Download The Phosphoproteome of a Chlamydomonas reinhardtii Eyespot

The Phosphoproteome of a Chlamydomonas reinhardtii
Eyespot Fraction Includes Key Proteins of the Light
Signaling Pathway1[W]
Volker Wagner, Katharina Ullmann, Anne Mollwo, Marc Kaminski, Maria Mittag2, and Georg Kreimer2*
Institut für Allgemeine Botanik und Pflanzenphysiologie, Friedrich-Schiller-Universität Jena, 07743 Jena,
Germany (V.W., M.K., M.M.); and Department Biologie, Friedrich-Alexander-Universität Erlangen, 91058
Erlangen, Germany (K.U., A.M., G.K.)
Flagellate green algae have developed a visual system, the eyespot apparatus, which allows the cell to phototax. In a recent
proteomic approach, we identified 202 proteins from a fraction enriched in eyespot apparatuses of Chlamydomonas reinhardtii.
Among these proteins, five protein kinases and two protein phosphatases were present, indicating that reversible protein
phosphorylation occurs in the eyespot. About 20 major phosphoprotein bands were detected in immunoblots of eyespot
proteins with an anti-phosphothreonine antibody. Toward the profiling of the targets of protein kinases in the eyespot fraction,
we analyzed its phosphoproteome. The solubilized proteins of the eyespot fraction were treated with the endopeptidases LysC
and trypsin prior to enrichment of phosphopeptides with immobilized metal-ion affinity chromatography. Phosphopeptides
were analyzed by nano-liquid chromatography-electrospray ionization-mass spectrometry (MS) with MS/MS as well as
neutral-loss-triggered MS/MS/MS spectra. We were able to identify 68 different phosphopeptides along with 52 precise in
vivo phosphorylation sites corresponding to 32 known proteins of the eyespot fraction. Among the identified phosphoproteins
are enzymes of carotenoid and fatty acid metabolism, putative signaling components, such as a SOUL heme-binding protein, a
Ca21-binding protein, and an unusual protein kinase, but also several proteins with unknown function. Notably, two unique
photoreceptors, channelrhodopsin-1 and channelrhodopsin-2, contain three and one phosphorylation sites, respectively.
Phosphorylation of both photoreceptors occurs in the cytoplasmatic loop next to their seven transmembrane regions in a
similar distance to that observed in vertebrate rhodopsins, implying functional importance for regulation of these directly
light-gated ion channels relevant for the photoresponses of C. reinhardtii.
Many flagellate green algae possess a single primitive visual system, the eyespot apparatus, for detecting
light direction and intensity. The design of the eyespot
apparatus in conjunction with the helical movement of
the cell produces a highly directional optical device
allowing effective tracking of the light direction. In
Chlamydomonas reinhardtii, the eyespot apparatus is usually
composed of two layers of highly ordered carotenoid-rich
lipid globuli that are situated at the periphery of the
chloroplast. The globuli layers are subtended by thylakoid membranes. Additionally, the outermost globule
layer is attached to specialized areas of the chloroplast
envelope membranes and the adjacent plasma membrane. The photoreceptors are generally considered to
be localized in this plasma membrane patch belonging
1
This work was supported by the Deutsche Forschungsgemeinschaft (grant no. Kr 1307/7–1 to G.K. and grant nos. Mi 373/7–3 and
Mi 373/8–3 to M.M.).
2
These authors contributed equally to the article.
* Corresponding author; e-mail [email protected].
The authors responsible for distribution of materials integral to
the findings presented in this article in accordance with the policy
described in the Instructions for Authors (www.plantphysiol.org)
are: Georg Kreimer ([email protected]) and Maria
Mittag ([email protected]).
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.107.109645
772
to the functional eyespot apparatus (for review, see
Kreimer, 2001). Light causes two major behavioral
responses, phototaxis, where the cells swim toward or
away from the light source, and photoshock. The latter
is observed when cells experience a large and sudden
change in light intensity, which causes a transient stop
in movement, followed by a short period of backward
swimming (for review, see Witman, 1993). The phototactic behavior is not only controlled by light, but also
by the circadian clock that is entrained by light-dark
cycles (Bruce, 1970; for review, see Mittag et al., 2005).
Due to the elaborate structures of algal eyespot apparatus and the known presence of rhodopsins in some
lineages, algae are thought to play an important role in
the evolution of photoperception and eyes (Gehring,
2004). Therefore, the structural components forming
this early visual system and its associated signaling cascades are not only of special interest to plant biologists.
Until 2005, only six components of the eyespot of
C. reinhardtii were known at the molecular level. These
included EYE2 and MIN1, two proteins important for
eyespot assembly (Roberts et al., 2001; Dieckmann, 2003),
two splicing variants of the abundant retinal binding
protein COP (Chlamydomonas opsin), and two unique
seven-transmembrane domain (TMD) photoreceptors,
COP3 and COP4 (Deininger et al., 1995; Fuhrmann
et al., 2001; Nagel et al., 2002; Sineshchekov et al., 2002;
Suzuki et al., 2003). COP3 and COP4, widely known as
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Phosphoproteomic Analysis of the Chlamydomonas Eyespot
channelrhodopsin-1 and channelrhodopsin-2 (ChR-1
and ChR-2), are directly light-gated cation channels
that contain a planar all-trans, 6-S-trans retinal chromophore, which undergoes 13-trans to cis isomerization
upon illumination. They are involved in the phototactic and photophobic behavior of C. reinhardtii. However, details of the signaling cascades initiated upon
their excitation are still obscure (Sineshchekov et al.,
2002; Nagel et al., 2002, 2005b; Govorunova et al., 2004;
Kateriya et al., 2004). Notably, ChR-2 has been successfully expressed for light stimulation of different
systems, including Caenorhabditis elegans and mammalian neurons (Boyden et al., 2005; Nagel et al., 2005a).
ChR-2 was even delivered to retinal ganglion cells in a
rodent model of inherited blindness (Bi et al., 2006). In
this way, genetically engineered surviving retinal neurons were generated to take on the lost photoreceptive
function.
A recent conducted proteomic analysis of a fraction
enriched in eyespot apparatuses of C. reinhardtii, including the lipid globuli and the associated parts of
chloroplast and plasma membranes, resulted in the
identification of 202 proteins that were covered by at
least two peptides in the mass spectrometry (MS) analysis (Schmidt et al., 2006). Besides the already abovementioned proteins, this analysis revealed the presence
of proteins from diverse functional groups in the eyespot. These include, for example, calcium-sensing and
binding proteins, channels, membrane-associated/
structural proteins such as proteins with PAP-fibrillin
domains, proteins involved in retinal, carotenoid, and
chlorophyll biosynthesis as well as in lipid metabolism,
and thylakoid membrane-associated proteins. Care
must be taken to conclude that thylakoid membraneassociated proteins of the chloroplast, which are also
present in the eyespot, have the same localization
and/or function there. The a-, b-, and g-subunits of
the soluble CF1 complex of the ATP synthase, for example, probably have specialized localization within the
eyespot and are thermolysin resistant (Schmidt et al.,
2007). Of special interest is also the presence of a SOUL
heme-binding protein (SOUL/HBP) in the eyespot. The
first member of this group of HBPs was found to be
specifically located in the retina and pineal gland in
chicken (Zylka and Reppert, 1999). Because Descartes
considered the pineal gland as the soul, Zylka and
Reppert named the protein accordingly. Further on,
kinases and phosphatases were found in the eyespot
proteome, indicating that light signaling cascades(s)
may involve regulation by reversible protein phosphorylation. The two detected protein phosphatases
(PPs) belong to the PP2C family of Ser/Thr PPs. In
higher plants, it was shown that PP2Cs often regulate
signaling negatively (Schweighofer et al., 2004). The
five identified kinases in the eyespot proteome include
a cyclic nucleotide-dependent kinase II, two unusual
protein kinases with AarF domains, the blue light photoreceptor phototropin with its Ser/Thr kinase domain, and casein kinase1 (CK1). CK1 was functionally
analyzed by an RNAi approach (Schmidt et al., 2006).
Its silencing has multiple effects and results in severe
disturbances in hatching, flagellum formation, and
circadian control of phototaxis. These first data already
point out the significance of reversible phosphorylation events in the signaling pathways within the eyespot and toward the flagella. Therefore, we decided to
find the targets of these kinases and phosphatases in
the eyespot by a phosphoproteome approach.
In C. reinhardtii, several cellular subfractions have been
unraveled in the past years by large-scale proteome
approaches, such as the flagella (Pazour et al., 2005),
centrioles (Keller et al., 2005), or the eyespot (Schmidt
et al., 2006). First phosphoproteome approaches have
also been carried out, for example, with crude extracts
(Wagner et al., 2006) or thylakoids isolated from C.
reinhardtii cells grown under different environmental
conditions (Turkina et al., 2006a, 2006b). However,
phosphoproteome analysis has been and still is a challenging task (for review, see Mann et al., 2002; Reinders
and Sickmann, 2005). This is due to a few facts. (1)
Phosphoproteins can have more than one phosphorylation site and the phosphorylation status of these
sites can fluctuate, depending on the physiological conditions of the cells. (2) Only a small portion of a given
protein in the cell can be phosphorylated. (3) Furthermore, phosphoproteins, especially those of signaling
pathways, are often low-abundance proteins anyway.
Therefore, enrichment of phosphoproteins/peptides
from the cell or a subcellular compartment is a prerequisite for efficient phosphoproteome analysis. Different methods can be used for this purpose (for review,
see Reinders and Sickmann, 2005). One of them, immobilized metal-ion affinity chromatography (IMAC),
is based on the presence of the negatively charged phosphate groups and enriches for phosphorylated Ser,
Thr, and Tyr. This method has already been applied for
phosphoproteome analyses in different systems like
lymphoma cells (Shu et al., 2004), higher plants (Nühse
et al., 2004), and C. reinhardtii (Turkina et al., 2006a,
2006b; Wagner et al., 2006). It relies on the direct identification of phosphopeptides in MS in contrast to other
methods that chemically substitute phosphate residues.
However, in tandem MS (MS/MS; hereafter MS2),
phosphopeptide precursor ions can exhibit neutral loss
of phosphoric acid (298 D). The reason for this loss is
that phosphopeptides (phospho-Ser and phospho-Thr)
can undergo gas-phase b-elimination when subjected
to collision-induced fragmentation (Mann et al., 2002).
Because mass/charge (m/z) values and not absolute
masses are measured in a mass spectrometer, doubly
and triply charged peptide ions show an apparent loss
of 49 and 32.66, respectively. In the MS2 spectrum, the
presence of neutral loss therefore indicates phosphorylation and can be used as a selection parameter for
phosphopeptides. There are some types of electrospray
ionization (ESI)-MS (e.g. linear ion trap LTQ [Thermo
Electron]) that allow the acquisition of data-dependent
neutral loss. Thus, neutral-loss analysis can be executed during MS measurements (MS2 scan). If a pair of
peaks for one of the most prominent ions of the MS2
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Wagner et al.
spectrum versus the full-scan MS spectrum is found
with a mass difference of 98, 49, or 32.66 (depending
on the charge of the peptide ion), a phosphorylationspecific neutral loss is indicated. This prominent ion of
the MS2 spectrum (representing the dephosphorylated
peptide) will then be selected for an automatically triggered MS/MS/MS (MS3) scan so that sufficient fragment ion information can be obtained if not provided
by the MS2 spectrum. Thus, data from MS2 spectra can
be additionally confirmed by MS3 spectra. This method
was used, for example, for phosphoproteomics for the
yeast (Saccharomyces cerevisiae) pheromone signaling
pathway (Gruhler et al., 2005), in the recent whole-cell
phosphoproteome approach in C. reinhardtii (Wagner
et al., 2006), but also for a selected protein (Ouelhadj
et al., 2007).
In this work, we analyzed the phosphoproteins of
eyespot preparations to get information about its in
vivo kinase targets and thereby insights into its signaling network. Due to the elaborate structure of the
eyespot and the rather hydrophobic character of many
of its proteins, we had to apply a special protocol to
bring the proteins in proper solution for efficient proteolytic digest followed by IMAC. Multiple liquid
chromatography (LC)-ESI-MS analyses from independent eyespot preparations were then conducted via MS2
and neutral-loss-triggered MS3 spectra. Thus, 68 phosphopeptides, belonging to 32 proteins that were already identified in former eyespot proteome analyses,
as well as 15 phosphopeptides that do not correspond
to yet-known proteins from this fraction, were identified. Analysis of phosphorylation sites revealed a bias
toward certain amino acids in their surroundings
and a tendency to occur outside of known functional
domains. The eyespot phosphoproteome includes
proteins of (potential) light signaling pathways, chloroplast and thylakoid components, carotenoid and
fatty acid metabolism, but also several proteins with
unknown function. Notably, two photoreceptors, ChR-1
and ChR-2, were also found with three and one phosphorylation sites, respectively. Localization of these
sites in a cytoplasmatic loop with close proximity to
the channel-forming region implies functional relevance for the regulation of these unique directly lightgated ion channels.
RESULTS
The Eyespot Fraction Contains a Significant Number of
Thr-Phosphorylated, But Only a Few
Tyr-Phosphorylated Proteins
Detection of five kinases and two Ser/Thr PPs of the
PP2C family in the eyespot proteome of C. reinhardtii
(Schmidt et al., 2006) underlined the potential importance of reversible protein phosphorylation for signaling pathways in this complex cell organelle. For
phosphoproteome analyses, preparation of the eyespot fraction was basically done according to Schmidt
et al. (2006). Additionally, a set of seven phosphatase
inhibitors (microcystin LR, cantharidin, (2)-p-bromotetramisole, vanadate, molybdate, tartrate, imidazole)
was used during cell rupture to reduce potential dephosphorylation of the proteins. For analysis of phosphoproteins, we first prepared immunoblots with two
different commercially available phospho-amino acidspecific antibodies. Western analysis of the proteins
from the purified eyespot fraction with a polyclonal
anti-phospho-Thr antibody revealed about 20 major
and some minor phosphorylated protein bands in the
apparent molecular mass range $25 kD (Fig. 1A). The
labeling pattern was reproducibly observed in several
independent eyespot isolations. Coomassie staining of
protein bands transferred to the polyvinylidene difluoride (PVDF) membrane showed that several phosphoproteins (e.g. .116 kD) belong to low-abundance
proteins that are slightly, if at all, detectable by
Coomassie staining (Fig. 1A). Specificity of the labeling was confirmed by alkali treatment of the blotted
proteins prior to immunoanalysis, which caused massive reduction in both labeling intensity and amount of
detected phosphoproteins (Fig. 1B). This treatment is
known to significantly reduce the amount of phosphorylation at Ser and Thr residues of blotted proteins
without significant protein loss from the PVDF membrane (Kamps and Sefton, 1989; Rudrabhatla et al.,
2006). To analyze whether Tyr-phosphorylated proteins
were also present in the final eyespot fraction, a monoclonal anti-phospho-Tyr antibody was used. In contrast to the results obtained with the anti-phospho-Thr
antibody, in this case only two major protein bands at
apparent molecular masses of 37 and 40 kD were consistently labeled (Fig. 1C). These results clearly demonstrate that (1) considerable amounts of phosphoproteins
are still present in the eyespot fraction after the isolation procedure; and (2) phosphorylation apparently
occurs mainly at Thr and probably also at Ser residues,
whereas phosphorylation at Tyr is minor.
Purification Procedure for Enrichment of Eyespot
Phosphopeptides Involves Digestion with LysC
and Trypsin Prior to IMAC
Because only a small fraction of the proteins prone
to phosphorylation exists in the phosphorylated state
at any given time and proteins regulated by phosphorylation can be very low abundant, enrichment of the
phosphopeptides is an essential initial step prior to
their subsequent analysis. Removal of the nonphosphorylated peptides increases selectivity and detection sensitivity of phosphopeptides in the MS analysis.
Different approaches are currently used for phosphopeptide enrichment from complex mixtures of peptides
(for review, see Reinders and Sickmann, 2005). We
applied IMAC based on Ga31 as a metal ion. Application of the membrane-shaving method with trypsin,
which is often used to release surface-exposed phosphopeptides of membrane proteins prior to IMAC (e.g.
Vener et al., 2001; Turkina et al., 2006a, 2006b), was not
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Phosphoproteomic Analysis of the Chlamydomonas Eyespot
hydrochloride solution containing the solubilized proteins down to a concentration of 0.6 M. However, in the
eyespot sample, massive protein precipitation occurred
upon dilution of guanidine hydrochloride prior to tryptic digestion. Therefore, a new protocol was developed
for getting a representative phosphopeptide yield from
eyespot fractions. For this purpose, the precipitated
proteins were solubilized in 4 M urea instead of 6 M
guanidinium hydrochloride and then treated with the
endoproteinase LysC, which specifically cleaves peptide bonds C-terminally at Lys and still has an enzyme
activity of 86% in 4 M urea (www.roche-applied-science.
com, datasheet LysC). Because the proteolytic digest
with LysC results in peptides with relatively high
molecular masses, which are not suited for routine LCESI-MS analysis, the reduced and desalted peptides
were subjected to a second digest with trypsin prior to
enrichment of the phosphopeptides with IMAC. By
using this modified procedure (summarized in Fig. 2),
aggregation and precipitation of the proteins of the
eyespot fraction, which is enriched in proteins with a
moderate hydrophobic and amphipathic character
(Schmidt et al., 2006), were avoided to a large extent.
Identification of Phosphopeptides from the Eyespot
Fraction by Nano-LC-ESI-MS
Figure 1. Western-blot analysis of proteins from the eyespot fraction of
C. reinhardtii with phospho-amino acid-specific antibodies. A, Proteins
(4 mg) were separated by 11% SDS-PAGE, transferred to a PVDF
membrane, and either analyzed with a polyclonal anti-phospho-Thr
antibody (Anti-pThr; 1:1,000) or stained with Coomassie Brilliant Blue
R250 (Coomassie). B, The membrane, to which proteins (4 mg) had
been blotted, was treated with 1 M KOH according to Kamps and Sefton
(1989) prior to incubation with the antiserum to reduce the amount of
phosphorylated Ser and Thr residues. The developing time was identical to that in A (3 min). C, Proteins (4 mg) from two independent
eyespot isolations were separated by SDS-PAGE (11%), blotted, and
probed with a monoclonal anti-phospho-Tyr antibody (Anti-pTyr;
1:1,000, developing time 15 min). Arrowheads indicate the two major
labeled protein bands. Positions of molecular mass markers are indicated on the left (kD).
chosen here for the following reason. We could not
exclude that the eyespot globules form a steric barrier
that prevents complete hydrolysis of the proteins. Because the eyespot fraction has a low protein and high
carotenoid/lipid content, samples were extracted by
chloroform:methanol:water (4:8:3 [v/v/v]) to concentrate the proteins and to extract the lipids/carotenoids
in parallel. Precipitated proteins were solubilized with
6 M guanidine hydrochloride. We then applied the
protocol of Wagner et al. (2006), which has successfully
been used for phosphoproteins of crude extracts from
C. reinhardtii. In this protocol, proteins are digested
with trypsin and afterward phosphopeptides are enriched with Ga31-based IMAC. For efficient digestion
with trypsin, it is necessary to dilute the guanidine
Phosphopeptides from the eyespot fraction were
enriched and analyzed in three independent experiments according to the protocol presented in Figure 2.
To increase the confidence of the final phosphoproteinpeptide assignment, the following four criteria had to
be fulfilled. (1) Phosphopeptides could be identified
within the protein sequence of a predicted gene model
of the analyzed genome. We used version 3 (Vs3) and
the older version 2 (Vs2) of the C. reinhardtii genome in
our analysis because the eyespot proteome was based
on Vs2 (Schmidt et al., 2006). (2) Phosphopeptides belong to proteins that have already been identified in
former eyespot analyses. An exception was only made
in one case, which is explained later. (3) They showed a
significant Xcorr factor and probability score (see
‘‘Materials and Methods’’ for details). (4) Phosphoproteins were identified in at least two of the three
independent experiments. Thus, one phosphopeptide
purification experiment always covers the proteins
from two independent eyespot isolations. In total, we
identified 68 phosphopeptides fulfilling these criteria.
Among them, 19 peptides (indicated by an arrow in
Table I) had overlapping sequences or the same, but
existed with a different number of phosphorylation
sites. The latter has most likely functional implications
for the respective protein. Table I summarizes the
phosphopeptides along with the protein ID numbers
and biological function (if known) of the corresponding protein models. One further phosphopeptide belonging to a protein of yet-unknown function (protein
ID 187713; Vs3) was also included in the list despite
the fact that it was not found in our former eyespot
analyses (Schmidt et al., 2006; M. Mittag and G.
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Figure 2. Schematic overview of phosphopeptide enrichment from the
eyespot fraction of C. reinhardtii for LC-ESI-MS analysis. Details are
described in ‘‘Materials and Methods.’’ Nano-LC-ESI-MS (MS2 and MS3)
analysis was carried out in a mass spectrometer with a linear ion trap
that permits the acquisition of data-dependent neutral loss (Finnigan
LTQ; Thermo Electron).
Kreimer, unpublished data). This protein has a distribution of trypsin sites that results in peptides being
either too small or too large for routine LC-ESI-MS
analysis and thus could have easily been missed in
those analyses. It is specially marked in Table I. In two
cases, the exact protein model could not be identified
with certainty because the same peptide was present
in different protein models. This concerns, for example, a peptide found in different members of the lightharvesting complex II (LHCII) family, which are highly
conserved. In these cases, all possible protein IDs and
National Center for Biotechnology Information (NCBI)
BLASTp results are listed. In statistical analyses, these
cases were taken as one protein. Further details (charges,
Xcorr values, and probability scores) of the identified
69 phosphopeptides are listed in Supplemental Table
S1. Moreover, the MS2 and MS3 spectra of these
phosphopeptides are publicly available under the following Web site (http://www.uni-jena.de/Protein_
network_of__C__reinhardtii-lang-en.html), where spectra
from each peptide resulting in the highest Xcorr are
shown.
In addition, 14 phosphopeptides were found in these
analyses, which belong to proteins that have not been
depicted in former eyespot analyses (Supplemental
Table S2). They have theoretically determined trypsin
sites that should be suitable for LC-ESI-MS analysis.
They might thus represent very low-abundance proteins, where the enrichment of phosphopeptides by
IMAC is needed to allow MS identification. Many of
them (seven) have an as-yet-unknown function.
The 69 phosphopeptides presented in Table I belong
in total to 33 different proteins. They correspond to
27 gene models of Vs2 and 29 models of Vs3 of the
C. reinhardtii genome. For three models present in Vs2,
but missing or reduced in Vs3, ESTs as well as proteomic support for the Vs2 model exist (Table I; Schmidt
et al., 2006; Wagner et al., 2006; this article). Seventeen
of the phosphoproteins were identified by a single
phosphopeptide and 16 were covered by more than
one, with up to nine phosphopeptides (Table I), where
slightly different peptides with overlapping sequences
or identical peptides with different phosphorylation
sites are also counted. From the identified 33 phosphoproteins in this study, only six were previously
described in phosphoproteomic approaches to wholecell extracts and thylakoids of C. reinhardtii (Table
II; Turkina et al., 2006b; Wagner et al., 2006) and an
additional three were found when comparing the
phosphoproteins from Supplemental Table S2. Thus,
most phosphorylation sites described here are novel.
Additionally, none of the proteins known or putatively
involved in light signaling is among the previously
identified phosphoproteins, whereas at least two key
elements of the phototactic signaling cascade have
been identified in this study (see below). Thus, subcellular fractionation prior to specific phosphoprotein
enrichment increases the probability of identifying
signaling-related and low-abundance phosphoproteins.
Twenty-seven phosphopeptides (Table I) were identified solely by MS2 spectra. Twenty-two phosphopeptides were identified by MS2 spectra and additionally
by the corresponding MS3 spectra of their neutral-loss
peptide (indicated in bold in Table I). An additional 20
phosphopeptides were solely identified by their MS3
spectra (indicated in bold and italics in Table I). Figure
3 illustrates the identification of the exact phosphorylation site for a peptide from the photoreceptor ChR-1.
The MS2 spectrum of the prominent peptide ion m/z
942.02 showing the neutral loss led to identification of
the phosphopeptide ASpLDGDPNGDAEANAAAGGK
with an Xcorr factor of 5.44 (Fig. 3A; see Supplemental
Fig. S1 for full MS scan in the m/z range between 300
and 2,000). Analysis of the MS3 fragmentation spectrum from the peptide ion m/z 892.6 revealed the same
peptide with the dehydroalanine residue (Dha) as a
result of the neutral-loss event with an Xcorr factor of
5.04, showing nearly a complete set of the corresponding y- and b-ion series (Fig. 3B).
In the 69 different phosphopeptides identified in
this study, 55 in vivo phosphorylation sites could be
determined precisely. Most peptides have one or two
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Phosphoproteomic Analysis of the Chlamydomonas Eyespot
Table I. Functional categorization and characterization of identified phosphoproteins from the eyespot fraction
Tp, Phosphorylated Thr or methyldehydroalanine; Sp, phosphorylated Ser or dehydroalanine; Yp, phosphorylated Tyr; Mo, oxidized Met; n, exact
phosphorylation sites within the peptide cannot be specified with certainty. Bold indicates that the phosphopeptide was identified by MS2 and, in
addition, by MS3 spectra. Bold and italics indicates that the peptide was solely identified by MS3 spectra. If a peptide is marked with an arrow, the
peptide above shows overlapping or identical sequences, but the number of phosphorylation sites may be different.
Protein ID Vs2
Photoreceptors
166415
164481
Vs3
95849
ChR-1 precursor [C. reinhardtii] ChR-1
182032
Retinal-binding protein [C. reinhardtii]
Cop-4/ChR-2
Retina-related proteins
171757
185703
158846b
Kinases
153985
–
Function and/or Homologies of Depicted
Proteins Determined by NCBI BLASTp
–
192323c
194793
SOUL/HBP [A. thaliana]
MORN repeat protein 1
[Bigelowiella natans]
Predicted unusual protein
kinase (ISS) [O. tauri]
Ser/Thr protein kinase stt7, chloroplast
precursor [C. reinhardtii]
Calcium-binding protein
–
162748 b
Similar to calmodulin 2
[Strongylocentrotus purpuratus]
Protein with PAP-fibrillin domain
161417
176214
No significant hit in NCBI BLASTp,
PAP-fibrillin domaind
Carotenoid and fatty acid metabolism-related proteins
158558
194594
1-Deoxy-D-xylulose-5-P synthase
[C. reinhardtii]
164164
161904
161263
56237
Sterol-C-methyltransferase [A. thaliana]
Omega-3 fatty acid desaturase [C. reinhardtii]
Thylakoid and chloroplast membrane-related proteins
163724
98021
Hypothetical protein
[Chlamydomonas sp. HS-5],
PSII 10-kD polypeptide PsbR
161722
187077
Tic62 protein [Pisum sativum]
–
182959
165731
143879
155786
155818
162239
138069
138110
184490
191690
185971
152995
PSI reaction center subunit VI,
chloroplast precursor (PSI-H);
(LHCI, 11-kD protein; P28 protein)
[C. reinhardtii]
Chloroplastic outer envelope membrane
protein (OEP75) [P. sativum]
LhcbM8, LhcbM4, LhcbM6; LHCII
protein precursors [C. reinhardtii]
Cytochrome b6-f complex subunit petO,
phosphoprotein precursor [C. reinhardtii]
Phosphopeptide
GFETpRASpLDGDPNGDAEANAAAGGK
/ASpLDGDPNGDAEANAAAGGK
YANRDSpFIIMR
YASRESpFLVMR
APFCPPLKTpSSpYDEAPAVEER
/TSSpYDEAPAVEER
RWPEDGINTpEDMVWLAEEANK
/WPEDGINTpEDMVWLAEEANK
VFESpEAGEPEAK
SpSVDSpNVDWFYSEAAQK n
/SpSVDSNVDWFYSEAAQK n
DAGLASpMEEAILK
TMDsa
1
1
2
(1)
1
(1)
VSFDEEEEEDGSpNKGSVLGR
/VSFDEEEEEDGSpNK
1
LPSEVLASpGDPYAAR
1
AGPAGSYSGEWDKLSpVEEIDEWR n
/AGPAGSYpSGEWDKLSpVEEIDEWR n
/AGPAGSYSpGEWDK n
/LSpVEEIDEWR
AMSpYTNYFADALTAEAER n
DKPTTLQLSGGSpIDSSK n
VKDEFTAYADSpYGK
SFTpNDHYVADSGDIVYYQK n
VGLNSpIEDPVVK
(1)
(1)
1
1
GTpVSLGALFGGLPK
GSpAQIFGFFGK
YFDLQDMENTpTpGSWDMoYGVDEK
/YFDLQDMoENTTpGSWDMoYGVDEK n
(1)
(1)
LTGDSpELEYSpVYDEGK n
NLVTpDSpEDLEEKPGER
SSpGVEFYGPNR
(1)
IDGSpFNDWFMoEAVVR
(1)
2
(Table continues on following page.)
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Wagner et al.
Table I. (Continued from previous page.)
Protein ID Vs2
Vs3
Others
164774
169475
169526
190424
182408
76715
170535
–
195087
126835
Contaminants
168463
–
–
Function and/or Homologies of Depicted
Proteins Determined by NCBI BLASTp
Kelch repeat: Kelch (ISS) [O. tauri]
S-adenosyl-Met synthetase [Pinus contorta]
Cobalamin-dependent Met synthase
(ISS) [O. tauri]
Os10g0455900 [Oryza sativa]
Heat shock protein 70B [C. reinhardtii]
123042
133334
Putative RNA helicase (ISS) [O. tauri]
External rotenone-insensitive NADPH
dehydrogenase (ISS) [O. tauri]
175482
Predicted protein [Ostreococcus
lucimarinus] with pre-mRNAprocessing factor 3 domain
Conserved and novel proteins of yet unknown function
165154
183448
Unknown protein [A. thaliana]
Phosphopeptide
AGSpFTNLDGTDQK
VLVHIEEQSPDIGQGVHGMGTpK
CENATpERMoLEYAASpLDPK
1
2
(1)
AESpLGDAANLVLEGK
KDDDVIDAEFTpDK
RMSpEVGSESTQVPYR
/RMoSpEVGSESTQVPYR
/MoSpEVGSESTQVPYR
(1)
(1)
AASpEDSpEEPVKPPTK
SESSSPVYTpTMSpLDGQNLK n
/SESSSPVYTpTpMoSpLDGQNLK
/SESSSPVYTTMSpLDGQNLK n
EATpPVPEAEPVPAVEWWDR
(1)
(1)
162350
149502
No significant hit in NCBI BLASTp
167609b
–
No significant hit in NCBI BLASTp
152999
157516
154464
–
No significant hit in NCBI BLASTp
No significant hit in NCBI BLASTp
193998
181146
180840e
190330
187713f
No significant hit in NCBI BLASTp
KDDGYISpEDEGLGNVAADYCAIDGAGK
/DDGYISpEDEGLGNVAADYCAIDGAGK n
RSpSpSGGGPAAAPAAAPEAAAVPLPVLEPK n
LPSplDSTLPLSAATR n
/LPSpIDSTpLPLSAATR
SpGDDAIAAALSAITGEER
GEASAPAAASASpEEDAVLR
HTGYTSDTpSLDHQQHPPR n
KGTpGFSFFGTpGK
/KGTpGFSFFGTGK n
KTGTpGFSFAFGAPK n
/TGTpGFSpFAFGAPK
AGSpGFNFFGTpGK
/AGSpGFNFFGTGK n
TGSpGFSFFGTGK
AAGFFGTpGR
KGTpGFSALFATPK
LCADIAAAGDGDDASpAAIVR
RSSpVEADDMEEPKDPAAEEEEEDGEQR n
RAGFSTDSpSPEQQEEEEEEVEAAALGAEAEEAR n
/AGFSTpDSpSPEQQEEEEEEVEAAALGAEAEEAR n
RDTpFIMR
No significant hit in NCBI BLASTp
No significant hit in NCBI BLASTp
ALEDVDAGLVTpDEINHEK
QTpSpRASpVDDEVLPTDEIAK
–
164273
170226f
TMDsa
(1)
1
(1)
(1)
2
2
(1)
(1)
1
a
TMDs, predictions done with TMHMM, TMpred, and TopPred [1, TMDs predicted by all three programs; (1), TMDs predicted by two programs;
b
c
EST support for the Vs2 model.
Sequence of Vs3 model differs significantly from sequence
2, TMDs predicted by only one or no program].
d
Vs2, peptides were present in both.
An E-value limit, usually 1e-05, was set. In one case of a special functional implication, an E value below is
e
f
also listed.
Protein was not present in former eyespot proteome analyses.
Protein was not present in former eyespot proteome analyses, but
lacks suitable tryptic sites for routine LC-ESI-MS.
phosphorylation sites. Three phosphorylation sites within
one peptide were detected only in two peptides (Table
I). However, in certain cases, the exact phosphorylation site within a given peptide could not be specified
with certainty. These peptides are marked by the
addition of an ‘‘n’’ at the end of the peptide sequence
in the tables. All peptides whose phosphorylation sites
were precisely predicted were phosphorylated at Ser
and/or Thr residues (Table I). Phosphorylation solely
at Tyr was observed only in two cases with proteins
listed in Supplemental Table S2. This reflects well the
results obtained with the anti-Thr and anti-Tyr antibodies in immunoblots with proteins from the eyespot
(Fig. 1).
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Phosphoproteomic Analysis of the Chlamydomonas Eyespot
Table II. Proteins from the eyespot phosphoproteome that were also detected in the C. reinhardtii
phosphoproteomic approaches with whole-cell extracts or thylakoids
Function and/or Homologies of Depicted Proteins
Determined by NCBI BLASTp
Protein ID Vs2
Vs3
158846a
161722
165731
–
187077
143879
169475
163724
182408
98021
167609a
160451
–
180708
163816
170226
–
187713
a
EST support for the Vs2 model.
present in Supplemental Table S2.
MORN repeat protein 1 [Bigelowiella natans]b,c
Tic62 protein [Pisum sativum]b
Chloroplastic outer envelope membrane protein
(OEP75) [P. sativum]b
S-adenosyl-Met synthetase [Pinus contorta]b
Hypothetical protein [Chlamydomonas sp.
HS-5], PSII 10-kD polypeptide, PsbRc
No significant hit in NCBI BLASTpb
Hypothetical protein [Paramecium tetraurelia];
FAP 157b,d
No significant hit in NCBI BLASTpb,d
No significant hit in NCBI BLASTpb,d
b
Wagner et al. (2006).
Characterization and Functional Analysis of the
Phosphoproteins from the Eyespot
To get functional information about the identified
phosphoproteins, we did NCBI protein BLAST homology searches and analyzed for putative TMDs (Table
I). Predictions using three different programs (see
‘‘Materials and Methods’’) revealed that the majority
of the phosphoproteins in the eyespot fraction have at
least a partial hydrophobic character. For 30% of the
phosphoproteins, all three programs predicted at least
one TMD. For an additional 54%, two of the used programs predicted TMDs.
The results of the NCBI searches were complemented by additional functional information from
the Joint Genome Institute (JGI) Web site and annotation notes (Supplemental Table S1). As already detailed above, with one exception, all proteins in Table I
were identified in large-scale proteome analyses of
eyespot fractions carried out previously by our laboratories. A significant group of the proteins on the list
(10) represents novel and conserved proteins of as-yet
unknown function. The highest phosphorylated protein identified in our analyses falls in this group (ID
167609; Vs2). It was identified with six different phosphopeptides along with nine phosphorylation sites.
Among the proteins with functional clues (23), those
(potentially) involved in light signaling show significant representation (five) as will be discussed later. The
most interesting candidates among these are the two
photoreceptors ChR-1 and ChR-2, which are involved
in phototactic and photophobic responses of C. reinhardtii.
ChR-1 was identified by three different and ChR-2 by a
single phosphopeptide. A detailed analysis will be
presented in the next paragraph. A small number of
phosphoproteins represent most likely contaminants,
such as a putative RNA helicase and a putative
mitochondrial external rotenone-insensitive NADPH
dehydrogenase.
c
Turkina et al. (2006b).
d
Proteins are
Analysis of Phosphorylation Sites Reveals a Bias for
Surrounding Amino Acids and a Tendency for Clustering
Outside Known Functional Domains
Specificity of the active sites of kinases toward their
substrates is primarily based on the linear sequence
surrounding the phosphorylation site and several
typical motifs are known for plant and animal kinases
(e.g. Blom et al., 1999; Nühse et al., 2004). To detect the
general frequency of selected single amino acid residues in the surrounding phosphorylation sites and to
find a potential bias, we analyzed the 26 to 16 positions of all unambiguously assigned phospho-Ser and
phospho-Thr sites from phosphopeptides in Table I,
including those of Supplemental Table S2. Our analysis was restricted to the group of acidic, basic, and
aromatic amino acids. Additionally, Gly was included
in this analysis. In total, 135 acidic, 79 basic, 69 aromatic amino acids, and 71 Gly residues were found to
surround the phosphorylation sites. The frequency of
these amino acids in positions 26 to 16 relative to
their total frequency in these 12 positions is shown in
Figure 4. Clear indications of a bias were found for
acid and basic residues as well as for Gly. Whereas
acidic residues cluster in the 11 to 16 positions with a
peak at the 12 position, basic residues are overrepresented in positions 22, 23, and 26. A disproportionate frequency was also found for Gly. Overall, 35.2% of
the total Gly residues are either present at the 21 or the
11 position, but were virtually excluded from the 22
and 15 positions. Except for clear exclusion from position 24, clear bias for the aromatic residues is less
evident. Indication of a bias toward acidic and basic
residues is in agreement with the observation that acidicor basic-directed site specificity is common among
characterized kinases. Examples are the basic motif
[R,K]-X1-2-Sp or the acidic motifs Sp-[D,E] and [D,E]Sp-[D,E] (e.g. Blom et al., 1999; Nühse et al., 2004, and
refs. cited therein).
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Wagner et al.
Figure 3. Identification of a phosphopeptide from ChR-1 (ID 166415; Vs2) by nano-LC-ESI-MS2 and by neutral-loss-triggered
MS3. A, Identification of the phosphopeptide ASpLDGDPNGDAEANAAAGGK by the MS2 fragmentation pattern of peptide ion
942.02; ‘‘p’’ indicates the phosphorylation site on Ser. B, Identification of the peptide ADhaLDGDPNGDAEANAAAGGK by the
MS3 fragmentation pattern of the detected neutral-loss fragment 892.6; ‘‘Dha’’ indicates the site of the neutral loss of phosphoric
acid from phospho-Ser. Only prominent y- and b-fragment ions have been labeled.
780
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Phosphoproteomic Analysis of the Chlamydomonas Eyespot
Figure 4. Frequency of selected
amino acids in single positions relative to their total frequency in
positions 26 to 16 surrounding
the Ser/Thr phosphorylation sites.
Only those phosphorylation sites,
which were unambiguously assigned, were included in the analysis. For the analyses, sequences of
Vs2 were used when models were
present in both genome versions.
We analyzed our data also with respect to the location of the phosphorylation sites relative to the functional domains of the proteins predicted by CD Search
(Marchler-Bauer and Bryant, 2004) of NCBI protein
BLAST. In the majority of cases, the sites were outside
the predicted domains. This situation was observed in
total for 23 proteins. Only for four proteins were the
phosphorylation sites observed inside the domain
regions. An additional three proteins have phosphorylation sites both inside and outside the predicted
domains. Thus, the phosphorylation sites clearly tend
to cluster outside known functional domains. For those
proteins with no functional domains, we found that
the phosphorylation sites were frequently located in
regions with a more hydrophilic character and predicted exposed locations. An additional outcome of this
analysis was that most of the phosphorylation sites
were either C-terminally (13 proteins) or N-terminally
(19 proteins) located. In only four proteins was phosphorylation detected either in the C and N termini or
in the C/N termini and the central region. Phosphorylation sites that are solely situated in the central
region were observed for 10 proteins.
Functional importance of the location of the phosphorylation sites can be deduced for the two ChRs. In
both vertebrates and invertebrates, rapid phosphorylation and dephosphorylation of rhodopsin critically
controls the visual transduction cascade (Ohguro et al.,
1998; Shukolyukov, 1999; Kennedy et al., 2001; Lee
et al., 2002). Thus, the identification of phosphopeptides from both ChRs of C. reinhardtii in this study is of
special interest. Phosphorylation of the vertebrate rhodopsins occurs at multiple sites at the C terminus.
Besides two Tyr phosphorylation sites directly at the
end of the last TMD, five additional sites are clustered in
a distance of 28 to 37 amino acids away from it (Ser-334
to Ser-343; PhosphoSite; http://www.phosphosite.org/
Login.jsp). We therefore analyzed the localization of
the phosphorylation sites in the rhodopsins of C.
reinhardtii in detail. Three sites were identified in
ChR-1 (Ser-358, Thr-373, Ser-376; Fig. 5A). Two of
these sites are also predicted by NetPhos2.0 (http://
www.cbs.dtu.dk/services/NetPhos) with high scores.
For ChR-2, we identified a single site at Ser-321. In
both photoreceptors, phosphorylation occurred in the
first cytoplasmic loop after the seven TMDs (Fig. 5, A
and B), which form the light-gated channel (Nagel
et al., 2002, 2003; Sineshchekov et al., 2002). Additionally, the distance from the last TMD to the first phosphorylation site is almost identical in both ChRs (54
amino acids in ChR-1 and 55 amino acids in ChR-2).
Both ChRs have unusually long C-terminal extensions.
Thus, the relative position of the phosphorylation sites
with respect to the seven TMD regions is remarkably
similar to those of the vertebrate rhodopsins, underlining the potential functional significance of phosphorylation at these sites also in the algal ChRs. The
amino acid sequences surrounding the first phosphorylated Ser residue in ChR-1, ChR-2, and the closely
related ChR-1 of Volvox are highly conserved (Fig. 5C).
No additional putative ortholog sequences of this
motif were found by NCBI Protein BLAST searches
against the nonredundant protein sequence database
and the Chlamydomonas genome using the 60-amino
acid sequence motif surrounding the phosphorylated
Ser-358 of ChR-1. Because specificity of the active sites of
kinases toward their substrates is primarily based on
the linear sequence surrounding the phosphorylation
site (e.g. Nühse et al., 2004), this might indicate that
green algal ChRs are targets of a specialized kinase.
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Wagner et al.
Figure 5. Identified phosphorylation sites in the two photoreceptors
ChR-1 and ChR-2. A, Partial sequences of ChR-1 and ChR-2 are
depicted. In both cases, parts of the C-terminal sequences are missing.
For ChR-1, only the first 420 and for ChR-2 only the first 328 amino
acids are shown, respectively. Peptides identified by MS analyses have
been labeled in bold and phosphorylation sites are marked by a ‘‘p’’
with gray background. All TMDs are underlined. B, Localization of the
phosphorylation sites in relation to the TMD arrangement of ChR-1 and
ChR-2 (modified from Kateriya et al., 2004). Phosphorylation sites are
marked by an asterisk. C, ClustalW alignment of the experimentally
determined first phosphorylation site of the two ChRs from C. reinhardtii (CrChr-1 and CrChR-2) and the orthologous sequence of the
ChR of Volvox carterii (VcChR-1). *, Perfect matches; :, high similarity;
, low similarity. Phosphorylated Ser is highlighted by a gray background and charged amino acid residues (1/2) at identical positions
are indicated above the sequence.
DISCUSSION
One of the key modifications of proteins, which is
crucial in the control of many regulatory pathways and
can affect protein function, activity, stability, localization, and interactions, represents phosphorylation.
About one-third of all proteins are thought to be
phosphorylated (Hubbard and Cohen, 1993). To get
insights into the phosphoproteome of the eyespot, we
first isolated eyespot fractions according to Schmidt
et al. (2006). The isolated eyespots contain, at least
partially, the two layers of carotenoid-rich lipid globuli
subtended by thylakoids, and the specialized areas of
the chloroplast envelope and plasma membrane overlaying the eyespot globules in this region. We then
extracted the proteins from the eyespot fraction and
used the IMAC procedure along with Ga31 as metal ion
to enrich phosphopeptides from the digested peptides.
Due to the elaborate structure of the eyespot, which is
dominated by hydrophobic components, many of its
proteins possess a rather hydrophobic and amphipathic
character (Schmidt et al., 2006). To bring the extracted
proteins back in solution, relatively high concentrations of guanidinium hydrochloride or urea were thus
necessary. These concentrations were in clear conflict
with working solutions of the tryptic digest employed
in the previous cellular phosphoproteome analysis of
C. reinhardtii (Wagner et al., 2006). Therefore, we had to
establish an alternative procedure. The endoproteinase
LysC tolerates concentrations up to 4 M urea without
significant loss in activity. It had already been used
successfully for digesting brain membrane proteins
prior to trypsin digest and subsequent MS analysis
(Nielsen et al., 2005). The proteins of the eyespot fraction can be rather well dissolved in 4 M urea. Thus, we
first digested the proteins with LysC. The resulting
peptides were then reduced, desalted, and subjected to
a tryptic digest to get peptide sizes suitable for routine
LC-ESI-MS analysis. In this way, we could prevent
solubilization and precipitation problems.
In large-scale proteomics using peptide-enrichment
strategies, such as IMAC, protein identification often
has to rely on identification by a single peptide. Thus,
it is very important that the single peptide is identified
with high stringency. We therefore did three independent isolations with two pooled eyespot fractions for
each (Fig. 2) and chose only peptides that were present
in at least two of the three isolations fulfilling the Xcorr
and P values (see ‘‘Materials and Methods’’). In the
central list (Table I), we included only proteins (with
one exception, discussed below) that had also been
depicted in former proteome analyses of eyespot fractions (Schmidt et al., 2006; M. Mittag and G. Kreimer,
unpublished data). From the 33 identified phosphoproteins that we found in this way, 16 were covered
with more than one phosphopeptide. In the case of 13
phosphoproteins, there is only information available
from one phosphopeptide based either on MS2 (eight)
or on neutral-loss-triggered MS3 (five) spectra. Four
phosphoproteins were depicted by one phosphopeptide based on information from both MS2 and MS3
spectra. Combining the information obtained in MS2
and MS3 spectra further increases confidence in individual peptide identification (Nielsen et al., 2005).
In total, 22 phosphopeptides are based on information
from both MS2 and MS3 spectra in our approach.
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Phosphoproteomic Analysis of the Chlamydomonas Eyespot
By this procedure, we were able to obtain insight into
the phosphoproteome of the eyespot of C. reinhardtii, a
subcellular compartment that is complex structured,
as mentioned before, and involves thylakoid membranes subtending the two lipid globule layers. Comparisons to the currently known phosphoproteins from
C. reinhardtii obtained by phosphoproteomic approaches
with whole-cell extracts (Wagner et al., 2006) or thylakoid membranes (Turkina et al., 2006b) revealed that
only a few of these phosphoproteins (six) are overlapping with the eyespot phosphoproteome (Table II).
Thus, a significant number of phosphoproteins (27)
could only be identified when the subcellular eyespot
fraction was enriched prior to enrichment of its
phosphopeptides by IMAC. These data further underline the importance of purifying subcellular fractions,
such as the eyespot, as an efficient and necessary approach to get a rather complete set of phosphoproteins
from any subcellular fraction. However, one should
not neglect the possibility that the significant increase
in phosphoproteins from the eyespot might in parallel
be due to application of the modified phosphopeptide
enrichment procedure involving LysC treatment. This
is especially valid for the rather hydrophobic proteins
that may simply not have been brought in solution in
the former whole-cell phosphoproteomic approach
(Wagner et al., 2006). Still, we cannot be sure whether
several phosphoproteins from the eyespot may have
been missed. Immunoblots with anti-Thr and anti-Tyr
antibodies, along with proteins from purified eyespot
apparatuses, revealed about 20 and two protein bands,
respectively, that were significantly and reproducibly
labeled. These analyses indicate that Tyr phosphorylation is rare in the eyespot fraction and Ser/Thr phosphorylation dominates. This agrees notably well with
the outcome that all precisely determined phosphorylation sites (55) in the central list (Table I) are found
on Ser and Thr. Only three Tyr phosphorylation sites
were found with proteins, which were not depicted in
former eyespot proteome analyses (listed in Supplemental Table S2). This is additionally in agreement
with the currently identified kinases of the eyespot
fraction, which are all predicted Ser/Thr kinases. Only
some of them (e.g. cyclic nucleotide-dependent protein kinase II) have, in addition, predicted Tyr-kinase
activity. Further, dominating PPs identified in the
large-scale proteomic approach to the eyespot fraction
are type PP2C Ser/Thr PPs (Schmidt et al., 2006).
Immunoblots also revealed the presence of very lowabundance phosphoproteins hardly detectable by this
technique. Such phosphoproteins are of special interest because signaling components often occur in a very
low amount in the cell. Among the identified 33 phosphoproteins, at least some of them should have been
detected. The functional classification of the identified
phosphoproteins indeed shows several (putative) components of light signaling pathways. Among them is a
Ca21-binding protein with an EF hand motif (ID 162748;
Vs2) with a C-terminally located single phosphorylation site. In the behavioral responses of C. reinhardtii to
light, extracellular Ca21 and Ca21 fluxes are intricately
involved (Schmidt and Eckert, 1976; Morel-Laurens
1987; Pazour et al., 1995; for review, see Witman, 1993).
Both photoreceptors ChR-1 and ChR-2 are directly lightgated ion channels, which are able to conduct Ca21
under physiological conditions. Their excitation initiates fast inward-directed complex currents in the region
of the eyespot, which finally produce a Ca21 influx into
the flagella (Harz and Hegemann, 1991; Holland et al.,
1996; Nagel et al., 2002, 2003; Sineshchekov et al.,
2002; for review, see Sineshchekov and Govorunova,
2001; P. Hegemann, personal communication). Notably, changes in the free concentration of Ca21 have
been shown to strongly affect rapid protein phosphorylation and dephosphorylation in isolated eyespot
apparatuses of the green alga Spermatozopsis similis
(Linden and Kreimer, 1995). No Ca21-dependent protein kinases have been detected in the core eyespot
proteome of C. reinhardtii (Schmidt et al., 2006). However, in an extended eyespot proteome analysis, two
kinases of this type have been detected recently (M.
Mittag and G. Kreimer, unpublished data). Their calculated molecular masses (67 and 54 kD) are similar to
proteins exhibiting Ca21-dependent autophosphorylation in eyespot preparations of S. similis (77, 48, and 47
kD; Linden and Kreimer, 1995; Schlicher et al., 1995).
Due to the outlined central role of Ca21 in eyespotrelated signaling, it seems likely that the identified
Ca21-binding phosphoprotein might be one member
of this light-driven signaling cascade.
Another protein that might be involved in this cascade represents a SOUL/HBP (ID 185703; Vs3) annotated as SOUL3. A SOUL/HBP was found in a screen
for chicken mRNAs specifically expressed in the retina
and pineal gland, where it might play a role in light
signaling or in protecting the retina from damage by
reactive oxygen species (Zylka and Reppert, 1999; Sato
et al., 2004). In the meantime, several HBPs have been
found in bacterial, animal, and plant systems and
several proteins of this family have been annotated in
Vs3 of the Chlamydomonas genome (Merchant et al.,
2007). Functional information on plant HBPs is still
lacking, whereas the former ones are involved in different processes. For example, mammalian NPAS2 has
two heme-binding sites and is functionally similar to
CLOCK. Both are transcription factors involved in
the oscillatory loop of the circadian clock that is entrained by light-dark cycles (DeBruyne et al., 2007). In
Escherichia coli, a heme-regulated phosphodiesterase
was characterized (Sasakura et al., 2005). In cultured rat
hepatocytes, HBP23 is an antioxidant protein, which is
induced by various stress stimuli (Immenschuh et al.,
2002). Phosphorylation of the C. reinhardtii SOUL/HBP
at two sites of its N terminus (Thr-42 and Ser-44) might
play a role for its heme-binding capacity, since His and
Cys possibly involved in the necessary complexation of
Fe21 in the heme are also situated at the N terminus
(T. Schulze and M. Mittag, unpublished data). A
second Chlamydomonas SOUL/HBP (ID 154433; Vs2),
which is not present in the eyespot proteome, is also
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Wagner et al.
phosphorylated at the N terminus (Ser-2 and Ser-3;
Wagner et al., 2006).
Also a predicted unusual protein kinase (ID 153985)
could be involved in signaling events. It is well known
that protein kinase activity can be regulated by phosphorylation or dephosphorylation (Krupa et al., 2004).
The relatively strong bias of amino acids surrounding
the phosphorylation sites that has been found in eyespot phosphoproteins (Fig. 4) might indicate that a
limited set of kinases was active under the chosen physiological conditions. This would be in concert with the
identification of only a few kinases in the eyespot proteome (Schmidt et al., 2006). The second kinase identified
here (stt7) is clearly not involved in the photoresponses.
It regulates state transitions in C. reinhardtii (Fleischmann
et al., 1999).
The most striking candidates that are clearly involved
in light signaling are the directly light-gated ChR-1
and ChR-2. In both cases, the phosphorylation sites are
located after the seven TMDs in the cytoplasmatic loop
region at the C termini. Whereas ChR-2 has only one
phosphorylation site, ChR-1 has three sites that are
situated close together. In vertebrates, the C terminus
of rhodopsin has a cluster of Ser and Thr residues that
are considered hallmarks for multiple phosphorylations. Thus, phosphate groups are mostly found at Ser334, Ser-338, and Ser-343 (Lee et al., 2002; Maeda et al.,
2003). In vertebrates, phototransduction starts with the
absorption of light, which causes photoisomerization
of 11-cis-retinal to all-trans-retinal and initiates a
phototransduction signaling cascade. Signal transduction in rods is terminated by a series of molecular events.
These are initiated by phosphorylation of rhodopsin
by a G-protein-coupled receptor kinase followed by
binding of a regulatory protein, arrestin (Maeda et al.,
2003). As detailed before in ‘‘Results,’’ the relative
position of the phosphorylation sites of the C. reinhardtii ChRs with respect to the seven TMD regions is
remarkably similar to those of vertebrate rhodopsins.
This might point to some evolutionarily conserved signaling mechanism with regard to the phosphorylation
sites, albeit the involved kinases and components of
the pathways are probably quite different. Because the
amino acid sequences surrounding the first phosphorylated Ser residue in ChR-1 and ChR-2 are highly conserved and cannot be found in other proteins from
C. reinhardtii, the green algal ChRs might well be targets
of a specialized kinase. From the five kinases identified
so far in the eyespot fraction, the two predicted unusual kinases with AarF domains could be involved.
The other identified kinases (CK1, the cyclic nucleotidedependent kinase II, and phototropin) are not likely
candidates for such a specialized function because they
are not restricted to the eyespot. All three can also be
found, for example, in the flagellum (Pazour et al., 2005)
and may be involved in eyespot flagellum-directed
signaling processes. In the case of CK1, there are
already data available supporting this hypothesis
(Schmidt et al., 2006). However, we cannot also rule
out that the responsible kinase has been lost during the
eyespot isolation procedure and is not among the
currently known kinases of this fraction.
Among the identified phosphoproteins, there were
also some enzymes that are involved in carotenoid and
lipid metabolism, components of thylakoid membranes,
or chloroplast outer membranes and other membranerelated components. 1-Deoxy-D-xylulose-5-P synthase
(DXS) catalyzes the first step in the methylerythritol
phosphate pathway leading finally to isopentenyldiphosphate, a precursor of carotenoids (Lohr et al.,
2005). Several phosphorylation sites were found in
this protein, indicating that phosphorylation might
play a role (e.g. for the activity or targeting of the
enzyme). At least one of these sites (MSYT) is conserved
in several other DXSs from different organisms. Furthermore, two proteins (Vs2; IDs 158846 and 161417)
grouped in the eyespot proteome by Schmidt et al.
(2006) under membrane-associated/structural proteins
were identified in this study as phosphoproteins. One
(Vs2; ID 161417) contains a motif with weak homology
to the PAP/fibrillin domain and the other has homologies to MORN repeat proteins. These proteins carry
multiple membrane occupation and recognition nexus
motifs, which function in attaching proteins to membranes and forming junctional complexes between
membranes (Takeshima et al., 2000; Shimada et al.,
2004). In the MORN repeat protein 1 found in the eyespot preparation, two phosphorylation sites were detected. One of them was already found in the cellular
phosphoproteome and could be confirmed here. The
other one is novel. Interestingly, Drosophila retinophilin,
which contains MORN repeats, is conserved in humans
(Mecklenburg, 2007). Drosophila retinophilin is expressed in fly photoreceptor cells and the human homolog was demonstrated to be expressed in the retina.
Despite the fact that significant homology of the C.
reinhardtii protein to these proteins is restricted to the
MORN motif region, the presence of proteins with such
motifs in visual systems from algae to humans is notable.
Several of the phosphoproteins found in this study
show no significant hit in NCBI BLASTp so their
potential function is unknown. Some of them have
multiple phosphorylation sites. One example is a
protein with nine phosphorylation sites (ID 167609).
Such candidates are also relevant for future functional
analysis because these proteins could be specific to
green algal eyespots and, thus, no homology to other
organisms may yet be available in the databases.
Our phosphoproteome analysis has also identified
proteins that have not been found in previous eyespot
proteome analyses (one protein in Table I and 13 in
Supplemental Table S2). In these analyses, the proteins
from the eyespot were separated on one-dimensional
SDS-PAGE and slices from the gels were used for in-gel
tryptic digestion. Thus, there was no specific enrichment for phosphopeptides. Within these 14 proteins,
13 were identified by only one phosphopeptide. Three
of them were also found in former phosphoproteome
analysis (Turkina et al., 2006b; Wagner et al., 2006).
Seven of them possess more than one phosphorylation
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Phosphoproteomic Analysis of the Chlamydomonas Eyespot
site. As reported previously, multiple phosphorylated
peptides are preferentially enriched by IMAC (Mann
et al., 2002). Therefore, it seems possible that very lowabundance phosphoproteins/peptides of the eyespot,
which have not been detected in recent proteome analyses, were significantly enriched by the IMAC procedure,
especially when they have multiple phosphorylation
sites. Another possibility would be an unfavorable
distribution of tryptic sites in these proteins, resulting
in peptides too long or short for routine LC-ESI-MS
analysis. We checked the tryptic sites in silico for all 14
proteins, but only in one case (ID 170226; Vs2) were the
tryptic sites found to be very unfortunate. Therefore,
this one protein was included with a special mark in
Table I. Notably, for most of the proteins in Supplemental Table S2, the function is fully unknown.
The phosphoproteins detected in our analyses represent only one analyzed physiological condition. As
pointed out earlier, the phosphorylation state of a given
protein can strongly depend on physiological conditions. For example, it was shown that state 1 to 2
transitions induced phosphorylation of the PSII core
components D2 and PsbR and quadruple phosphorylation of a minor LHCII antennae subunit, CP29, as well as
phosphorylation of constituents of a major LHCII complex, Lhcbm1 and Lhcbm10 in C. reinhardtii (Turkina
et al., 2006b). Examinations of various physiological
conditions, especially with respect to various light
conditions (e.g. intensity and quality), will be interesting to study in the future. However, this will be time
consuming and work intensive. For this identification of
the eyespot phosphoproteome, 40 L of cells were grown
for one enrichment of phosphopeptides (two pooled
samples). To get reliable results, we repeated this procedure two times and used only candidates that came
up in at least two of the three experiments. Thus, 120-L
cell cultures and six eyespot isolations were needed in
total for the final experiments for only one physiological
condition. Furthermore, only analyses of steady-state
conditions will be amenable by the chosen experimental
approach. Expected transient and/or rapid changes in
the in vivo phosphorylation status of eyespot proteins
(e.g. in response to sudden changes in the light conditions) cannot be analyzed in this way due to the timeconsuming concentration of large volumes of cells and
the subsequent isolation of eyespots. Nevertheless,
these data underline that phosphorylation plays an
important role in eyespot signaling. Having a significant number of targets of the eyespot kinases in hand
creates an efficient basis for studying the molecular
components of the eyespot signaling pathways in detail.
For example, silencing of the eyespot kinases, as was
already done with CK1 (Schmidt et al., 2006), can show
which targets and phosphorylation sites in the eyespot
are specific for a given kinase. Of special interest will be
the identification of the most likely specialized kinases
involved in phosphorylation of the two ChRs because it
might have a critical role in guiding phototransduction
in the eyespot. It is striking that the relative position of
the functional sites of phosphorylation within the algal
and vertebrate rhodopsins are so much conserved. The
identification of these sites paves the way for future
studies aimed to dissect the exact role of phosphorylation of these unique directly light-gated ion channels in
the phototactic signaling cascade.
MATERIALS AND METHODS
Isolation of Eyespot Apparatuses
Growth of Chlamydomonas reinhardtii strain cw15 and isolation of fraction A
enriched in eyespot apparatuses was done as previously described (Schmidt
et al., 2006), except that the homogenization buffer was supplemented with
phosphatase inhibitor cocktails 1 and 2 (Sigma; dilution 1:50). The concentrated eyespot fraction was extracted overnight with chloroform:methanol:
water (4:8:3 [v/v]) at 220°C. Precipitated proteins were washed several times
with methanol:chloroform (2:1 [v/v]) and finally dried in a speed-vac (1 min
at room temperature) and stored at 280°C.
Protein Digestion and Reverse-Phase Chromatography
The precipitated proteins of two independent eyespot isolations (0.5–0.7
mg) were digested with endoproteinase LysC (Roche Diagnostics) and the
resulting peptides were reduced and carboxyamidomethylated according to
Nielsen et al. (2005) prior to a tryptic digest of the purified peptides. Briefly,
proteins were resuspended in 250 mL of LysC buffer (4 M urea, 100 mM Tris-HCl,
pH 8.0). Five micrograms of endoproteinase LysC (in 50 mL of LysC buffer) were
added and the mixture was incubated overnight at 25°C. After addition of
further 200 mL of LysC buffer, 10 mL of 0.5 M dithiothreitol were added. The
solution was incubated for 30 min on ice. Subsequently, 9 mg of iodoaceteamide
were added to the solution to reach a final concentration of 100 mM. After 2 h of
incubation at 25°C, peptides were separated from the insoluble material by
centrifugation (25,000g; 15 min; 4°C). Peptides were fractionated on a reversephase column (SOURCE 15 RPC; GE Healthcare) on a FPLC system. After
adding acetonitrile (2% final concentration) and formic acid (0.1% final concentration) to the peptide mix, peptides were loaded onto the column and
eluted with 0.5 mL 80% acetonitrile and 0.1% formic acid. The eluent was dried
in a vacuum concentrator and the resulting pellet was dissolved in 100 mL of 100
mM NH4HCO3 followed by overnight incubation at 37°C with 10 mg of trypsin
(Promega). Tryptic peptides were again desalted with a SOURCE RPC 1-mL
column (see above) and applied to the IMAC procedure.
Enrichment of Proteins by IMAC
IMAC was done according to Wagner et al. (2006), using self-made microcolumns prepared according to Erdjument-Bromage et al. (1998). Briefly, 50 mL
of POROS 20 MC (Applied Biosystems) metal-chelating resin (66% [w/w]
slurry) was transferred into Eppendorf gel-loading tips. For sufficient column
packing, 2 3 50 mL of 0.1% acetic acid were passed through the column.
Charging of the micro-tip column was carried out by applying 150 mL of 100 mM
GaCl3, followed by a washing step (50 mL of 0.1% acetic acid). The following
procedure was performed according to Shu et al. (2004) with slight modifications. After loading the peptides onto the activated IMAC column, the column
was subsequently washed with 50 mL of 0.1% acetic acid, 50 mL of 50%
acetonitrile/0.1% acetic acid, 50 mL of 50% acetonitrile/0.1% acetic acid/100
mM sodium chloride, and, finally, with 50 mL of 0.1% acetic acid. The
phosphopeptides were eluted with 3 3 20 mL of 200 mM Na2HPO4 and desalted
using a ZipTip micro tip (Perseptive Biosystems) according to Stauber et al.
(2003). The ZipTip contained C18 reverse-phase chromatographic medium and
was additionally loaded with 50 mL of a POROS R2 (Applied Biosystems)
mixture (10 mL of POROS R2 1 50 mL of methanol). To improve binding of the
phosphopeptides to the reverse-phase column, formic acid (final concentration
5%) was added to the sample. After two washing steps with 5% methanol/5%
formic acid, the acidified sample was applied to the ZipTip, followed by two
washes with 50 mL of 5% methanol/5% formic acid. Peptides were eluted with
2 3 50 mL of 60% methanol/5% formic acid. The eluate was dried in a speed-vac
for 2 to 3 h and stored at 280°C.
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Wagner et al.
Peptide Identification by LC-ESI-MS
After dissolving the dried pellet in 5 mL of 5% acetonitrile/0.1% formic acid,
phosphopeptides were subjected to nano-LC-ESI-MS using an UltiMate 3000
nano-HPLC (Dionex) with a flow rate of 300 nL/min coupled online with a
linear ion trap ESI mass spectrometer (Finnigan LTQ; Thermo Electron).
Peptides were concentrated on a m-precolumn cartridge (LC Packings) using
a flow rate of 2 mL/min for 2 min. After applying the peptides to the analytical
reverse-phase C18 column (LC Packings), the successive steps of the gradient
for the elution of the peptides were as follows: 5 min 96% A/4% B (v/v); within
30 min gradually to 50% A/50% B (v/v); within 1 min gradually to 10% A/90%
B (v/v); 10 min 10% A/90% B (v/v); within 1 min gradually to 96% A/4% B (v/v);
and 15 min 96% A/4% B (v/v), whereby A consists of 5% acetonitrile/0.1% (v/v)
formic acid and B consists of 80% acetonitrile/0.1% (v/v) formic acid. The
instrument was run by the data-dependent neutral-loss method, cycling
between one full MS and MS2 scans of the four most abundant ions. After
each cycle, these peptide masses were excluded from analysis for 1 min.
Detection of a neutral-loss fragment (98, 49, or 32.66 D) in the MS2 scans immediately triggered a MS3 scan of the precursor ion representing the dephosphorylated peptide. Peptide mass tolerance was set to 1.5 D in MS mode. In MS2
and MS3 mode, fragment ion tolerance was set up to 1 D.
(Kreimer et al., 1991; Calenberg et al., 1998). Western-blot analyses followed
basically the protocol of Schlicher et al. (1995) with the following modifications: Skim milk powder was replaced by bovine serum albumin, the first
blocking step was reduced to 2 h, and incubations with the polyclonal antiphospho-Thr antibody and the monoclonal anti-phosphor-Tyr antibody (Cell
Signaling Technologies; both used at a dilution of 1:1,000) were done overnight at 4°C. Images of blots were taken with a Coolpix 990 (Nikon) and
processed with Photoshop (Adobe Systems).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Full MS scan in the m/z range 300 to 2,000 with
the prominent peptide ion 942.02
Supplemental Table S1. Detailed information about the identified phosphopeptides from the Chlamydomonas eyespot fraction.
Supplemental Table S2. Identified phosphopeptides from the Chlamydomonas eyespot fraction that belong to proteins that have not been
identified in former eyespot proteome analyses.
Data Analysis
Data analysis was done according to Schmidt et al. (2006) with some
modifications using Bioworks software (version 3.2) from Thermo Electron
including the SEQUEST algorithm (Link et al., 1999). Searches were done for
tryptic peptides allowing two missed cleavages. Software parameters were set
to detect a modification of 79.96 D in Ser, Thr, or Tyr in MS2 and MS3 spectra.
When phospho-Ser and phospho-Thr undergo gas-phase b-elimination, Dha
and 2-aminodehydrobuturic acid (methyldehydroalanine), respectively, are
produced. Thus, modifications of 218.00 D on Ser and Thr residues were
additionally used for the database searches with MS3 data. Further, detection of
a modification of 16 D on Met representing its oxidized form was enabled.
Scores for the cross-correlation factor Xcorr (Eng et al., 1994) were set to the
following limits: Xcorr . 1.5 if the charge of the peptide is 1; Xcorr . 2 if the
charge of the peptide is 2; and Xcorr . 2.5 if the charge of the peptide is 3. Only
peptides that fulfilled the Xcorr limits and had, in addition, a probability score
P , 0.05 were included into the result tables. P was recently introduced with the
Bioworks (version 3.2) software from Thermo Electron and represents the
statistical likelihood of finding an equally good peptide match by chance.
By default, peptide probabilities are reported as probabilities normalized to
1 and a lower probability value represents a better match (Bioworks, version
3.2). Application of a P-value screening, in addition to the Xcorr settings,
strengthens the quality of positive hits. Furthermore, three independent
phosphoprotein purifications (one purification combines the proteins from
two independent eyespot isolations) were analyzed and for all databases a
significant hit was only considered when the identified phosphoprotein was
present in at least two of these samples.
Data were searched against the following C. reinhardtii databases: final
model database from the JGI (versions 2 and 3 [genome.jgi-psf.org/chlre2/
chlre2.home.html and genome.jgi-psf.org/chlre3/chlre3.home.html]), mitochondrial database available from the NCBI (NC001638 [gi:11467088]), and the
chloroplast database (www.chlamy.org/chloro/default.html). Data from all
runs were combined and further evaluated using a program developed inhouse (Schmidt et al., 2006). Additionally, EST information from the Kazusa
DNA Research Institute (Asamizu et al., 1999, 2000, 2004) and the JGI were used
for data evaluation as well as previous C. reinhardtii proteome data from the
eyespot and the cellular phosphoproteome deposited on a protein network site
of C. reinhardtii (available under http://www2.uni-jena.de/biologie/chlamy/
index.php?page5search&cmd5search). Protein sequences of the gene models
were compared to the NCBI protein database using BLAST (Altschul et al.,
1997). For positive identification of both protein and functional domain
prediction, an internal cutoff E value of 1e-05 was used. TMD information
was based on predictions by the programs TMHMM (Krogh et al., 2001),
TMpred (Hofmann and Stoffel, 1993), and TopPred (von Heijne, 1992). Theoretical predictions of phosphorylation sites were done with NetPhos 2.0
software (Blom et al., 1999).
Electrophoretic Methods
For SDS-PAGE analyses, a modified high-Tris system was used. Lipid
removal, protein precipitation, and SDS-PAGE were conducted as described
ACKNOWLEDGMENTS
We thank Wolfram Weisheit and Sascha Schäuble for their help with the
Web site containing the spectra. We appreciate the free delivery of EST and
genome sequences from the genome projects of C. reinhardtii in the U.S.
Department of Energy (JGI) and Japan (Kazusa DNA Research Institute).
Received September 24, 2007; accepted November 28, 2007; published December 7, 2007.
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