From the Cellular and Molecular Biology Program and
the Department of Horticulture, and the
University of Wisconsin
Biotechnology Center, University of Wisconsin,
Madison, Wisconsin 53706
Received for publication, October 6, 2000, and in revised form, December 7, 2000
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ABSTRACT |
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The use of mass spectrometry to characterize the
phosphorylome, i.e. the constituents of the proteome that
become phosphorylated, was demonstrated using the reversible
phosphorylation of chloroplast thylakoid proteins as an example. From
the analysis of tryptic peptides released from the surface of
Arabidopsis thylakoids, the principal phosphoproteins were
identified by matrix-assisted laser desorption/ionization and
electrospray ionization mass spectrometry. These studies revealed that
the D1, D2, and CP43 proteins of the photosystem II core are
phosphorylated at their N-terminal threonines (Thr), the
peripheral PsbH protein is phosphorylated at Thr-2, and the mature
light-harvesting polypeptides LCHII are phosphorylated at Thr-3. In
addition, a doubly phosphorylated form of PsbH modified at both Thr-2
and Thr-4 was detected. By comparing the levels of phospho- and
nonphosphopeptides, the in vivo phosphorylation states of
these proteins were analyzed under different physiological conditions.
None of these thylakoid proteins were completely phosphorylated in the
steady state conditions of continuous light or completely dephosphorylated after a long dark adaptation. However, rapid reversible hyperphosphorylation of PsbH at Thr-4 in response to growth
in light/dark transitions and a pronounced specific dephosphorylation of the D1, D2, and CP43 proteins during heat shock was detected. Collectively, our data indicate that changes in the phosphorylation of
photosynthetic proteins are more rapid during heat stress than during
normal light/dark transitions. These mass spectrometry methods offer a
new approach to assess the stoichiometry of in vivo protein
phosphorylation in complex samples.
The reversible phosphorylation of specific proteins participates
in the regulation of virtually all aspects of cell physiology and
development. The extent of its importance is illustrated by the
hundreds of conventional protein kinases and phosphatases detected
in various eukaryotic genomes (1-3). Whereas, serine, threonine, and
tyrosine residues are the typical targets of these kinases,
phosphorylation of at least six other amino acids is feasible,
potentially expanding even further the dimensions of this
post-translational modification (reviewed in Ref. 4). Despite the
importance of this pool of phosphorylated proteins, our understanding
of its depth and breadth remains sketchy. One barrier has been the lack
of methods to define en masse the "phosphorylome," i.e. the subset of proteins in the proteome that become
modified in vivo by phosphorylation. Precise
characterization of the phosphorylome will be essential to fully
understand how proteins are activated or inhibited, encouraged to
interact with other components in the cell, and selected for rapid
degradation. Certainly, the dynamic and transient nature of many
protein phosphorylation reactions underscores the difficulties of
resolving the complete phosphorylome for a given organism.
Nevertheless, the identification of even just the principal cellular
phosphoproteins under distinct physiological conditions should bring
significant biological insights.
The most common method for analysis of the phosphorylome involves the
use of radioactive labeling either in vivo or in
vitro. However, uneven uptake of the label in complex
multicellular organisms, the large pools of endogenous free phosphate,
and the presence of pre-existing bound phosphate often limit
conclusions. Phosphoamino acid antibodies have been exploited recently
but their use is restricted to tailor-made immunological applications
and because these antibodies cannot detect the nonphosphorylated form
they are unable to determine stoichiometry. More recently, mass
spectrometry (MS)1 has been
applied to analyses of protein phosphorylation (5-10). This highly
sensitive technique offers the advantage of scanning complex mixtures
for phosphoproteins that became modified in vivo. Moreover
with appropriate considerations, we show here that MS can also be used
to provide estimates of the phosphorylation state of specific proteins.
To demonstrate the utility of MS, we have applied this technique to the
analysis of the major phosphoproteins in the chloroplast thylakoid, the
membrane containing the photosynthetic light reactions of photosystem
(PS) I and II, light-harvesting chlorophyll a/b proteins (LHCII),
cytochrome b·f complex, and the ATP synthase (11, 12). Multiprotein complexes within the thylakoids are responsible
for light-driven oxidation of water with concomitant release of oxygen
and the production of energy and reducing potentials. During these
reactions, reversible phosphorylation is thought to play critical roles
in (i) the redistribution of excitation energy between PSI and II via
modification of LHCII (11-13), and (ii) the maintenance of the PSII by
controlling the turnover of its reaction center polypeptides (14-18).
Studies in spinach and pea using 32P labeling and
phosphoamino acid antibodies showed that a number of proteins are
phosphorylated, including threonine residues at or near the N termini
of LHCII (19) and PSII polypeptides, the D1 and D2 reaction center
proteins, chlorophyll-binding protein CP43 (20), and peripheral
polypeptide PsbH (21).
However, given the limitations of 32P labeling and
immunoassays, it remains unclear how important chloroplast protein
phosphorylation is to the normal function and regulation of the
photosynthetic light reactions. In vitro studies using
32P labeling have suggested that a number of thylakoid
proteins are extensively phosphorylated in the light (22) by a kinase controlled by the photosynthetic electron transport chain (12, 23, 24),
and dephosphorylated in the dark by phosphatase(s) that are not light
sensitive (12, 25). More recent studies using phosphothreonine
antibodies questioned the magnitude of this phosphorylation by showing
that some thylakoid phosphoproteins remain phosphorylated even in
dark-adapted plants (26-28). Furthermore, the maximal phosphorylation
of LHCII only occurs at low light and is drastically decreased at
higher irradiations (26). Phosphorylation of spinach LHCII was also
found to increase in darkness upon exposure of leaves to heat shock
(27).
Here, we report the identification of the major phosphoproteins in the
thylakoid membranes from Arabidopsis thaliana and map their
phosphorylation sites using both matrix-assisted laser
desorption/ionization time-of-flight (MALDI-TOF) and
electrospray ionization (ESI) MS. Measurements of the
phosphorylation level of each protein revealed that changes in the
steady state phosphorylation during normal light/dark growth cycles may
be less extensive and slower than previously thought and may be more
significant and rapid during a response to stress (e.g. heat
shock). The MS techniques described here should be applicable for many
analyses involving complex mixtures of phosphoproteins.
Preparation of Chloroplasts and Thylakoids--
A.
thaliana ecotype Columbia-0 was grown at 21 °C either in soil
or on 0.7% (w/v) agar containing one-half strength MS media (Life
Technologies, Inc.). Plants were irradiated with 100 µmol m
For direct preparation of thylakoids, 5 g of leaves were
homogenized with a Polytron (Brinkmann PT 10/35) in 25 ml of ice-cold extraction buffer, containing 300 mM sorbitol, 50 mM sodium phosphate (pH 7.5), 5 mM
MgCl2, 10 mM NaF. The suspension was filtrated through four layers of Miracloth and centrifuged for 3 min at 1500 × g. The pellet was resuspended in 7 ml of lysis buffer (10 mM sodium phosphate (pH 7.5), 5 mM
MgCl2, 1 0 mM NaF) and homogenized 10 times in
a Potter grinder. The suspension was diluted to 30 ml with the lysis
buffer and centrifuged for 5 min at 4000 × g. The
pellet was resuspended in 3 ml of the extraction buffer with the Potter
grinder and layered on the top of a sucrose step gradient, containing
(bottom to top): 10 ml of 1.8 M, 10 ml of 1.3 M, and 10 ml of 0.5 M sucrose in 50 mM sodium phosphate (pH 7.5) and 10 mM NaF.
After centrifugation in a swinging bucket rotor for 15 min at 5000 × g, the thylakoid fraction was collected from the 1.3 M, 1.8 M sucrose interface.
Thylakoids were diluted to 25 ml with extraction buffer and collected
by a 5-min centrifugation at 4000 × g. The thylakoid
pellet was resuspended in 1 ml of 25 mM
NH4HCO3 (pH 8.0), 10 mM NaF, and
pelleted again using a microcentrifuge.
In Vitro Phosphorylation Reactions--
Chloroplasts (0.2 mg of
chlorophyll) were gently resuspended in 1 ml of 20 mM
Tricine (pH 8.0), 330 mM sorbitol, 6.6 mM
MgCl2, 1 mM Na2HPO4 and
incubated at 22 °C. Phosphorylation was induced by a 10-20 min
irradiation with 100 µmol m Preparation of Thylakoid Peptides and
Phosphopeptides--
Isolated thylakoids were washed twice with 25 mM NH4HCO3 (pH 8.0), 10 mM NaF by centrifugation and resuspension in the same buffer to a concentration of 1.3-1.5 mg of chlorophyll/ml. The suspension was incubated with sequencing-grade modified trypsin (Promega) (8 µg of trypsin/mg of chlorophyll) at 22 °C for 90 min.
The digestion products were frozen, thawed, and clarified at 14,000 of
g. The supernatant containing released thylakoid peptides
was collected. Similar peptides were collected from spinach thylakoids
as described (30).
Phosphopeptides were affinity enriched from the thylakoid peptide
fraction by chromatography with immobilized Fe(III) or Ga(III) columns
(31, 32). Typically, columns containing 50 µl of chelating Sepharose
Fast Flow (Amersham Pharmacia Biotech) beads were washed with 0.3 ml of
water, 0.3 ml of 0.1% (v/v) acetic acid, charged with 0.3 ml of 0.1 M FeCl3 or GaCl3, and washed with
0.5 ml of 0.1% (v/v) acetic acid. Thylakoid peptides (0.2-0.3 ml)
were mixed with an equal volume of 20% acetic acid and loaded onto the
columns. After washing twice with 0.2 ml of 0.1% (v/v) acetic acid,
bound phosphopeptides were eluted with 300 µl of either 20 mM sodium phosphate buffer (pH 7.0) (for Fe(III) (31)), or
20 mM of nonbuffered Na2HPO4 (for
Ga(III) (32)).
Synthetic Phosphopeptides--
Synthetic phosphopeptides
included: APRTpPGGRR; CDGVTTKTpTAGTPD,
CDGVTTKTpFAGTPD, LIPQQSpINEAIK,
DRHDSGLDSpNKDE, DRHDSpGLDSpNKDE,
CDRHDSpGLDSpNKDE, and GRPRTTSpFAE
(where Tp and Sp indicate
phosphothreonine and phosphoserine, respectively). To obtain the
corresponding dephosphopeptides, 0.25 nmol of each phosphopeptide were
dissolved in the phosphatase buffer (25 mM NH4HCO3 (pH 8.0), 10 mM
MgCl2, 2 mM dithiothreitol) and
incubated for 2 to 24 h at 37 °C with the addition of 1 to 5 units of alkaline phosphatase (New England Biolabs, Beverly, MA). The
extent of dephosphorylation was monitored by MALDI-TOF MS.
MALDI-TOF MS Analyses--
Samples were prepared by mixing 1-2
µl of each mixture with 1-2 µl of LC-ESI MS Analyses--
Peptide mixtures were separated on 5 µm of C18 MetaChem 150 × 1.0-mm column at a flow rate 20 µl/min. A gradient of 0.1% (v/v) formic acid in water (A) and 0.1%
(v/v) formic acid in acetonitrile (B) was distributed as follow: 0% B
in first 3 min; 0-20% B in 3 to 20 min; 20-70% B in 20 to 105 min;
70-99% B in 105 to 115 min. The online detection with
positive/negative-ion mode switching was performed using an API 365 triple quadrupole MS with a standard ionspray source (Applied
Biosystems/MDS Sciex, Foster City, CA). Each 5.2-s positive-ion scan in
the m/z range from 320 to 1800 was followed by a
0.7-s pause for polarity switching and 1.5-s single ion-monitoring of
negative 79 and another 0.7-s pause to return to positive-ion mode. In
the positive-ion mode, ion source, orifice, and ring voltages were set
at 5 kV, 12 V, and 140 V, respectively, to minimize fragmentation.
However, in some cases a small amount of nonphosphorylated ion species
was generated from the phosphopeptide by partial skimmer-induced
fragmentation (see Fig. 3C, for example). In the
negative-ion mode, the ion source, orifice and ring voltages were set
at
For skimmer collision-induced dissociation (CID), the ion source,
orifice, and ring voltages were set at 5 kV, 95 V, and 200 V,
respectively, to maximize peptide fragmentation. Sequencing of high
performance liquid chromatography-purified peptides was performed by
tandem MS/MS using conditions recommended by Applied Biosystems/MDS
Sciex (Foster City, CA).
Previous studies with spinach and pea chloroplast thylakoids
showed that the primary sites for phosphorylation involve polypeptide regions exposed to the outer surface of the membranes (30, 33). To
enrich for these regions, thylakoid membranes were purified from
chloroplasts isolated from Arabidopsis leaves and then
"shaved" with trypsin to release surface-exposed peptides from the
various constituent proteins (Fig.
1A). Although a variety of
proteins were digested, several proteins and/or protein domains that we presume were protected by the membrane remained intact. LC-ESI MS
analyses of the released fraction revealed approximately a thousand
major peptides liberated by this protease treatment. By using the
characteristic decomposition products of phosphopeptides following the
breakdown of phosphoryl-peptide linkages as a signature (5, 6, 8),
numerous phosphopeptides were identified. We enriched for
phosphopeptides by immobilized metal [Fe(III) and/or Ga(III)] affinity chromatography (IMAC)
(31, 32). Because the binding specificity and elution properties of
Fe(III) and Ga(III) IMAC differ, both were used to isolate a range of
phosphopeptides (see Table I). Although
nonphosphorylated peptides were also present in the eluted fractions,
the partial purification of phosphopeptides by IMAC greatly simplified
their identification and initial analysis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2 s
1 of white light provided by
fluorescent lights with a photoperiod of 16-h light/8-h dark.
Chloroplasts and thylakoids were prepared from 3-week-old plants.
Chloroplasts were extracted from the leaves and purified by Percol
gradient centrifugation according to Ref. 29. To isolate thylakoids,
the chloroplasts were resuspended in 7 ml of 10 mM sodium
phosphate (pH 7.5), 5 mM MgCl 2, 5 mM NaCl, and homogenized 10 times in a Potter grinder. The
homogenate was diluted to 30 ml with the same buffer and thylakoids
were collected by centrifugation for 5 min at 4000 × g.
2 s
1 of white
light and terminated by addition of 10 ml of ice-cold 20 mM
Tricine (pH 8.0), 5 mM Na 4EDTA, 10 mM NaF. Thylakoids were prepared from chloroplasts as
described above. When isolated thylakoids were used, they were
resuspended in 20 mM Tricine (pH 8.0), 100 mM
sorbitol, 5 mM MgCl2, 1 mM ATP and
irradiated as described above.
-cyano-4-hydroxycinnamic acid
dissolved in 70% (v/v) acetonitrile with 2% (v/v) trifluoroacetic
acid. One µl of final mixture was spotted on the target. Linear and
reflector mass spectra were recorded using Biflex III MALDI-TOF mass
spectrometer (Bruker, Billerica, MA) operated in delayed extraction
mode using an accelerating voltage of 19 kV. Spectra were calibrated
externally. Post-source decay (PSD) spectra were recorded using
Bruker's FAST procedure.
4.5 kV,
200 V, and
300 V, respectively, to maximize the
phosphoryl-79 signal.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Preparation of peptides from
Arabidopsis thylakoid membranes and identification of
phosphopeptides by MALDI-TOF MS. A, SDS-PAGE separation
of the thylakoid membrane proteins before and after treatment with
trypsin. The thylakoids were prepared from isolated
Arabidopsis chloroplasts. B and C,
MALDI-TOF MS identification of the 1355.6 m/z
phosphopeptide (N terminus of CP43) in the peptide fraction enriched by
Ga(III) IMAC. The arrows indicate the signal of the
metastable ion (1269.6 m/z) in the reflector mode
spectrum (B) that is absent in the linear mode spectrum
(C). D and E,
identification of the 1355.6 m/z phosphopeptide
peptide of CP43 in a crude mixture of peptides before Ga(III) IMAC
enrichment. The arrows point toward the position of the
metastable ion signal, which is present in reflector mode spectrum
(D) but absent in the linear mode spectrum
(E).
Phosphorylation sites of the major phosphopeptides from Arabidopsis
thylakoids
indicate the type of IMAC that allowed
enrichment of the phosphopeptide as determined by subsequent MS
sequencing.
During MALDI-TOF-MS, phosphopeptides lose phosphoric acid as H3PO4 (98 Da) and HPO3 (80 Da) with the concomitant production of metastable ions (6). Whereas both the metastable and parent phosphopeptide ions arrive at the detector simultaneously and thus produce one coherent signal in the linear mode, they generate separate signals in the reflector mode, with the metastable ions arriving sooner than the parent ion. The appearance of this metastable ion is especially evident for peptides containing phosphoserine and phosphothreonine, which generate intense daughter signals upon losing H3PO4 (98 Da) (6). In our experimental settings, these metastable ions actually appeared as ions 86 m/z rather than 98 m/z smaller than the parent ions (Fig. 1B). This difference was a result of the metastable ion flying out of focus from the ion mirror, a phenomenon that also led to a broad ion signal that is characteristic of an ion lacking isotope resolution (Fig. 1B).
From the analysis of a series of synthetic phosphopeptides (see
"Experimental Procedures"), we found that all generated this metastable ion peak in the reflector mode regardless of the amino acid
sequence or position of the phosphoserine or phosphothreonine residue.2 In fact, doubly
phosphorylated peptides produced two metastable ions in the reflector
mode, 86 m/z and 172 m/z
smaller than the parent ion. We could reliably detect these metastable
ions with as little as femtomole amounts of these synthetic
phosphopeptides, indicating that they could be detected with high
sensitivity. Thus, we exploited the presence of metastable signal in
the reflector mode at 86 m/z and its unique
shape as a reliable indicator for phosphopeptides.
Using this metastable ion signature, numerous phosphopeptides were detected by MALDI-TOF MS in the IMAC-enriched fractions of peptides released from thylakoids of light-adapted plants. As an example, Fig. 1 shows the MALDI-TOF MS detection of a single ion cluster at 1355.6 m/z in the linear mode (Fig. 1C) that behaved as two ion clusters in the reflector mode, one for the parent ion at 1355.6 m/z and another for the metastable ion at 1269.6 m/z, which was 86 m/z smaller and devoid of isotope resolution (Fig. 1B). By this approach we detected eight phosphopeptides abundant in the IMAC-enriched fractions (Table I). In several cases, we also detected these phosphopeptides in the crude trypsin hydrolysates before IMAC enrichment. As shown in Fig. 1, D and E, the metastable 1269.6 m/z ion for the 1355.6 parent ion was readily detected in the reflector mode but absent in the linear mode.
The thylakoid phosphopeptides identified by MALDI-TOF MS were sequenced
using MALDI-TOF PSD, ESI MS/MS, and LC with online ESI-skimmer CID MS.
MALDI-PSD MS of the 1355.6 m/z phosphopeptide identified its sequence as Ac-TLFNGTLALAGR (Fig.
2A). A search of the
Arabidopsis protein sequence data base revealed that this sequence belonged to the chloroplast-encoded CP43 subunit of PSII, assuming that the first 14 amino acids of the initial translation product were removed and the resulting N-terminal threonine residue was
acetylated. Because the phosphate moiety is readily lost from phosphopeptides during MALDI-TOF-MS, we used complementary ESI-CID MS
sequencing to unambiguously identify the phosphorylation site(s). The
most efficient method was the ESI-skimmer CID MS when used online with
LC separation of IMAC-enriched peptides. Fig. 2B shows the
mass spectrum containing mostly y (C-terminal) and
b (N-terminal) ion fragments of the N-terminal
phosphopeptide from CP43. The fragmentation pattern was consistent with
the N-terminal threonine being both N-acetylated and
O-phosphorylated.
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The LC with online ESI-skimmer CID MS also revealed the presence of an unexpected isoform of CP43 in which the amino acid at position 4 was aspartic acid not asparagine (Fig. 2C). This aspartate isoform was found in all thylakoid preparations isolated from plants under a variety of conditions and comprised ~15% of the total CP43 pool. Given that the Arabidopsis CP43 gene encodes asparagine at this position (34), it is likely that this isoform was created by a deamidation reaction, the nature of which is currently unknown.
By similar analysis, we determined the sequence of the seven other phosphopeptides and identified the corresponding proteins in the Arabidopsis sequence data bases (Table I). Besides CP43, the D1 and D2 proteins of the PSII reaction center were identified and found to also contain an N-terminal threonine that was both N-acetylated and O-phosphorylated. We identified two phosphopeptides that corresponded to the mature LHCII polypeptides phosphorylated at the Thr-3 (Table I). One represented the expected tryptic fragment (TPVAKPK) whereas the other was two amino acids longer and N-acetylated (Ac-RKTPVAKPK). The second form likely represented an incomplete digestion product caused by the phosphate at Thr-3 blocking trypsin cleavage after Lys-2. The PsbH protein of PSII was phosphorylated at Thr-2. Notably, we also found a doubly phosphorylated form of this peptide containing a second phosphate bound to Thr-4 (Fig. 2D and Table I). The doubly phosphorylated form of PsbH was also detected in spinach thylakoids, suggesting that its presence is widespread in higher plants.2
To study the phosphorylation state of these principal
Arabidopsis thylakoid proteins in different physiological
conditions, we developed an ESI MS method to compare the levels of the
phospho and nonphospho forms for each. First, we determined the LC
elution positions of the eight phosphopeptides present in the complete tryptic peptide mixtures without IMAC enrichment. This LC separation did not completely resolve these complex mixtures but did make spectrometric identification of separate peptide ions in each fraction
possible (Fig. 3A). Following
LC, the peptides were detected online by ESI-MS and their masses were
determined from full scan data in the positive-ion mode.
Phosphopeptides were concurrently identified by switching to the
negative-ion mode every 6 s; this mode led to peptide
fragmentation and production of ions of 79 m/z,
which are diagnostic for PO
79
m/z ion monitoring in the negative-ion mode, the
LC retention times of all thylakoid phosphopeptides described in Table
I were determined (Fig. 3, A and B). The
intensity of the
79 m/z ion signal depended on
the nature of a parent phosphopeptide and its propensity to decompose.
For example, the
79 m/z signals produced by
fragmentation of N-terminal phosphothreonines of D1, D2, and CP43
peptides were more intense as compared with the internal phosphothreonine from the LHCII and PsbH phosphopeptides (Fig. 3B). We could also detect this set of phosphopeptides in the
negative-ion mode, using the characteristic fragmentation ions of
97
m/z
(H2PO
63
m/z (PO
79
m/z and gave higher noise during LC-ESI MS, thus
reducing their reliability.2
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The intensity of ionic species detected by MS depends on the nature of a particular peptide and its ionization properties. Consequently, quantitative comparisons of different peptides is generally not allowed. However, because phosphorylation of a peptide adds just 80 m/z (HPO3) and does not change the ionization state of the peptide under acidic conditions, quantitation of a phosphopeptide relative to its parent peptide could be possible using LC-ESI MS in the positive-ion mode. In this approach, the total peptide mixture (without IMAC enrichment) is separated by LC and the phosphopeptides and peptides then are detected in the same run by ESI MS. Because both forms would be detected simultaneously in the same sample, the stoichiometry of phosphorylation could be measured for individual proteins. To test this approach, we subjected equimolar ratios of six different synthetic phosphopeptides and their corresponding dephospho forms (see "Experimental Procedures") to LC-ESI MS. Under our LC conditions, the phosphopeptides eluted ~1 to 2 min earlier than the dephospho-forms, which simplified detecting the ions derived from the dephosphorylated forms. In all cases, the sum of the peak intensities of each phosphopeptide ionic species determined by ESI MS in the positive-ion mode was near equal to that of the dephosphorylated peptide.2
Using this semi-quantiative MS method, we determine the stoichiometry of phosphorylation for D1, D2, CP43, PsbH, and LHCII under several physiological conditions (Table II). Following the in vivo or in vitro treatments, the thylakoids were shaved by trypsin and the amount of the resulting peptides and phosphopeptide ions were measured following LC by ESI MS in the positive-ion mode. For the D1, D2, and CP43 peptides (Table I), the level of phosphorylation was expressed in percent after dividing the intensities of phosphopeptide ions by the sum of the intensities for both phospho- and nonphosphopeptide ions. For PsbH, the intensities of the single and double phosphopeptide ions were divided by the sum of the intensities for both the phosphopeptide and nonphosphopeptide ions. The alternative proteolysis of phospho-LHCII polypeptides precluded the objective quantitation of the total ion pool (see above and Table I). Instead, the amount of each phosphopeptide ion (TPVAKPK and AcRKTPVAKPK) was normalized relative to the amount of the corresponding nonphosphorylated peptide ion (TVAKPK) in each preparation. Thus, the data for LHCII phosphorylation reflected changes in the level of phosphorylation for these polypeptides rather than the stoichiometry of their phosphorylation.
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As a first test, we isolated thylakoids by a conventional protocol from plants either grown in continuous light or in continuous light followed by a 48-h dark-adaptation. The basal level of endogenous phosphorylation for D1, D2, CP43, LHCII, and PsbH was then measured by LC-ESI MS. None of the PSII proteins (D1, D2, CP43, and PsbH) were found to be completely phosphorylated or dephosphorylated in either preparation (Table II). The only exception was the doubly phosphorylated form of PsbH, which was absent in tissue harvested after dark adaptation. This result provided an initial indication that light is not strictly required for phosphorylation of these PSII proteins as had been previously proposed (reviewed in Refs. 12 and 13). However, for the LHCII polypeptides, these long-term light/dark treatments did significantly affect their phosphorylation state. Whereas a substantial portion of the LHCII pool was phosphorylated in the light-grown samples, the phosphorylated forms were undetectable after an extended dark adaptation (Table II).
Previous in vitro studies with spinach thylakoids, using either 32P labeling and/or phosphoamino acid antibodies, showed that heat appears to cause a rapid dephosphorylation of PSII core proteins D1, D2, and CP43, presumably by a heat-activated phosphatase (27). Here, we demonstrated a similar effect for Arabidopsis thylakoids by MS. Isolated thylakoid membranes were incubated in the light with or without a 15-min exposure to 40 °C and then the associated peptides were released by trypsinization. MS analysis of the released fraction revealed a significant dephosphorylation of the PSII reaction center proteins D1, D2, and CP43 after exposure to 40 °C. This was observed as a dramatic decrease in the phosphopeptide ion intensities as compare with those from their nonphosphorylated forms (Fig. 3, C and D, and Footnote 2). In contrast, the levels of LHCII and PsbH phosphopeptides were unaffected by this heat treatment.2 Collectively, these data imply that a heat-activated phosphatase is also present in Arabidopsis thylakoid membranes. Because only the PSII core proteins were dephosphorylated, we suggest that this phosphatase prefers substrates bearing N-terminal N-acetyl-O-phosphothreonines.
In vitro 32P labeling of pea and spinach thylakoids showed that light rapidly stimulates the phosphorylation of both LCHII proteins and the components of PSII (12, 22). To measure the extent of this reaction by MS, we prepared intact chloroplasts or thylakoid membranes from dark-adapted Arabidopsis plants, added inorganic phosphate or ATP, respectively, and then initiated the kinase reaction by irradiating the preparations with white light for 10 to 20 min. Studies with spinach and pea thylakoids indicated that this kinase reaction is complete by this time (22, 23, 25), although more recent studies with Arabidopsis thylakoids suggested that additional time is required for completion (35). LC-ESI MS quantitation of the resulting phosphopeptides revealed that these short light treatments had little effect on the stoichiometry of phosphorylation for the D1, D2, CP43, LHCII, and PsbH proteins; light increased the amount in the phosphorylated form by only a few percent over that observed for the protein from dark-adapted plants without the in vitro light treatment.2 As a result, these well characterized in vitro kinase reactions may reflect only a small fraction of that which occurs in vivo.
By LC-ESI MS, we then attempted to quantitate the in vivo changes in phosphorylation state of the D1, D2, CP43, LHCII, and PsbH proteins when the Arabidopsis leaves were subjected to several environmental conditions. In an attempt to more effectively capture the phosphorylated forms, we developed a method to rapidly prepare thylakoid membranes directly from Arabidopsis leaves. It involved collecting a crude chloroplast preparation by centrifugation, osmotic shock to release the thylakoid membranes, and then enrichment of the membranes by sucrose step gradient centrifugation. All buffers contained 50 mM phosphate and 10 mM NaF to inhibit endogenous phosphatases. Previous studies showed that these levels of inhibitors used singly are sufficient to completely block most, if not all, protein phosphatases in thylakoid preparations from spinach and various other plants species (12, 13, 26, 30). The thylakoid protein patterns prepared by this method were identical to those prepared conventionally (Figs. 1A and 4A). Moreover, the LC-ESI MS analysis revealed similar levels of phosphorylation for the five phosphoproteins prepared from light-adapted samples (Table II). The only major change was an enrichment of the doubly phosphorylated form of PsbH and a concomitant reduction in the singly phosphorylated form using the new method.
For the environmental analysis, the Arabidopsis plants were
grown in a 16-h light/8-h dark photoperiod and then harvested: 1) at
the end of the 8-h dark period; 2) 4 h into the 16-h light period;
3) 4 h into the light period followed by a 30-min dark treatment;
and 4) 4 h into the light period followed by a 15-min heat shock
at 40 °C (Table II). After harvest, thylakoids were prepared and the
shaved peptides were subjected directly to LC-ESI MS. As an example,
the MS spectra of the fractions containing the D1 phospho- and
nonphosphopeptides and the influence of the four treatments on their
signal intensities are shown in Fig. 4,
B and C. As can be seen in Table II, the
phosphorylation states of all five proteins were not radically altered
by any of the treatments. A modest decrease in LHCII phosphorylation
and slight decreases in D1, D2, and CP43 phosphorylation were evident
in the dark period as compared with the light period. However, this change was not yet evident after a 30-min dark adaptation, suggesting that the kinetics of this dephosphorylation is slow. The only major
light-dependent change was for PsbH, which revealed a near complete loss of the doubly phosphorylated form when the plants were
transferred to darkness. This response was rapid, being complete within
30 min of transfer. A significant decrease in phosphorylation of D1,
D2, and CP43 was evident upon heat shock, but was much less pronounced
than that observed in vitro (see above). Collectively, the
data indicate that none of the principal phosphoproteins in the
Arabidopsis thylakoids are completely phosphorylated or
nonphosphorylated under normal photoperiodic growth conditions.
They also suggest that significant and rapid changes in the overall
phosphorylation state of the photosynthetic complexes may occur in
heat-stressed plants.
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DISCUSSION |
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Attempts to assess the importance of phosphorylation in many cellular processes have been hampered by the dynamic and reversible nature of this process and by the lack of reliable methods to quantitate phosphorylation in intact cells. We show here that MS can be helpful in these studies by allowing the detection and mapping of phosphoproteins even in complex mixtures and by providing a semi-quantitative measure of the phosphorylation state for individual proteins. This approach improves upon current techniques using phosphoamino acid antibodies or 32P labeling because it can detect with high sensitivity both the phosphorylated and nonphosphorylated forms simultaneously and because it measures stoichiometry directly without the need for an exogenous tracer.
Using the regulatory phosphorylation system from Arabidopsis chloroplast thylakoids as an example, we detected a number of abundant phosphorylated proteins, easily mapped their phosphorylation sites, and estimated the effects of a range of environmental conditions on their phosphorylation state. In this case, the availability of the chloroplast and near-complete nuclear genomic sequences from Arabidopsis were instrumental for the unequivocal identification of the corresponding proteins following MS-MS sequencing of the peptides. Given the complexity of these thylakoid preparations, containing numerous proteins either peripheral or integral to the membrane, the success of MS analysis underscores its potential ability to dissect much more complex cell structures and organelles and may even be adapted to whole cell studies.
Several conditions helped simplify our characterization of the thylakoid system. First, the straightforward isolation of plant photosynthetic membranes allowed us to study protein phosphorylation in a well defined subcellular compartment rather than using the total cellular constituents. Second, the restriction of phosphorylation to surface exposed segments of the thylakoid proteins helped limit the number of peptides by focusing only on those accessible to trypsin. The remaining hydrophobic segments of membrane proteins were easily removed after proteolysis by centrifugation. This characteristic should also help simplify studies with other membrane protein phosphorylation cascades, including those involved in membrane receptor signaling. For more soluble systems, it is possible that complete trypsinization will generate many more peptides, thus precluding extensive MS analysis. And third, the use of both Fe(III) and Ga(III) IMAC allowed us to focus initially on preparations enriched for phosphopeptides. However, the ability of either IMAC to differentially discriminate among various phosphopeptides (Table I) suggests that these purification steps should be used with caution.
The key for our selective identification of phosphopeptides was the inherent instability of phosphoester bonds in a number of MS conditions (5-9). We found that the reflector mode detection of metastable ions produced by MALDI-TOF MS during phosphopeptide decomposition (6) was extremely useful. Both the presence of the metastable ion signals 86 m/z smaller than the parent ions and their lack of isotope resolution facilitated recognition of phosphopeptides among the signals of "normal" peptides. Moreover, the presence of two metastable ions of 86 and 172 m/z smaller (as observed for PsbH) allowed easy detection of potentially doubly phosphorylated forms. Subsequent MS analysis in the linear mode then identified the parent phosphopeptide ions. It remains possible that the distinct shape of metastable ion signals and their apparent mass difference from parent ions depend on the experimental conditions and on the particular MALDI-TOF spectrometer used. But once these conditions are established, this simple MS approach may be able to identify minute amounts of phosphopeptides in any complex peptide mixture.
MS analysis of the Arabidopsis thylakoid phosphopeptides in conjunction with the Arabidopsis genome sequence database allowed us easily to map for the first time the phosphorylation sites of central photosynthetic proteins in this plant species. These phosphoproteins included the chlorophyll-binding proteins LHCII and the D1, D2, CP43, and PsbH polypeptides of PSII. In all cases, the phosphate was bound to threonines at or near the N terminus. Coincidentally, the analysis of the corresponding peptides also allowed identification of the mature N-terminal residue of each of the five proteins and showed that three are both N-acetylated and O-phosphorylated (D1, D2, and CP43). The results obtained here with Arabidopsis are similar to those obtained earlier with spinach using conventional phosphate mapping, suggesting that this photosynthetic phosphorylation system is conserved among higher plants (19-21).
A novel finding of our study was the identification of a second phosphorylation site in PsbH protein at Thr-4. In contrast to phosphorylation at Thr-2, modification of Thr-4 is highly sensitive to the ambient light conditions, being rapidly dephosphorylated when plants are placed in darkness. In the green alga Chlamydomonas reinhardtii, PsbH is essential for assembling PSII in thylakoids (36); psbH mutants have a PSII-deficient phenotype and lack a functional PSII complex (36, 37). O'Connor et al. (37) concluded from studies showing that C. reinhardtii strains expressing a PsbH mutant lacking Thr-2 (Thr-2 to Ala) behave as wild-type, that phosphorylation of PsbH may not be important for its function. However, our finding of a second phosphorylation site in PsbH and the demonstration that modification of this site is light responsive in vivo reopens the role of phosphorylation in PsbH function.
Mapping of phosphopeptides using LC-ESI MS along with detection of both phosphorylated and nonphosphorylated forms obtained from the same physiological context shows that this approach can be used to estimate the in vivo phosphorylation state of many proteins simultaneously. When applied to the five thylakoid proteins characterized here, we demonstrated that both the phosphorylated and nonphosphorylated forms are present under various light/dark conditions in planta. Consequently, our data indicate that the current model of photosynthetic regulation in which thylakoid proteins are phosphorylated in light and dephosphorylated in darkness requires reconsideration at least in Arabidopsis (12, 22). Despite extensive dark adaptation, we found that only LHCII was completely dephosphorylated whereas a substantial percentage of D1, D2, CP43, and PsbH protein of PSII remained phosphorylated in Arabidopsis plants. Only the second phosphorylation site of PsbH followed the original model, being rapidly and completely dephosphorylated following the transition from light to dark. Whether these effects are universal to all plants remains to be demonstrated. Several studies suggest that thylakoid protein phosphorylation patterns can vary considerably among plant species (26, 28).
The recent use of phosphothreonine antibodies revealed previously unanticipated patterns of thylakoid protein phosphorylation in vivo (26-28). Irradiance-dependent phosphorylation of PSII and LHCII phosphopeptides was observed and connected to a regulatory mechanism involving in vivo changes in the thiol-disulfide redox state (26, 38). Moreover, significant phosphorylation of thylakoid proteins in the dark was found in plants after light and heat stress (27, 28). In a few plant species, high-light stress led to sustained phosphorylation of D1 and D2 proteins in the dark, the extent of which correlated with a sustained xanthophyll cycle-dependent dissipation of energy (28). In spinach, high temperature stress induced fast and specific dephosphorylation of PSII reaction center proteins (27). Using MS, we confirm here the effects of high temperature stress in Arabidopsis and show that heat shock conditions induce specific dephosphorylation of PSII core phosphoproteins D1, D2, and CP43. For each of these proteins, dephosphorylation involves the N-terminal N-acetyl-O-phosphothreonyls, suggesting that the responsible heat-activated phosphatase prefers this type of substrate.
Collectively, our MS study shows that reversible regulatory protein
phosphorylation in Arabidopsis photosynthetic membranes overall may be more rapid during stress than during the normal light-dark cycles. In this respect, larger and faster changes in the
protein phosphorylation may be anticipated in response to high light
stress, short light pulses, or limited light intensities when the
influence of dominant wavelengths would be important. In contrast with
all prior methods used to study thylakoid phosphorylation, our MS
approach allowed objective quantitation of in vivo
phosphorylation of multiple proteins obtained from plants exposed to
different physiological conditions. The further application of MS
should allow us to understand the role(s) of reversible phosphorylation in photosynthetic regulation and reveal additional components that are
affected by this modification. Of particular interest will be a
re-examination of the short-term effects of light previously shown by
other methods to have rapid and substantial effects on thylakoid phosphorylation.
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ACKNOWLEDGEMENTS |
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We thank Gary Case (University of Wisconsin-Madison) for providing the synthetic phosphopeptides, Shaun Snyders (Duke University, NC) for advice on preparation of Arabidopsis thylakoids, and Barbara Demmig-Adams (University of Colorado-Boulder) for forwarding us a manuscript before publication.
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FOOTNOTES |
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* This work was supported by Dept. of Energy Div. of Basic Energy Sciences Grants DE-FG02-88ER13968 (to R. D. V.) and DE-FG02-88ER13938 (to M. R. S.), National Science Foundation Grant DBI-9977525 (to M. R. S.), and the University of Wisconsin at Madison, Graduate School.
§ Present address: Div. of Cell Biology, Linköping University, SE-581 85 Linköping, Sweden. Tel.: 46-13-22-4050; Fax: 46-13-22-431-4; E-mail: aleve@ibk.liu.se.
¶ To whom correspondence may be addressed.
** To whom correspondence may be addressed: Dept. of Horticulture, 1575 Linden Dr., University of Wisconsin, Madison, WI 53706. Tel.: 608-262-8215; Fax: 608-262-4743; E-mail: vierstra@facstaff.wisc.edu.
Published, JBC Papers in Press, December 11, 2000, DOI 10.1074/jbc.M009394200
2 A. V. Vener, A. Harms, and R. D. Vierstra, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: MS, mass spectrometry; CID, collision-induced dissociation; ESI, electrospray ionization; IMAC, immobilized metal affinity chromatography; LC, liquid chromatography; LHCII, light-harvesting chlorophyll a/b complex II; MALDI, matrix-assisted laser desorption/ionization; m/z, mass over charge ratio; PSD, post-source decay; PS, photosystem; TOF, time-of-flight; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
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