membrane of
chloroplasts. This second electron path, driven by PSI only, is the cyclic electron flow, and it
1
Thylakoid Lumen
Thylakoid
Grana
Stacks
Stroma
Outer and
Inner Envelope
Membrane
Stroma Lamellae
Figure 1. Schematic representation of chloroplast. The
thylakoid membrane is divided into the appressed grana stacks
and non-appressed interconnecting stroma lamellae. The
thylakoid lumen is enclosed by thylakoid membrane.
produces neither O2 nor NADPH. Electrons from PSI can be recycled to plastoquinone, and
subsequently to the cytochrome b6f complex (Heber and Walker, 1992; Joliot and Joliot,
2002). Such a cyclic flow generates pH and thus ATP without the accumulation of reduced
species. The role of cyclic electron flow is less clear than linear, while it is proposed, that the
linear flow itself cannot maintain the correct ratio of ATP/NADPH production. The absence
of cyclic flow will ultimately lead to excessive accumulation of NADPH in the stroma and
thereby, its over-reduction (Munekage et al., 2004).
Both photosystems consist of two closely linked components: reaction centers and light
harvesting complexes (LHCs), a superfamily of chlorophyll and carotenoid-binding proteins
which absorb the sunlight. Light energy captured by LHCs is funneled to the minor antenna
proteins (Jansson, 1999; Yakushevska et al., 2003) and then transmitted to the reaction
2
H+
NADP+
+H+ NADPH
Light
PQ
Stroma
LHCI
PQH2
H+
H2O
PSI
Cyt b6f
LHCII
H+
PSII e-
ATP
PC
ATP
synthase
Light
ADP +Pi
Lumen
O2 + H+
Figure 2. The scheme of photosynthetic complexes in the
thylakoid membrane. The linear electron (e-) and proton (H+)
flow are indicated with dashed and solid lines respectively.
centers. The LHCs associated with PSII and PSI contain different proteins and called LHCII
and LHCI, respectively. The photosystems are differently distributed in thylakoids: PSI is
located in the stroma lamellae, PSII is found almost exclusively in the stacked grana regions,
the ATPase is concentrated in nonappresed thylakoid regions, and the cyt b6f complex is
evenly distributed in grana and stroma (Anderson, 2002; Andersson and Anderson, 1980;
Staehelin, 2003).
1.2. Role of protein phosphorylation in adaptive responses of the
photosynthetic apparatus
1.2.1. State transitions and phosphorylation of LHCII
Photosystems are working in sequence within the photosynthetic electron transport
chain. The antenna systems of PSII and PSI preferentially absorb light at 650 and 700 nm,
respectively. Due to this difference in light absorption properties the fluctuations of light can
lead to unequal excitation of the photosystems and thus decrease the photosynthetic yield.
Balance between the two photosystems is achieved through the reversible association of the
major antenna complex (LHCII) with the two photosystems in response to changing
3
NADP+
+H+ NADPH
e-
PQH2
PQ
LHCI
PSII
Cyt b6f
LHCII
State 1
LHCII
Light
PSI
PC
Phosphatase
O2 +
Kinase
H2O
H+
Light
PQ
PSI
LHCII
PQH2
LHCI
PSII
Cyt b6f
State 2
LHCII
PPP
PC
Figure 3. The scheme of state transitions. The linear and
cyclic electron flow are indicated with dashed lines.
conditions (Allen and Forsberg, 2001). This phenomenon called “state transitions” was first
described in a red alga (Murata, 1969) and a green alga (Bonaventura and Myers, 1969)
almost 40 years ago. In State 1 the non-phosphorylated LHCII is bound to PSII, while
phosphorylated part of LHCII migrates to PSI in State 2 (Figure 3) (Allen, 1992b; Allen,
2003; Allen et al., 1981). State transitions are redox-controlled: LHCII proteins are
phosphorylated when plastoquinone (PQ), an electron carrier, is reduced (Vener et al., 1995;
Vener et al., 1997). Plastoquinone is involved in the electron transport chain of
photosynthesis; it accepts electrons from photosystem II and donates them to cytochrome b6f
complex. When the photosystems are balanced, the rate of electron flow into plastoquinone is
4
equal to the rate of electron flow out. If light favors one of the photosystems, then this
photosystem works faster than the other and the plastoquinone pool redox state changes
(Allen, 2005). State 1 corresponds to the oxidized and State 2 to the reduced state of
plastoquinone in the thylakoid membranes. Reduction of the plastoquinone pool activates the
LHCII-kinase, which increases phosphorylation status of LHCII (Vener et al., 1997; Zito et
al., 1999). Dephosphorylation of LHCII is provided by a protein phosphatase, which is
supposed to be constitutively active (Allen, 1992a). The protein kinase, which phosphorylates
a part of the LHCII light-harvesting complexes, has recently been identified in the green alga
Chlamydomonas reinhardtii (Depege et al., 2003). The Chlamydomonas thylakoid-associated
Ser/Thr kinase Stt7, which is required for state transitions, has an orthologue named STN7 in
the plant Arabidopsis thaliana (Bellafiore et al., 2005). The loss of Stt7 or STN7 blocks state
transitions and LHCII phosphorylation (Bellafiore et al., 2005). Beside this, it was shown that
growth of stn7 mutants was reduced under changing light conditions and proposed that STN7,
and probably state transitions, have an important role in the response to environmental
changes (Bellafiore et al., 2005).
Despite the similarity in thylakoid protein phosphorylation between plants and green
algae and a high homology between the plant STN7 and the algal Stt7 protein kinases
(Bellafiore et al., 2005), the extent of photosynthetic state transitions differs between these
species. As much as 80% of the LHCII antenna can be redistributed from PSII to PSI in
Chlamydomonas during state transitions (Delosme et al., 1996), whereas in plant thylakoids
the mobile fraction of LHCII comprises only 15–20% (Allen, 1992b).
Because of this
dramatic difference in LHCII redistribution, it was proposed that the state transitions in
Chlamydomonas reinhardtii are necessary not only for maximizing of photosynthetic
efficiency in changing light conditions by balancing the excitation of two photosystems, as in
plants (Finazzi, 2005; Rochaix, 2007). During state transitions in alga the switch between
linear and cyclic electron flow occurs (Vallon et al., 1991). In state 1 electron transport is
linear and the two photosystems are working in series generating reducing power (NADPH)
and ATP. In state 2 PSII is largely disconnected from the electron transport chain and cyclic
electron flow around PSI produces exclusively ATP (Aro and Ohad, 2003; Finazzi et al.,
2002; Wollman, 2001). It was proposed, that the major role of state transitions in alga is the
adjustment/restoration of the intracellular ATP levels, required for the carbon fixation (Aro
and Ohad, 2003; Bulte et al., 1990) by a reorganization of the photosynthetic electron
transport chain (Finazzi, 2005; Rochaix, 2007).
5
The light-harvesting antenna transmits the absorbed light energy to the reaction centers
of photosystems and migrates between photosystems in response to changing light conditions
(Allen and Forsberg, 2001). If the light intensity is too high, photosynthesis becomes
saturated and much of the absorbed energy is not needed. Moreover, excited states of
chlorophyll and the presence of molecular oxygen are the reasons for irreversible damage to
proteins, lipids, and pigments in the photosynthetic membrane. To maintain optimal
photosynthetic capacity alga and plants evolved several photoprotection mechanisms (Horton
and Ruban, 2005). To prevent photodamage, LHCII can rapidly and reversibly switch into a
specific antenna state, which safely dissipates the excess energy as heat. This process is called
non-photochemical quenching (Cogdell, 2006). The structural changes in the major lightharvesting complex LHCII are responsible for switching between efficient light-harvesting
and the dissipative state where excess light energy is converted into heat (Horton and Ruban,
2005; Pascal et al., 2005).
1.2.2 PSII turnover and phosphorylation under high light stress
Photosynthetic apparatus endure structural and functional alterations, which are required
both at low and high light conditions. Despite this, high light is the major cause of
photosynthetic apparatus damage (Barber and Andersson, 1992; Kanervo et al., 2005;
Tyystjarvi and Aro, 1996). The most extensive injury occurs on the PSII reaction centre and
leads to damage and degradation of its central functional subunit D1. PSII is one of the central
parts of oxygenic photosynthesis and performs light-driven extraction of electrons from water
with a concomitant production of oxygen. The chemistry of PSII involves formation of highly
oxidizing radicals and toxic oxygen species that damage PSII itself. The frequency of this
damage is relatively low under normal conditions but becomes a significant problem for the
plants under increasing light intensity, especially when combined with other environmental
stress factors. Algae, plants and cyanobacteria developed repair mechanism called PSII
repare cycle which involves partial disassembly of inactivated PSII; proteolytic degradation of
the photodamaged reaction centre protein D1; D1-polypeptide synthesis and reassembly of
active complexes recycling undamaged subunits (Aro et al., 1993; Barber and Andersson,
1992; Mattoo and Edelman, 1987; Nishiyama et al., 2006; Yokthongwattana and Melis,
2006). Three thylakoid proteases, Deg P2 (Haussuhl et al., 2001), FtsH (Lindahl et al., 2000)
and Deg1 (Kapri-Pardes et al., 2007), have been implicated in proteolytic degradation of the
photo-damaged D1. However, recent works suggest a model in which FtsH proteases alone
can be responsible for the removal of damaged D1 in cyanobacteria and plants (Komenda et
6
al., 2006; Komenda et al., 2007; Nixon et al., 2005). FtsH proteases are involved at an early
stage of D1 degradation (Bailey et al., 2002), but how the damaged subunit is recognized by
protease, degraded and replaced is still poorly understood. The degradation of damaged D1
by FtsH protease proceeds from its N-terminus, exposed to the chloroplast stroma (Komenda
et al., 2007; Nixon et al., 2005). It has been found that N-terminal phospho-threonine
dephosphorylation of the light-damaged D1 protein is required for its degradation and
replacement (Koivuniemi et al., 1995; Rintamäki and Aro, 2001; Rintamaki et al., 1996).
Thus, the N-terminal phosphorylation of D1 subunit in PSII protects this protein from
degradation (Nixon et al., 2005), as has been documented experimentally in several studies
(Aro et al., 1992; Ebbert and Godde, 1996; Elich et al., 1992; Koivuniemi et al., 1995;
Rintamäki et al., 1996). The phosphorylated N-terminus of D1 may have a very low affinity
towards the active site pore of FtsH and by that protect the protein from proteolysis (Ebbert
and Godde, 1996; Koivuniemi et al., 1995; Rintamäki et al., 1996) or dephosphorylation
affects the conformation of the protein (Yoshioka et al., 2006). The exact mechanism
underlying this effect is not yet known.
1.2.3. Chlamydomonas reinhardtii as a model organism for the adaptive
response studies of the photosynthetic apparatus
Acclimation to changing conditions is crucial for cell survival and growth. Organisms
are able to adapt to small fluctuations in their environment, or can resist even dramatic
changes due to development of complex sensing and signaling acclimation mechanisms.
Adaptive responses of cells involve different cellular compartments and structures, several
kinds of biomolecules and various signaling systems. Escherichia coli traditionally has been
the major model prokaryotic microorganism for molecular level studies of the adaptive
responses.
In eukaryotic organisms, the yeast Saccharomyces cerevisiae, plants and
mammals have for a long time been the organisms of choice for studies at the molecular level
(Mendez-Alvarez et al., 1999). Currently, the unicellular green alga Chlamydomonas
reinhardtii, the “green yeast” (Goodenough, 1992; Rochaix, 1995) has developed into a
model organism for studying adaptive responses in photosynthetic organisms. Off all algae
the Chlamydomonas reinhardtii is the most studied.
Chlamydomonas reinhardtii is a unicellular green alga approximately 10 micrometers in
diameter that swims with two flagella (Figure 4). It has a glycoprotein cell wall; the nucleus;
a large cup-shaped chloroplast, which has a single large pyrenoid where the starch formed
from photosynthetic products is stored. Two small contractile vacuoles, which have an
7
F
Ch
ES
C
V
N
M
St
T
P
F - flagella
C - cytosol
V - vacuole
N - nucleus
Ch - chloroplast
M - mitochondria
St - stroma
S - starch granula
T - thylakoid membrane
ES - eye-spot
P - pyrenoid
S
Figure 4. Schematic representation of Chlamydomonas reinhardtii cell
excretory function, are located near the flagella. There is also a red pigment "eyespot" which
is light-sensitive. The eyespot apparatus allows the cells to sense light direction and intensity
and respond to it by swimming either towards or away from the light. This helps the cells in
finding an environment with optimal light conditions for photosynthesis. Chlamydomonas
reinhardtii has been extensively used (Harris, 2001) in studying photosynthesis, chloroplast
biogenesis, circadian rhythms (Mittag et al., 2005; Mittag and Wagner, 2003), flagellar
function and assembly (Keller et al., 2005; Pazour et al., 2006). A number of polypeptides
associated with flagella assembly or function are similar to proteins altered in diseased
mammalian cells (Li et al., 2004; Pennarun et al., 2002; Snell et al., 2004). Therefore, C.
8
reinhardtii is serving as an important model system for elucidating the biology of both
photosynthetic and nonphotosynthetic eukaryotes.
The Chlamydomonas genome project reported the complete sequence of the chloroplast
genome (Maul et al., 2002) and recently the whole genome ((Grossman, 2005; Shrager et al.,
2003) accomplished by Joint Genome Institute of the US Department of Energy,
http://www.jgi.doe.gov/chlamy). A comparative phylogenomic analysis of the nuclear
genome of Chlamydomonas was performed, identifying genes encoding uncharacterized
proteins that are likely associated with the function and biogenesis of chloroplasts or
eukaryotic flagella (Merchant et al., 2007). Expressed sequence tags (ESTs; (Shrager et al.,
2003)), microarray information and other genomic resources can be found at
http://www.chlamy.org and http://genome.jgi-psf.org/.
Since various molecular and genetic tools are available for this organism and several
mutant strains can be relatively easily created or are already available, Chlamydomonas
reinhardtii is extremely useful to study adaptive responses at cellular, physiological,
biochemical and molecular levels. C. reinhardtii is haploid during vegetative growth,
mutations are almost immediately expressed and specific mutant phenotypes can be readily
observed as colonies on solid medium and the cells behave homogeneously in terms of
physiological and biochemical characterization.
C. reinhardtii cells grow quickly with
doubling time less than ten hours and can be grown for most experiments in liquid, usually
with moderate shaking (bubbling), or on agar plates.
Chlamydomonas can be grown heterotrophically with acetate as a sole source of carbon
and this alga can synthesize chlorophyll and assemble the complete photosynthetic apparatus
in the dark. These remarkable properties allow isolation of mutants that are unable to perform
photosynthesis, as well as maintaining light-sensitive mutants in complete darkness.
Chlamydomonas is the only known eukaryote in which the nuclear, chloroplast and
mitochondrial genome can all be transformed, which is extensively used in photosynthetic
studies (Dent et al., 2001).
1.2.4. Adaptation of alga to nutrient limitation: carbon concentrating
mechanism
Chlamydomonas has a world wide distribution: species have been isolated not only
from freshwater and soils, but marine environments and even snow. Chlamydomonas cells
can successfully
adapt to various kinds of environmental stresses such as temperature,
nutrient limiting conditions of carbon, nitrogen, sulfate, and phosphate (Grossman et al.,
9
2007) and light stress. Efficient utilization of light and carbon is most critical for the growth
of all photosynthetic organisms. Photosynthesis in aquatic environments may be limited due
to the low solubility and slow diffusion rate of CO2 in water (Badger and Spalding, 2000).
Alga have adapted to the variable and often-limiting availability of CO2, and inorganic carbon
(Ci) in general, by inducing CO2-concentrating mechanism (CCM) that allows them to
optimize carbon acquisition. The main function of CCM is to increase internal Ci
accumulation by stimulation of inorganic carbon transport through the cellular membranes
and generation of elevated levels of HCO3¯ in the chloroplast stroma, utilizing the pH gradient
across the thylakoid membrane. CO2 accumulation in the cell is performed by Rubisco
(Ribulose-1,5-bisphosphate carboxylase/oxygenase; EC 4.1.1.39), which catalyzes the first
major step of carbon fixation (Badger and Price, 2003; Badger and Spalding, 2000; Giordano
et al., 2005; Kaplan and Reinhold, 1999; Moroney and Somanchi, 1999; Spalding, 2007).
CO2-concentrating mechanism requires considerable structural changes in the cell (Badger
and Price, 2003; Badger and Spalding, 2000; Kaplan and Reinhold, 1999). The CCM
initiation leads to fast changes in gene expression and is associated with transcriptional
regulation of a few dozen CO2-responsive genes (Im et al., 2003; McGinn et al., 2003; Miura
et al., 2004; Woodger et al., 2003). Certain genes are transcribed only under low CO2, for
others the level of transcript increases during acclimation (Eriksson et al., 1998).
Identification of the genes involved in the acclimation is complicated by multiple
consequences of changing CO2 concentration on cell metabolism, leading to modulation of
the expression of many different genes, only some of which are directly involved in the
acclimation (Kaplan and Reinhold, 1999). Moreover, many other low CO2-inducible genes
are predicted to encode proteins of unknown function. The proteins of known identity include
carbonic anhydrases (Eriksson et al., 1998; Fukuzawa et al., 1990), photorespiratory pathway
enzymes (Chen et al., 1996; Mamedov et al., 2001), chloroplast carrier protein (Chen et al.,
1997) and inorganic carbon transporters (Miura et al., 2004; Spalding, 2007).
The cellular signals initiating and triggering these acclimation mechanisms are
unknown. However, induction of CO2-responsive genes has been found to be controlled by a
transcription regulator called Ccm1 (Cia5) (Fukuzawa et al., 2001; Xiang et al., 2001).
Disruption of the Ccm1 gene prevented the induction of almost all low-CO2-inducible genes,
which demonstrated that this protein is a master regulator of CCM. A posttranslational
modification of the C-terminal domain in Cia5 has been postulated to control the transcription
of CO2-responsive genes, and reversible protein phosphorylation has been suggested as a link
connecting sensing of CO2 levels with activation of Ccm1, but no specific protein
10
phosphorylation during the adaptation of Chlamydomonas cells to low CO2 has been
demonstrated (Kaplan et al., 2001; Xiang et al., 2001). Phosphorylation in photosynthetic
membranes is implied in adaptive responses regulating photosynthetic process (Vener, 2007).
However, the molecular mechanisms underlying regulation of photosynthesis by
environmentally dependent thylakoid protein phosphorylation are still largely unknown.
Consequently, the analysis of the phosphoproteome of C. reinhardtii can significantly
enhance our understanding of various regulatory pathways.
2. Present investigation
2.1. Aim of the research
The aim of this project was to systematically identify and characterize phosphoproteins
and in vivo protein phosphorylation events involved in adaptive responses in unicellular green
alga Chlamydomonas reinhardtii. The mass spectrometry approach was used to resolve the
regulatory and signaling network of protein phosphorylation, involved in acclimation of algal
cells to different environmental conditions including stress. It should be noted that before our
study there were only two phosphorylation sites identified in the proteins from photosynthetic
membranes of Chlamydomonas reinhardtii (Dedner et al., 1988; Fleischmann and Rochaix,
1999).
2.2. Metodological aspects
2.2.1. Mapping of protein phosphorylation
Reversible protein phosphorylation is a fundamental regulatory cellular mechanism and
a crucial part of signaling pathways (Cohen, 2000; Huber, 2007; Pawson and Scott, 2005).
Approximately one-third off all proteins are phosphorylated in vivo at any given time (Cohen,
2000; Knight et al., 2003; Manning et al., 2002). Phosphorylation at specific serine, threonine
and tyrosine residues is the most ubiquitous specific post translational modification that
occurs in complex eucaryotic systems (Mann and Jensen, 2003). These modifications are able
to change many properties of protein, such as interaction with other proteins, stability,
localization and activity. Reversible protein phosphorylation plays an important role in the
regulation of many different processes, such as cell growth, differentiation, migration,
metabolism, apoptosis and stress responses (Baena-Gonzalez et al., 2007; Huber et al., 1989;
11
Huber, 2007; Hunter, 2000; Tran et al., 2004). Phosphorylation of thylakoid proteins in plants
has been implicated in adaptive responses to a number of environmental stress factors (Vener,
2007), such as high light (Ebbert et al., 2001; Xu et al., 1999), cold stress (Bergantino et al.,
1995), combined high light and cold treatment (Pursiheimo et al., 2001), heat shock (Rokka et
al., 2000), combined magnesium and sulfur deficiency (Dannehl et al., 1995), and waterdeficient conditions (Giardi et al., 1996).
The protein phosphorylation in photosynthetic membranes of plants and algae has been
studied by traditional techniques, such as analysis of potential protein phosphorylation sites
with site-directed mutagenesis (Andronis et al., 1998; Fleischmann and Rochaix, 1999;
O'Connor et al., 1998) or techniques based on electrophoretic separation of proteins:
detection of the shift in the electrophoretic mobility of individual proteins (Aro et al., 2004; de
Vitry et al., 1991; Elich et al., 1992; Rintamäki et al., 1997); radioactive labeling (Bellafiore
et al., 2005; Bennett, 1977; Depege et al., 2003; Owens and Ohad, 1982); immunological
analyses with anti-phosphoamino acid antibodies (Aro et al., 2004; Kargul et al., 2003;
Vainonen et al., 2005); N-terminal protein sequencing by Edman degradation of
electrophoretically-separated proteins (Dedner et al., 1988; Michel and Bennett, 1987).
During the last few years proteomic methods based on mass spectrometric sequencing of
proteins have been established as powerful tools for identification of novel phosphorylation
sites, especially for the detection of in vivo protein phosphorylation (Carlberg et al., 2003;
Gomez et al., 2002; Hanson et al., 2003; Vener et al., 2001), (Paper II-IV).
Phosphoproteomics have as its object the comprehensive study of protein
phosphorylation by identification of phosphoproteins, mapping of phosphorylation sites,
quantitation of phosphorylation and, finally revealing the role of protein phosphorylation in
signaling/regulatory networks. The analysis of the entire phosphorylome, i.e. the complete set
of all phosphorylated proteins in a cell, is challenging (Cox and Mann, 2007; Goshe, 2006;
Olsen et al., 2006) despite of the optimization of enrichment protocols for phosphoproteins
and phosphopeptides, improved fractionation techniques and development of methods to
selectively visualize phosphorylated residues using mass spectrometry. Phosphoproteomics is
a powerful tool for understanding various biological problems (Mann, 2006; Rossignol, 2006;
Rossignol et al., 2006; Stern, 2005) but, as any other technique, it has limitations. Classical
proteomic approaches imply first proteins dissolving (extraction) and digestion before
submission to mass spectrometry analysis (or analysis directly from the digest). In many cases
relevant proteins were missed from the analyses since no extraction condition is suitable for
extraction of all proteins (especially membrane proteins) from complex samples. The
12
extraction of membrane proteins means use of detergent for membrane solubilization, which
is not compatible with subsequent mass spectrometry analysis. Proteins with low
stoichiometry of phosphorylation, in very low abundance, or phosphorylated to become a
target for rapid degradation, are often lost during analysis. Sequencing of individual
phosphopeptides from very complex mixtures is technically difficult because the signal
suppression of phosphate-containing molecules in the commonly used positive ion detection
mode (Mann and Jensen, 2003; Mann et al., 2002; McLachlin and Chait, 2001).
Recent efforts have focused on developing technologies for enriching and quantifying
phosphopeptides. To avoid some of the problems in membrane protein phosphorylation
research the new strategy, named “vectorial proteomics”, was developed (Aboulaich et al.,
2004; Vener and Stralfors, 2005).
2.2.2. Vectorial proteomics approach
Vectorial proteomics is a methodology for the differential identification and
characterization of proteins and their domains exposed to the opposite sides of biological
membranes by proteolysis and mass spectrometry (Aboulaich et al., 2004; Vener and
Stralfors, 2005). In mathematics and physics vector is an object defined by both magnitude
and direction; in contrast to a scalar, an object with magnitude only. Vectorial proteomics
allow detection of not only the “magnitude” of the protein phosphorylation in particular
phosphopeptide by mass spectrometry but at the same time “direction”: orientation and
topology of membrane proteins at the opposite membrane surfaces and subsets of extrinsic
proteins associated with the distinct membrane surfaces. This approach was originally
introduced for characterization of protein phosphorylation in thylakoid membranes of
Arabidopsis thaliana (Vener et al., 2001). In 2004 the methodology termed “vectorial
proteomics” was introduced (Aboulaich et al., 2004).
Phosphorylation of membrane proteins is an intrinsically hydrophilic process restricted
to the surface-exposed domains of membrane proteins and to peripheral proteins attached to
the membrane surface. Vectorial proteomics methodology utilizes the natural membrane
morphology. To avoid long and difficult membrane protein purification steps prior to
biochemical characterization, vectorial proteomics uses proteolytic shaving of the hydrophilic
domains exposed to the surface of uniformly oriented membrane vesicles. The trypsin
treatment of isolated thylakoids removes from the membrane phosphorylated peptides of all
major phosphorylated proteins, which was confirmed
by Western blotting with anti-
phosphothreonine antibodies (Turkina et al., 2006; Turkina and Vener, 2007; Vener et al.,
13
2001). The relatively short soluble peptides are collected in the supernatant after
centrifugation and usually enriched for phosphorylated peptides by chromatographic
techniques (Turkina and Vener, 2007) prior to final mass spectrometric sequencing. Mass
spectrometry analysis determines exactly which amino acid(s) of the phosphorylated peptide
is (are) phosphorylated as well as their modification by acetylation or deamidation (Carlberg
et al., 2003; Turkina et al., 2006; Turkina et al., 2004). Application of this methodology
allowed mapping of most of the presently known in vivo protein phosphorylation sites in the
photosynthetic membranes.
2.3. Exploring the phosphoproteome of photosynthetic membranes in
Chlamydomonas reinhardtii
2.3.1. Specific protein phosphorylations induced at limited environmental CO2
Photosynthetic alga induces CO2-concentrating mechanism (CCM) in response to
environmental CO2 limitations that allows cells to acclimate to unfavorable conditions by
optimization of carbon acquisition. CO2-concentrating mechanism is energy-dependent
(Palmqvist et al., 1990). CO2 limitation creates the initial stress situation where the low
concentration of inorganic carbon is limiting photosynthesis, which decreases consumption of
ATP and NADPH and causes reduction of electron carriers in photosynthetic thylakoid
membranes (Palmqvist et al., 1990). It was shown that CCM responses are blocked by
inhibitors of photosynthetic electron transport (Fukuzawa et al., 1990) and in photosynthetic
mutants (Spalding and Ogren, 1985). The redox state of thylakoid membranes has been shown
to be critical for induction of CCM and expression of Ccm1 (Cia5)-controlled genes
(Fukuzawa et al., 2001; Im and Grossman, 2002). Reduction of the plastoquinone (PQ) in the
membranes activates thylakoid protein kinases and induces phosphorylation of photosystem II
and its light-harvesting antenna membrane proteins (Allen, 2003; Aro and Ohad, 2003; Vener
et al., 1998), (Paper I), which are involved in the state transitions process (Allen, 2003;
Wollman, 2001; Wollman and Delepelaire, 1984).
It was found that the protein kinase Stt7 of C. reinhardtii and its ortholog STN7 from
Arabidopsis thaliana are responsible for phosphorylation of a few LHCII proteins and critical
for state transitions (Bellafiore et al., 2005; Depege et al., 2003). STN8 protein kinase from
Arabidopsis thaliana was shown to be specific in phosphorylation of N-terminal threonine
residues in D1, D2, and CP43 proteins, and Thr-4 in the PsbH protein of photosystem II
(Vainonen et al., 2005). However, the nature of other redox-dependent kinases, triggering the
14
molecular mechanisms of acclimation of aquatic algae to limitations in environmental CO2
and the physiological significance of phosphorylation of other thylakoid proteins are largely
unknown (Aro and Ohad, 2003; Depege et al., 2003).
Reversible protein phosphorylation is involved in the triggering of CCM (Fukuzawa et
al., 2001; Kaplan et al., 2001; Xiang et al., 2001), (Paper I). Specific redox-dependent
phosphorylation in cells exposed to low-CO2 conditions was recently revealed in the two,
previously unknown, extrinsic proteins associated with thylakoid membranes (Paper I).
Phosphorylation, which occurred in the early stage of low CO2-acclimation, was found
dependent on both limiting CO2 and the reduced state of the electron carriers in the
photosynthetic membrane, but it was independent of protein kinase Stt7. The first
phosphoprotein that we identified has not been annotated before, thus we named it UEP
(unknown expressed protein) and found it encoded in the genome of C. reinhardtii. The
second, Lci5 protein, is encoded by the low-CO2-inducible gene 5 (lci5) (Im et al., 2003;
Miura et al., 2004), which is controlled by Ccm1 (Cia5) regulator of CO2-responsive genes
(Miura et al., 2004). The phosphorylation sites were mapped in the tandem repeats of Lci5
ensuring phosphorylation of four serine and three threonine residues in the protein.
Both Lci5 and UEP have striking similarities to the plant specific protein Tsp9
undergoing multiple phosphorylation at the surface of thylakoids (Carlberg et al., 2003). All
these three extrinsic proteins are phosphorylated by redox-dependent thylakoid protein
kinases in response to changing environmental conditions: either light (Carlberg et al., 2003)
or limiting CO2 (Paper I). The multiple Tsp9 phosphorylation leads to the release of phosphoTsp9 from the plant thylakoid membranes (Carlberg et al., 2003). An association of TSP9
with LHCII, as well as with the peripheries of the photosystems, suggests its involvement in
regulation of photosynthetic light harvesting (Hansson et al., 2007). The exact functions of
TSP9, Lci5 and UEP are yet unknown, however, these proteins envisage a previously
unknown class of basic proteins undergoing environmentally-induced redox-dependent
phosphorylation at the surface of oxygenic photosynthetic membranes. These novel extrinsic
thylakoid phosphoproteins can probably participate in cellular signalling and regulation via
environmentally induced phosphorylation (Vener, 2007).
There is an example of another, similar to UEP and Lci5, protein phosphorylation that is
controlled by metabolically dependent redox changes in thylakoid membrane. The serine
phosphorylation of PII protein (Forchhammer and Tandeau de Marsac, 1994) regulates
acclimation of cyanobacteria to changing carbon–nitrogen regimes (reviewed in
(Forchhammer, 2004)). Phosphorylation of PII was found at the photosynthetic membranes
15
(Harrison et al., 1990). The phosphorylation was controlled by photosynthetic electron
transport (Harrison et al., 1990; Tsinoremas et al., 1991) and rapidly changed depending on
either reduction or oxidation of the electron acceptor of photosystem I (Hisbergues et al.,
1999). Serine phosphorylation and dephosphorylation of PII in vivo reflected the cellular
nitrogen status and also depended on addition of ammonium or limitation in CO2
(Forchhammer and Tandeau de Marsac, 1995). The exact role of PII phosphorylation in
cyanobacteria is not clear, no is the nature of the protein kinase responsible for serine
phosphorylation of this regulator (Forchhammer, 2004).
Thus, phosphorylation of proteins UEP and Lci5, catalyzed by still unknown specific
low- CO2- and redox-dependent protein kinase, acts as an early adaptive response of the green
alga to limitation in inorganic carbon. The discovery of this phenomenon suggests that both
sensing of limiting CO2 and initiation of signal transduction pathways, triggering CCM in
green algae, may occur in the photosynthetic membranes. The mechanisms of these signal
transduction pathways are still to be revealed, particularly by characterization of the algal
cells deficient in UEP and Lci5.
2.3.2. Light-dependent protein phosphorylations
Photosynthetic organisms have to adjust their molecular machinery to the permanently
changing quality of light in natural environments. In response to fluctuations of light and
redox conditions green algae and plants induce the mechanism of state transitions, which
maintain the balance between the two photosystems. During state transitions the protein
phosphorylation events regulate dynamic lateral migration of light harvesting proteins in the
thylakoid membranes. However, the molecular details of this mechanism remain largely
unknown. In Chlamydomonas reinhardtii the mobile part of LHCII is four times bigger than
in plants (Delosme et al., 1996), and thereby Chlamydomonas is a better model organism to
study the state transition mechanism. However, the exploration of redox-dependent protein
phosphorylation of thylakoid membranes first started in plants (Carlberg et al., 2003; Michel
et al., 1988; Michel et al., 1991; Rinalducci et al., 2006; Vener, 2007; Vener et al., 2001).
Only the recent advances in vectorial proteomics allowed mapping of in vivo phosphorylation
sites in thylakoids from Chlamydomonas reinhardtii (Table 1) (Paper II-IV); while before our
publications (Paper II-IV) only a couple of the phosphorylation sites were known from this
alga (Dedner et al., 1988; Fleischmann and Rochaix, 1999).
The protein domains containing functionally important phosporylation sites are exposed
to the surface of photosynthetic thylakoid membranes of the Chlamydomonas.
16
Table 1. Phosphorylation sites identified in thylakoid proteins from green alga
Chlamydomonas
reinhardtii
exposed
to
different
environmental
conditions.
The
phosphorylated amino acid residues are numbered according to their positions in the initial
translation products of the proteins. The sequences of phosphorylated peptides from the
proteins that were not annotated are shown in single amino acid code.
Protein
Phosphorylated residue
Environmental conditions
Reference
D1
Thr-2
Light
Paper IV
D2
Thr-2
State2/light
(Fleischmann and Rochaix 1999),
Paper IV
CP43
Thr-3
Light
Paper IV
PsbH
Thr-3
PsbR
Ser-43
State2/light
Paper IV
Lhcbm1
Thr-27
State2/light
Paper IV
Lhcbm4
Thr-19 and Thr-23
High light
Paper IV
Lhcbm6
Thr-18 and Thr-22
High light
Paper IV
Lhcbm9
Thr-19 and Thr-23
High light
Paper IV
Lhcbm10
Thr-26
State2/light
Paper IV
CP26 (Lhcb5)
Thr-10
High light
Paper IV
CP29 (Lhcb4)
Thr-7
Darkness/light
Paper IV
Thr-17
State 2/light
Paper IV
Thr-33
Darkness/light
Paper IV
Ser-103
State 2/light
Paper IV
Thr-11
High light
Paper IV
Thr-18
High light
Paper IV
Thr-20
High light
Paper IV
Thr-116, Thr-176, Thr-237
Low CO2
Paper I
Ser-136 and Ser-137
Low CO2
Paper I
Ser-196 and Ser-197
Low CO2
Paper I
UEP
AAAGADsADEEAEAR
Low CO2
Paper I
Unknown protein A
VFEsEAGEPEAK
Darkness/light
Paper IV
Unknown protein A
DVDsEEAR
Light
Paper IV
Unknown protein B
GEIEEADsDDEAR
State2/light
Paper IV
Lci5
(Dedner, Meyer et al. 1988)
17
Phosphorylation events occurred either in the PSII or LHCII of this photosystem, which
catalyzes the first step in photosynthetic electron transport (Nield and Barber, 2006) providing
electrons by photooxidation of water. Using vectorial proteomics we established that the State
1-to-State 2 transition induced phosphorylation of the proteins D2 and PsbR (both PSII core
components) and quadruple phosphorylation of a minor LHCII antennae subunit, CP29, as
well as phosphorylation of constituents of a major LHCII complex, Lhcbm1 and Lhcbm10
(Paper III, IV). Exposure of the algal cells to either moderate or high light caused additional
phosphorylation of the D1 and CP43 proteins of the PSII core. The high light treatment led to
specific hyperphosphorylation of CP29 at seven distinct residues, single phosphorylation of
another minor LHCII constituent, CP26, and double phosphorylation of additional subunits of
a major LHCII complex, including Lhcbm4, Lhcbm6, Lhcbm9, and Lhcbm11. Besides these,
two other previously unknown phosphorylated proteins A and B have been discovered (Table
1) (Paper II-IV). Thus, almost all phosphorylation events occurred in the algal cells exposed
to the specific environment, as indicated in Table 1. Mapping of in vivo protein
phosphorylation sites in photosynthetic membranes revealed that the major environmentally
dependent changes in phosphorylation are clustered at the interface between the PSII core and
its light-harvesting antennae (Figure 5) (Paper IV).
Reversible phosphorylation of the PSII reaction center proteins has been found essential
for the maintenance of active PSII under high light stress (Baena-Gonzalez et al., 1999; Ebbert
and Godde, 1996; Rintamäki and Aro, 2001). Accordingly, reversible N-terminal
phosphorylation of the PSII subunits could be involved in regulation of biodegradation of
these polypeptides (Table 1) (Vener, 2007). However, until publication of Paper IV reversible
phosphorylation of the D1 protein has been considered as plant-specific only (Pursiheimo et
al., 1998; Rintamäki and Aro, 2001; Rintamäki et al., 1996).
2.3.3. Multiply phosphorylated light-harvesting phosphoprotein CP29 is an
integral part of the stress-response mechanism
The light energy captured by the light harvesting antenna is transferred to the reaction
centers. For maximal efficiency of photosynthesis and safety of the photosynthetic apparatus,
the reaction centers should remain balanced in their rates of light energy conversion and lightdriven electron transport. The redistribution of light harvesting antenna proteins with its lightabsorbing chlorophylls between the two photosystems is the major strategy which is used by
photosynthetic organisms to provide this balance.
18
CP26
LHCII
trimer
CP43
D1
CP29
D2
sites phosphorylated in State2
CP47
CP47
D2
sites phosphorylated additionally
at high light
CP29
D1
LHCII
trimer
CP43
CP26
Figure 5. Mapping of the environmentally-induced protein
phosphorylation sites in the model of dimeric photosystem II
supercomplex.
In C. reinhardtii, the LHCI and LHCII proteins are encoded by multigene families, but
the minor PSII light-harvesting polypeptides, CP26 and CP29, are each encoded by one gene
(Elrad and Grossman, 2004; Teramoto et al., 2002). The LHCs genes are nuclear encoded,
translated in the cytosol, and their products are then posttranslationally directed to the
chloroplasts where they associate with pigments and insert into the thylakoid membrane. The
major LHCII proteins form trimers, while monomeric minor LHCII proteins CP29 and CP26
provide a connection between the LHCII trimers and the reaction center of PSII (Elrad and
Grossman, 2004; Yakushevska et al., 2003). Migration of LHCII in the thylakoid membranes
between PSII and PSI is controlled by reversible protein phosphorylation (Allen, 1992a;
Allen and Staehelin, 1994; Bonaventura and Myers, 1969; Vener, 2006; Vener, 2007), (Paper
II-IV). Conformational changes occur within the LHCII upon phosphorylation (Nilsson et al.,
1997). The protein kinase responsible for the phosphorylation of LHCII was identified both in
19
alga and in plants: Stt7 in Chlamydomonas reinhardtii (Depege et al., 2003) and its ortholog
STN7 in Arabidopsis thaliana (Bellafiore et al., 2005). Phosphorylation of several LHCII
subunits and the minor antenna protein CP29 was drastically reduced in the stt7 and stn7
kinase mutants, but still it was not clear if Stt7 and STN7 directly phosphorylated LHCII or if
they operated via a kinase cascade (Bellafiore et al., 2005; Depege et al., 2003; Tikkanen et
al., 2006). Despite the fact, that the mobile pool of LHCII is significantly smaller in vascular
plants than in green algae (Allen, 1992b), state transitions are thought to function similarly in
all LHCII-containing organisms (Vallon et al., 1991). In contrast to other LHCII proteins and
plant LHCII, which have a single phosphorylation site each (Vener, 2007), the CP29 of
Chamydomonas reinhardtii was shown to be heavily phosphorylated (Table 1) (Paper II-IV),
(Allen and Staehelin, 1994; Vener, 2007). CP29 has been found specifically phosphorylated
in several plants under cold and high light stress (Bergantino et al., 1995; Bergantino et al.,
1998; Pursiheimo et al., 1998). Multiple phosphorylation sites of CP29 were identified and
sequenced by mass-spectrometry and characterized under different light and redox conditions
(Paper II-IV). Two sites are phosphorylated (Thr-7 and Thr-33) under state 1 condition, two
additional sites (Thr-17 and Ser-103) are phosphorylated under state 2 conditions and three
additional sites (Thr-11, Thr-18 and Thr-20) are phosphorylated under high light (Table 1).
Beside this, in contrast to all known nuclear-encoded thylakoid proteins, the transit peptide in
the mature algal CP29 is not removed but processed by methionine excision, N-terminal
acetylation and phosphorylation on Thr-7 (Paper II). This processing of the nuclear-encoded
thylakoid protein is similar to the processing of chloroplast-encoded reaction center proteins
of photosystem II. We proposed that retention of the transit peptide in CP29 is a result of
evolutional compromise that allows keeping both the transit peptide and the functionally
important phosphorylation site in the same amino acid sequence (Paper II).
We characterized the structural changes of C. reinhardtii photosynthetic apparatus
during state transitions. The supercomplex of PSI and its antenna, LHCI (LHCI–PSI) was
isolated from the cells of C. reinhardtii exposed to either State 1 or State 2 and analyzed by
electron microscopy and mass spectrometry (Paper III). The minor LHCII protein CP29 was
shown to be strongly associated with PSI-LHCI supercomplexes under State 2 conditions
(Paper III).
Biochemical analyses performed later by another research group has also
confirmed binding of phosphorylated CP29 to PSI in the algal cells exposed to State 2
(Takahashi et al., 2005). Electron microscopy suggests that the binding site for this highly
phosphorylated CP29 is close to the PsaH protein. PsaH was previously found in green plants
to be important in establishing State 2 and it was suggested that PsaH/I/O region of the PSI
20
could be the docking site for phosphorylated LHCII in State 2 conditions (Jensen et al., 2007;
Jensen et al., 2004; Lunde et al., 2000; Zhang and Scheller, 2004). Mass spectrometric
analyses revealed that CP29 was doubly phosphorylated (Thr-7 and Thr-33) in State 1 and
quadruple phosphorylated (Thr-7, Thr-17 and Thr-33, and Ser-103) in State 2 exposed cells
(Table 1) (Paper III).
Previously, CP29 has been well-characterized as a linker protein required for binding of
LHCII to PSII (Yakushevska et al., 2003). We demonstrated that CP29 did not belong
exclusively to PSII, but shuttling between PSII and PSI during state transitions (Paper III, IV).
Isolation of pigment-containing phosphorylated and non-phosphorylated forms of CP29 and
their spectroscopic and biochemical analyses revealed reversible conformational changes in
this protein upon phosphorylation (Croce et al., 1996). We suggested that at state 1 doubly
phosphorylated CP29 acts as a linker between the PSII core dimer and the trimeric LHCII,
while in state 2 the quadruple phosphorylation of CP29 linker protein uncouples it from PSII
and leads to migration and binding of CP29–LHCII complex to PSI, as schematically outlined
in Figure 6. The quadruply phosphorylated CP29 migrates to PSI at the PsaH side and provide
the docking of LHCII trimers to the PSI complex. The phosphorylation-induced
conformational changes in CP29 could significantly affect the affinity of this linker protein to
PSII or PSI (Paper III).
Moreover, we found that exposure of Chlamydomonas cells to high light stress caused
hyperphosphorylation of CP29 at seven distinct residues (Table 1). Under high light
conditions, photoprotective mechanisms minimize the damaging effects of excess light:
excess of excitation energy absorbed by the thylakoid membrane is dissipated as heat. This
non-photochemical quenching in the green alga takes place within the LHCII trimers
peripherally associated with PSII and was shown to be dependent mostly on the peripherally
associated trimeric LHCII polypeptides (Elrad et al., 2002). The recent structural studies
detected specific changes in the configuration of LHCII and its pigment population, which
provides LHCII with a capability to regulate energy flow directed either for photosynthesis or
thermal dissipation (Horton and Ruban, 2005; Pascal et al., 2005). Thus, taking into account
phosphorylation-dependent detachment of CP29-LHCII complex from PSII, we suggested
that high light-induced hyperphosphorylation of CP29 in Chlamydomonas may uncouple this
protein together with LHCII from both photosystems and preclude the transfer of excitation
energy from LHCII (Figure 6).
Besides the multiple phosphorylation of CP29, high light induces in green alga the
single phosphorylation of the other PSII–LHCII linker protein, CP26 (Paper IV). It was
21
P
P
State 1
PSII
P
PSI
P
CP29
LHCII
State 2
P
P
PSI
PP
PSII
P
P
High Light
PSI
PSII
P
PP
P
PP
PP
PP
PP
PP
Figure 6. The proposed mechanisms for photosynthetic state
transitions and for thermal energy dissipation under high
light stress
22
proposed, that CP26 (and Lhcbm5 protein) may also contribute to dissociation of LHCII from
PSII (Takahashi et al., 2005). An analysis of antisense or knockout mutants of plant LHCII
has indicated that the formation of PSII–LHCII supercomplexes is completely prevented by
the absence of CP29, while the absence of CP26 leads to formation of less stable PSII–LHCII
in comparison to wild type. The absence of either CP29 or CP26 does not lead to its
replacement by another protein (Ruban et al., 2003; Yakushevska et al., 2003). The authors
proposed that CP29 and CP26 each have a unique role and location in the oligomeric structure
of PSII. It therefore appears that the phosphorylation of linker protein CP26, occurring at the
interface between the PSII core and LHCII, is probably also involved in the regulation of
thermal dissipation in Chlamydomonas. Thus, uncoupling of LHCII from PSII via the high
light-induced phosphorylation of linker proteins CP29 and CP26 should preclude the transfer
of excitation energy from LHCII to the PSII core and result in the thermal dissipation within
LHCII (Figure 6) (Paper IV).
Threfore, environmentally induced protein phosphorylations at the interface of PSII core
and the associated antenna proteins, particularly multiple differential phosphorylations of
CP29 linker protein, suggests the mechanisms for control of photosynthetic state transitions
and for LHCII uncoupling from PSII under high light stress to allow thermal energy
dissipation.
23
3. Concluding remarks
This thesis presents the first systematic identification and characterization of
phosphoproteins and in vivo protein phosphorylation events involved in adaptive responses in
unicellular green photosynthetic alga Chlamydomonas reinhardtii. The results and
conclusions of the research published in papers I-IV are summarized as follows:
•
31 in vivo protein phosphorylation sites were identified within photosynthetic
membranes from the alga subjected to distinct environmental conditions known to
affect the photosynthetic machinery.
•
Acclimation of the alga to limiting environmental CO2 induced specific
phosphorylation of Lci5 and UEP proteins. Lci5 is associated with the stromal side of
chloroplast thylakoids. The low-CO2-dependent phosphorylation acts as an early
adaptive response of alga to limitation in inorganic carbon.
•
The reversible phosphorylation of D1 protein is discovered in the alga, which until this
work has been considered as plant specific.
•
Multiple phosphorylations occur in the minor light harvesting protein CP29 under
different light and redox conditions. Two sites (Thr-7 and Thr-33) are phosphorylated
under state 1 condition, two additional sites (Thr-17 and Ser-103) are phosphorylated
under state 2 conditions and three more sites (Thr-11, Thr-18 and Thr-20) are
phosphorylated under high light.
•
In contrast to all known nuclear-encoded thylakoid proteins, the transit peptide in the
mature algal CP29 is not removed but processed by methionine excision, N-terminal
acetylation and phosphorylation on Thr-7. The importance of this phosphorylation site
is a probable reason for the transit peptide retention.
•
CP29 does not belong exclusively to PSII, as it was postulated before this work, but
shuttling between PSII and PSI during state transitions. The LHCI-PSI supercomplex
isolated from the alga in State 2 contains strongly associated CP29 in phosphorylated
form in the vicinity of the PsaH protein region. Structural changes of CP29, induced
by reversible phosphorylation, determine the affinity of LHCII trimers for either of the
two photosystems.
•
Environmentally induced dynamic changes in protein phosphorylation at the interface
between the PSII core and its associated LHCII may regulate photosynthetic light
24
harvesting and PSII dynamics in green algae as well as facilitate State 1-to-State 2
transitions.
•
High light-induced hyperphosphorylation of CP29 may uncouple this protein together
with LHCII from both photosystems to minimize the damaging effects of excess light.
•
This work provides the first comprehensive insight into the network of
environmentally regulated protein phosphorylation in algal photosynthetic membranes
and explains molecular differences in photosynthetic adaptive responses between
green algae and higher plants.
•
This thesis results provide basis for the future mutagenesis and reverse genetic studies
aimed at dissecting the exact role of thylakoid protein phosphorylation in regulation of
photosynthetic machinery.
25
4. Acknowledgments
I would like to thank all of you who have helped to make this thesis research.
Particularly, I thank my supervisors: Professor Alexander Vener, Professor Peter
Strålfors and Professor Bertil Andersson. Without them this work would have been
impossible. Thank you for guidance and encouragement, for patience and understanding.
Thank you for giving me the opportunity to work at Linköping University.
I would like to thank my co-authors: Arsenio Villarejo, Amaya Blanco-Rivero, James
Barber, Joanna Kargul, Jon Nield, Sam Benson and Julia Vainonen for the creativity and
excellent collaboration.
All the people from our plant group for creating the helpful, supportive and
intellectually stimulating atmosphere. Thank you for constructive comments and exiting
discussions on our group meetings. Particularly, I thank Rikard Fristedt and Alexey
Shapiguzov, my officemates, for the nice chats and serious talks, optimism, positive energy
and support. I thank Anna Edvardsson and Maria Hansson for lots of help during my first
years in LiU. I thank Cornelia Spetea-Wiklund for sharing her knowledge and enthusiasm.
Thanks for Björn Lundin, Björn Ingelsson, Lorena Ruiz-Pavón, Hanna Klang and Patrik
Karlsson for being friendly and cheerful colleagues.
I thank Julia Vainonen and Nabila Aboulaich for being very good friends and for the
good times we had shared. Miss you!
Many thanks for Torill and Kjell Hundal for their warm friendship and for the
wonderful and unforgettable time in Norway.
I thank Inger Carlberg for being so kind.
Thanks to Unn, Åsa, Anita, Karin, Siri, Elisabeth, Margareta, Lilian, Anna, Jana, Johan
and and all others from the “Strålforsgruppen” and at the floor 12 for being so helpful.
Thanks to Håkan Wiktander, Erik Mårdh, Ingrid Nord, Anette Wiklund, Monika
Hardmark, Ulf Hannestad and many others at IBK/IKE. It is your work that made my project
to run smoothly.
Thanks to Annette, Åsa, Lena, Anneli, Peter, Patiyan, Nils, Pia, Amanda, Elisavet, Anna
and to all my colleagues and friends at the floor 9 and 13 for the kindness and for the nice
company.
26
I thank everyone at Protein Chemistry Department of A.N. Belozersky Institute of
Physical-Chemical Biology, Moscow State University for teaching me and giving me good
start in science.
I would like to thank my family and my friends for all tremendous support all these
years.
The research underlying this thesis was performed with support from the Graduate
Research School in Genomics and Bioinformatics (FGB), the Swedish Research Council
(VR), the Swedish Research Council for Environment, Agriculture and Spatial Planning
(Formas) and Nordiskt Kontaktorgan för Jordsbruksforskning (NKJ).
27
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Turkina et al., Supplemental Data
Supplementary Figure Legends
Supplementary Fig.1. Mapping of the phosphorylation site in the peptide from unknown
protein A (found phosphorylated in all four tested environmental conditions). Collisioninduced fragmentation ion spectra of the doubly protonated ions of phosphorylated and methylesterified peptides. The major detected y (C-terminal) and b (N-terminal) ions are indicated in the
spectrum and in the corresponding amino acid sequence above the spectrum. The ions marked
with an asterisk indicate that corresponding fragments underwent neutral loss of phosphoric acid
(H3PO4, 98kDa). The selected parent peptide ion with m/z 721.8 is labeled in the spectrum along
with the fragment at 672.8 m/z, which was produced after characteristic neutral loss of
phosphoric acid. The phosphorylated serine residue in the peptide sequence is indicated with s.
For location of Chlamydomonas reinhardtii gene encoding this phosphorylated protein A see
Supplementary Table 1.
Supplementary Fig.2. Mapping of phosphorylation site in the peptide from unknown protein
B (found phosphorylated the algal cells exposed to State 2 conditions and light). Collisioninduced fragmentation ion spectra of the doubly protonated ions of phosphorylated and methylesterified peptides. The major detected y (C-terminal) and b (N-terminal) ions are indicated in the
spectrum and in the corresponding amino acid sequence above the spectrum. The ions marked
with an asterisk indicate that corresponding fragments underwent neutral loss of phosphoric acid
(H3PO4, 98kDa). The selected parent peptide ion with m/z 814.3 is labeled in the spectrum along
with the fragment at 765.3 m/z, which was produced after characteristic neutral loss of
phosphoric acid. The phosphorylated serine residue in the peptide sequence is indicated with s.
For location of Chlamydomonas reinhardtii gene encoding this phosphorylated protein B see
Supplementary Table 1.
1
Supplementary Fig.3. Mapping of phosphorylation site in the peptide from unknown protein
A (found phosphorylated in only in light-exposed algal cells). Collision-induced fragmentation
ion spectra of the doubly protonated ions of phosphorylated and methyl-esterified peptides. The
major detected y (C-terminal) and b (N-terminal) ions are indicated in the spectrum and in the
corresponding amino acid sequence above the spectrum. The ions marked with an asterisk
indicate that corresponding fragments underwent neutral loss of phosphoric acid (H3PO4, 98kDa).
The selected parent peptide ion with m/z 535.7 is labeled in the spectrum along with the fragment
at 486.7 m/z, which was produced after characteristic neutral loss of phosphoric acid. The
phosphorylated serine residue in the peptide sequence is indicated with s. For location of
Chlamydomonas reinhardtii gene encoding this phosphorylated protein A see Supplementary
Table 1.
Supplementary Fig.4. Sequence alignment of PsbR homologs. Alignment of mature PsbR
proteins from Chlamydomonas reinhardtii (PSBR_CHLRE), Brassica rapa (PSBR_BRACM),
Arabidopsis thaliana (PSBR_ARATH), Lycopersicon esculentum (PSBR_LYCES), Solanum
tuberosum
(PSBR_SOLTU),
Nicotiana
tabacum
(PSBR_TOBAC),
Spinacia
oleracea
(PSBR_SPIOL). Sequences were aligned with Clustal W (Higgins D., Thompson J., Gibson
T.Thompson J.D., Higgins D.G., Gibson T.J.(1994). CLUSTAL W: improving the sensitivity of
progressive multiple sequence alignment through sequence weighting, position-specific gap
penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.) The amino acid residues
in bold correspond to the peptide sequenced in this work. The arrow indicates the phosphorylated
serine residue. Note that mature C. reinhardtii PsbR sequence is 14 aminoacids longer then others
and part of the N-terminal sequence is absent in all higher plants PsbR sequences.
PSBR_CHLRE sequence was obtained from the Chlamydomonas reinhardtii genome version 3.0,
JGI (98021). Other sequences were from Genbank: PSBR_BRACM (P49108), PSBR_ARATH
2
(P27202), PSBR_LYCES (Q40163), PSBR_SOLTU (P06183), PSBR_TOBAC (Q40519) and
PSBR_SPIOL (P10690).
3
Supplementary Table 1. Location of nucleotides in Chlamydomonas reinhardtii genome
(version 3.0, JGI), encoding sequenced peptides from unknown proteins. Peptides
VFESEAGEPEAK and DVDSEEAR belong to the same unknown protein A.
Peptide
sequence
Nucleotide sequence
VFEsEAGEPEAK
DVDsEEAR
GEIEEADsDDEAR
GTCTTCGAGTCTGAGGCGGGCGAGCCGGAGGCGAAA
GACGTGGACAGCGAGGAGGCGCGC
GGCGAGATTGAGGAGGCTGACAGTGACGACGAGGCCCGC
Scaffold
number
4
scaffold_12
scaffold_12
scaffold_58
Location
Number of the
bases in scaffold
1514518 - 1514553
1515079 - 1515102
160845 - 160883
Turkina et al., Supplementary Fig.1
y4
11 10 9 8 7
y10*2+
a2 b2
y1
y6
60%
PEA
b4*
40%
6
4
2 3
4
721.8
672.8
b8*
y9*
b3
y7
y8
y10*
y10
y9
y11*
20%
200
400
1 y
VFEsEAGEPEAK
y102+
Intensity (%)
80%
b
600
800
m/z
1000
1200
Turkina et al., Supplementary Fig.2
9
8
7 6
3 2
1 y
GEIEEADsDDEAR
b
2 3 4 5
6
814.3
Intensity (%)
y2
b3
765.3
30%
y1
b2
b5
y8
y3
y9
b6
20%
y6
b4
y7
y9*
y7*
10%
200
400
600
800
m/z
1000
1200
Turkina et al., Supplementary Fig.3
6
5 4
3
2 1 y
DVDsEEAR
a2
b
2
3
4 5
6 7
Intensity (%)
80%
535.7
b2
60%
486.7
y6*2+
y3
y6*
y4
y62+
40%
y1
b5*
y5*
y2
b4*
b3
20%
200
400
600
m/z
y6
y5
b6*
b7*
800
Turkina et al., Supplementary Fig.4
PSBR_CHLRE
PSBR_BRACM
PSBR_ARATH
PSBR_LYCES
PSBR_SOLTU
PSBR_TOBAC
PSBR_SPIOL
** * . *
* . * :* *:**** .* ***:: *****
SGGGKTDITKVGLNSIEDPVVKQNLMGKSRFMNKKDWKDASGRKGKGYGVYRYEDKYGA
SGVKKIKTDKP--------------FGVNGSMDLRDGVDASGRKGKGYGVYKFVDKYGA
SGVKKIKTDKP--------------FGINGSMDLRDGVDASGRKGKGYGVYKYVDKYGA
SGVKKLKTDKP--------------YGINGSMALRDGVDASGRKPKGKGVYQYVDKYGA
SGVKKLKTDKP--------------YGINGSMALRDGVDASGRKPKGKGVYQYVDKYGA
SGVKKLKTDKP--------------YGINGSMSLRDGVDASGRKQKGKGVYQFVDKYGA
SGVKKIKVDKP--------------LGIGGGMKLRDGVDSSGRKPTGKGVYQFVDKYGA
PSBR_CHLRE
PSBR_BRACM
PSBR_ARATH
PSBR_LYCES
PSBR_SOLTU
PSBR_TOBAC
PSBR_SPIOL
*********. : *: :** * *..** ** : :** *. *: .** *.
NVDGYSPIYTPDLWTESGDSYTLGTKGLIAWAGLVLVLLAVGVNLIISTSQLGA
NVDGYSPIYNEDEWSASGDVYKGGVTGLAIWAVTLAGILAGGALLVYNTSALAQ
NVDGYSPIYNENEWSASGDVYKGGVTGLAIWAVTLAGILAGGALLVYNTSALAQ
NVDGYSPIYNTDEWSPSGDVYVGGTTGLAIWAVTLLGILAGGALLVYNTSALAQ
NVDGYSPIYNTDEWSPSGDVYVGGTTGLAIWAVTLVGILAGGALLVYNTSALAQ
NVDGYSPIYNTDDWSPSGDVYVGGTTGLAIWAVTLVGILAGGALLVFNTSALAQ
NVDGYSPIYNEEEWAPTGDVYAGGTTGLLIWAVTLAGLLAGGALLVYNTSALAQ
114
100
100
100
100
100
100
60
46
46
46
46
46
46