OF BIOLOGICAL

THEJOURNAL CHEMISTRY Vol. 267, No. 5, Issue of February 15, pp. 3523-3529,1992
Printed in U.S.A.

Identification, Characterization,and Resolution of thein Vivo

Phosphorylated Form of theD l Photosystem I1 Reaction Center
Protein*
(Received for publication, October 11, 1991)

Tedd D. ElichS, Marvin EdelmanQ, and AutarK. MattooSlI

From the $Plant Molecular Biology Laboratory, Beltsville Agricultural Research Center- West, United States Department of
Agriculture, Agricultural Research Service, Beltsville,Maryland 20705 and the §Department of Plant Genetics, The Weizmann
Institute of Science, Rehovot 76100,Israel

The chloroplast-encodedD l protein ofoxygenic pho-
tosynthetic organismsis a component of the photosys-
tem I1 reaction center.Previously, we detected anelec-
trophoretic variant ofD l which was generated in vivo
in granal-localized reactioncenters in a light depend-
ent manner (Callahan, F. E., Ghirardi, M. L., Sopory,
S. K., Mehta, A. M., Edelman, M., and Mattoo, A. K.
(1990)J. BioZ Chern. 266,16367-16360). In the pres-
ent study, we identify this modifiedform as phos-
In higher plants, D l is synthesized as a precursor polypep-
tide (Grebanier et al., 1978; Reisfeld et al., 1982) whichis
inserted into the stromal lamellae of the thylakoids where it
is subsequently processed toits mature size (Mattoo and
Edelman, 1987)by a C-terminalcleavage (Marder et al., 1984).
Following processing, D l is translocated to the grana stacks
of the thylakoids where the majority of photosynthetically
active PSII is located (Mattoo and Edelman, 1987). In this
membrane region, D l undergoes rapid, light-dependent deg-

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phorylated D l . The in vivophosphorylation occurson radation (Edelman and Reisfeld, 1978; Mattoo and Edelman,
a threonine residue(s) localized within 1 kDa from the
N terminus and is identical to the phosphorylation of 1987).
D l catalyzed in vitroby a redox-regulated thylakoid- In additionto proteolytic processing, D l is known to
bound protein kinase. While virtually all of the D l undergo several other structuralmodifications. Phosphoryla-
protein presentin thylakoids can be phosphorylated in tion of the blocked N-terminal threonine residue of D l by a
vitro, the steady-state level of phosphorylated D l in thylakoid-bound kinase has been demonstrated in uitro
vivo varies with light intensity and did notexceed 20% (Michel et al., 1988; Marder et al., 1988) while covalent pal-
of the total D l under the conditions ofthis study. mitoylation (Mattoo andEdelman, 1987) and putative protein
cross-linking (Ohad et al.,1990) have been reported to modify
this protein in uiuo. The modification attributed to protein
cross-linking was suggested to be related to the rapid degra-
Oxygenic photosynthesis is mediated by two membrane- dation of D l that occurs under photoinhibitory conditions
bound, pigment-proteincomplexes: Photosystem I (PSI)’ and (Ohad et al., 1990), whereas the physiological significance of
Photosystem I1 (PSII). PSIIcatalyzes the reactions resulting in vitro phosphorylation and in uiuo palmitoylation are un-
in theoxidation of water and thereduction of plastoquinone. known.
The reaction center of a photosystem is defined as themini- Recently, our laboratory detectedan in uiuo-generated elec-
mal unit necessary for primary charge separation and stabi- trophoretic variant of D l , designated 32*, in Spirodela oligor-
lization. The PSII reaction center consists of a heterodimer rhiza as well as four other higher plants (Callahan et al.,
of related proteins, D l and D2, along with cytochrome bSS9 1990). This form is generated specifically in granal-localized
and thepsbI gene product. The D1/D2 heterodimer is thought reaction centers and its appearance was correlated with the
to contain or ligand all the electron carriers and cofactors onset of D l degradation. Formation of this modified form was
necessary for electron transport through the reaction center: light-dependent and inhibited by DCMU, a PSII herbicide
P680; the YZ and YD electron donor tyrosines on D l and D2, known to inhibit D l degradation. Based on these results, it
respectively; pheophytin; the plastoquinone electron accep- was hypothesized that themodification which yields 32* may
tors QA and Q B ; and non-heme iron (for reviews, see Ruther- be involved in thelight-dependent degradation of this protein.
ford, 1989; and Mattoo e t al., 1989). In thepresent study we identify the electrophoretic variant
of D l as the in uiuo phosphorylated form of this reaction
* This research was supported by a United States-Israel BARD center protein. The phosphorylation occurs on a threonine
grant (to M. E. and A. K. M.), and United States Department of residue(s) located within 1 kDa of the N terminus. The i n
Agriculture Grant 91-37100-6623(to A. K. M.). The costs of publi- uiuo D l phosphorylation site(s) is shown to be identical to
cation of this article were defrayed in part by the payment of page thesite(s) modified in uitro by athylakoid-bound redox-
charges. This article must therefore be hereby marked “uduertise-
ment” in accordance with 18 U.S.C. Section 1734 solely to indicate regulated kinase, most likely the blocked N-terminal threo-
this fact. nine residue (Michel et al., 1988). To our knowledge, a rigorous
1To whom correspondence should be addressed: Plant Molecular identification and characterization of an i n viuo phosphoryl-
Biology Lab, USDA/ARS/BARC-West, Bldg. 006, 10300 Baltimore ation siteof a PSII-associated proteinhas not previously been
Ave., Beltsville, MD 20705-2350.Tel.: 301-504-5103;Fax: 301-504- reported.
5320.
’ The abbreviations used are: PS, photosystem; SDS-PAGE, so- EXPERIMENTALPROCEDURES
dium dodecyl sulfate-polyacrylamide gel electrophoresis; E, einstein;
Tricine, N-[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]glycine; PVDF, Plant Growth-Axenic cultures of S. oligorrhiza, an aquatic an-
polyvinylidine difluoride; DCMU, 3-(3,4-dichlorophenyI)-l,l-dime-giosperm, were grown in half-strength Huntner’s medium (Posner,
thylurea; FSBA, 5’-p-fluorosulfonylbenzoyladenosine. 1967) supplemented with 0.5% (w/v) sucrose under 20 pE m-’ s-’

3523
3524 Phosphorylation of D l in Vivo
white light (PPFD, cool white fluorescent light).
Thylakoid Isolation-All procedures were performed at 4 "C under
dim light. Ice-cold extraction buffer (100 mM Tricine-NaOH, pH 7.8,
400 mM sorbitol, 10 mM MgC12, 10 mM NaC1, 0.2% (w/v) polyvinyl-
pyrrolidone, 0.2% (w/v) ascorbate) was added to washed Spirodela
plants a t a 5:l ratio (v/w) and thetissue was ground with a Polytron
homogenizer (Brinkman Instruments). The resulting slurry was fil-
tered through two layers of miracloth (Behring Diagnostics) and the
filtrate centrifuged at 1500 X g for 10 min. The supernatant was
discarded, and the pellet was washed twice with extraction buffer
minus sorbitol and resuspended in a small amount of buffer A (10
mM Tricine-NaOH, pH 7.8, 100 mM sorbitol, 10 mM MgClz, 10 mM
NaCl). Chlorophyll concentrations were determined in 80% acetone
extracts of the resuspended pellets using the equations of Arnon
(1949).
In Vitro Phosphorylation-Isolated thylakoids were diluted to a
chlorophyll concentration of0.2 mg/ml in buffer A supplemented
with 10 mM NaF. An ATP stock solution containing [r-"P]ATP
(Amersham, 3000 Ci/mmol) was added to a final concentrationof 0.2
mM and 50 pCi/ml. For light-stimulatedphosphorylation, thylakoids
were incubated under 50 pE rn-' s-l white light at room temperature
for the times indicated in the text and figure legends. Phosphorylation
in darkness was carried out by addition of 3.5 p~ ferredoxin (Sigma)
and 0.5 mM NADPH (Sigma) followed bygentle stirring in complete
darkness at room temperature for the times indicated. Unless other-
wise indicated, phosphorylation reactions were quenched by addition
of 0.5 volumes of 3 X SDS sample buffer.
For in uitro dephosphorylation experiments, isolated thylakoids
were phosphorylated in the dark as described above except that NaF
was omitted from the buffers. The phosphorylated thylakoids were
collected by centrifugation, washed twice with buffer A, and resus-
pended to a chlorophyll concentration of0.2 mg/ml in buffer A
supplemented with MgClz to a final concentration of20mM. The
samples (100 pl) were then incubated in microfuge tubm in the
presence or absence of 10 mM NaF for up to20 h at room temperature
in darkness with gentle stirring. At the appropriate times, 50 pl of 3
X SDS sample buffer was added to quench the reactions and the
samples were analyzed by SDS-PAGE, immunoblotting, and autora-
diography.
In Viuo Phosphorylation-Monolayers of Spirodela plants in 45-
mm diameter culture dishes (Falcon) containing 4 ml of half-strength
Huntner's medium lacking phosphate were adapted to 50 pE m-' s"
white light for at least 24 h. For labeling, the plants were drained and
0.5 ml offresh medium containing 0.5 mCi/ml of [3'PP]orthophosphate
(New England Nuclear Du Pont) was added per plate. The plants
were incubated for 3 h under the same light intensity after which
they were drained, washed three times with ice-cold H20, andstored
at -80 "C until thylakoid isolations were performed.
Phosphoamino Acid Analysis-Acid hydrolysis of phosphoproteins
bound to PVDF membrane was carried out essentially according to
Kamps and Sefton (1989). I n vitro and in vivo phosphorylated thy-
lakoid proteins were prepared as described above, resolved by SDS-
PAGE (see below), and electrotransferred to PVDF membrane (Im-
mobilon, Millipore) (see below). The resulting blots were washed
extensively in Hz0 and allowed to dry. The blots were subjected to
autoradiography to visualize the phosphorylated D l bands which
were then excised. Upto six 1.5-cm wideblot bands from each sample
were pooled, cut into small pieces, and transferred to screw-top
microcentrifuge tubes. The blot pieces were wetted with MeOH,
washed three times with HzO, and covered with pH 5.7 constant
boiling HCl (Pierce Chemical Co). The tubes were purged with N,,
sealed, and incubated at 110 "C for 1.5 h. After hydrolysis, the acid
was collected and evaporated in a Speed-vac Concentrator (Savant).
The residue was resuspended in a small volume of Hz0 containing
phosphoamino acid standards (Sigma) and applied to 20 X 20-cm
cellulose thin layer plates (Kodak). Thin layer electrophoresis was
carried out at 1000 V for 30 min using a pH 3.5 buffer system
(pyridine:acetic acidHzO, 101001890 (v/v)) (Cooper et al., 1983).
Phosphoamino acid standards were visualized by ninhydrin staining
and theplates were then fluorographed as described below.
Two-dimensional Phosphopeptide Analysis-In situ trypsinization
of D l bound to nitrocellulose membrane was performed by a modifi-
cation of the procedure of Aebersold et al. (1987).In vivo and in vitro
phosphorylated thylakoid proteins were resolved on SDS gels and
electrotransferred to nitrocellulose membranes as described below.
The blots were exposed to x-ray film to visualize the phosphorylated
D l bands which were then excised. Up to six 1.5-cm wide blot bands
from each sample were incubated in a microcentrifuge tube with 1.2
ml of 100 mM acetic acid containing 0.5% polyvinylpyrrolidone (av-
erage molecular mass 40 kDa) for 30 min at 37 "C. The blot bands
were then washed with water, cut into small pieces, and incubated at
37 "C for approximately 18 h in a small volume of 50 mM NH~HCOI
containing trypsinat anenzyme to substrate ratio of 1:20 (w/w). The
amount of D l present in the in uitro phosphorylated samples was
estimated by determining the radioactivity in one blot band by
scintillation counting and assuming 1 mol of phosphate/mol of Dl.
The in vivo phosphorylated samples were estimated to contain ap-
proximately one-third this amount, per pg of chlorophyll, based on
immunoblot analysis. Following incubation, the tubes were briefly
centrifuged and the supernatants removed. The supernatants were
brought to dryness in a Speed-vac concentrator and then further
dried in vacuo over P205and NaOH. The residue was resuspended in
a small volume of 20% pyridine and applied to 20 X 20-cm thin layer
cellulose plates (Kodak). Thin layer electrophoresis was carried out
for 30 min a t 1000 V using a pH 3.5 buffer system (acetic acidformic
acidHzO, 195:65:1040) (Peter et al., 1990). The plates were allowed
to air dry and then subjected to thin layer chromatography in the
second dimension using a solvent consisting of 1-butano1:acetic
acidHZOpyridine, 15:3:12:10 (Coughlan and Hind, 1987). After air
drying the plates were analyzed by fluorography (see below).
Partial Proteolytic Mapping Procedures-Partial proteolytic map-
ping following Staphylococcusaureus V8 or papain digestion of "S-
and"P-labeled D l excised from SDS gels wasperformed as previously
described (Marder et al., 1987).
In situ trypsinization of D l in thylakoid preparations was per-
formed essentially as previously described (Marder et al., 1987).
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Washed thylakoids were diluted to 0.2 mg/ml in buffer A and a stock
solution of L-1-tosylamido-2-phenylethylchloromethyl ketone-
treated trypsin (Sigma) was added to a final concentration of 0.01
pg/ml. The samples were incubated for 1 h in the dark a t room
temperature with gentle stirringafter which the reactions were
quenched by addition of one-half volume of 3 X SDS sample buffer.
The samples were then subjected to SDS-PAGE, immunoblotting,
and autoradiography.
SDS-PAGE and Immunological Procedures-Procedures for re-
solving total thylakoid proteins on 10-20% linear gradient SDS-
polyacrylamide gels were carried out as previously described (Calla-
han et al., 1989). To specifically resolve phosphorylated D l from the
nonphosphorylated form, the same gel system was used except that
electrophoresis was carried out at 150 V until a 30-kDa prestained
molecular weight marker (BRL) was approximately 1 cm from the
bottom of the resolving gel (approximately 17 h). Gel sample loads
were determined by chlorophyll concentration (2 pglwell, unless
otherwise noted). Electrotransfer to nitrocellulose membrane was
performed as previously described (Callahan et al., 1989). Electro-
transfer to PVDF membrane was performed a t 800 mA for 3.5 h using
the same transfer buffer system. The anti-Dl antibodies used for
immunodevelopment of blots and immunoprecipitations came from
antiserum directed against a D l fusion protein expressed in E. coli
(Mullet et al., 1990), and was obtained as a kind gift from Dr. J. E.
Mullet. Immunoprecipitations were performed according to published
procedures (Mullet et al., 1990). Alkaline phosphatase-conjugated
goat anti-rabbit secondary antibodies used in immunoblotting exper-
iments were from Pierce. Radiolabeling of Spirodela plants with [%SI
methionine was performed as previously described (Callahan et al.,
1990). Unless otherwise specified, all blots and gels weresubjected to
direct autoradiography using Kodak X-Omat AR film. Where indi-
cated, fluorography of blots, gels, or cellulose plates containing '*P-
labeled samples was performed by exposure to x-ray film in the
presence of an intensifying screen (Du Pont Cronex Lightning Plus)
at -70 "C, while fluorography of gels containing 35S-labeledsamples
was performed by gel-impregnation with ENIHANCE (Du Pont-New
England Nuclear) according to the manufacturers instructions, fol-
lowed by exposure to x-ray film at -70 "C. Except for the experiment
shown in Fig. 3 where the "P-radiolabel in D l blot bands was
determined with a Betascope @-counter (Betagen),all quantification
of radiolabeled- or immunoreactive-Dl was by densitometry of direct
autoradiographs or immunoblots, respectively, using an LKB laser
ultroscanner.
RESULTS
In Vitro Phosphorylation of Dl-As a first step towards
identifying the modification giving rise t o 32* (Callahan et
al., 1990),we tested whethera similar form could be generated
 Phosphorylation of Dl in Vivo
in vitro. Incubation of isolated Spirodela thylakoids in the
light in the presence of [y-32P]ATPresulted in a time-de-
pendent virtually quantitative conversion of D l t o a slower
migrating form designated Dl-P (Fig. lA, Immunoblot). The
slower migrating Dl-Pband was observed to co-electro-
phorese with in vivo generated 32* (data not shown). Auto-
radiography of the immunoblot demonstrated a parallel ap-
pearance of a 32P-radiolabeledband which co-migrated with
the less mobile Dl-P band (Fig. lA,Autoradiogram, and Fig.
1B). This radiolabel could be immunoprecipitated with D1-
specific antibodies verifying that it was associated with this
protein (Fig. 2 ) . The 32P-labeledDl-P band was subsequently
shown to contain phosphothreonine confirming that the ra-
diolabel incorporation was due to protein phosphorylation
(see next section).
Reducing power in the form of ferredoxin and NADPH
could substitute for the light requirement of D l phosphoryl-
ation (Fig. lA). Thus, in vitro phosphorylation of Spirodela
D l is catalyzed by a redox-regulated protein kinase similar to
activities that have been characterized in the thylakoids of
other photosynthetic organisms (Bennett, 1977; Allen et al.,
1981). Furthermore, DCMU and atrazine, two PSII herbicides
that bind to D l and block photosynthetic electron transport
to plastoquinone, the putative redox effector of the kinase(s)
(Allen et al., 1981), and FSBA, an ATP analog known to
inhibit protein kinases including the redox-regulated thyla-
koid kinase(s)(Farchaus et al., 1985), inhibited D l phos-
phorylation as well as the concomitant mobility shift exhib-
ited by phosphorylated D l (data notshown).
Although phosphorylation of D l was correlated with an
observed mobility shift, it was still possible that each phenom-
enon represented a separate modification. Since thylakoids
are known to contain a membrane-bound protein phospha-
tase(s) (Bennett,1980),we tested whetherthe electrophoretic
mobility shift exhibited by phosphorylated D l could be re-
versed by such an activity. Isolated thylakoids were phos-
phorylated in the dark by addition of ATP, ferredoxin, and
NADPH, washed twice to remove excess ATP andreductant,
and further incubated in the dark at room temperature for up
A Light
ATP :
Fd/NADPH:
FIG. 1. In vitro phosphorylation Dl-P
of D l in isolated thylakoids. Isolated
thylakoids were incubated inthe light or
>-
Dl
darkness, in the presence or absence of T i m (em i n ) : 0 5 10 20 4 0 60 60 60 6 0
[yD2P]ATP ferredoxin
and (Fd)/
NADPH as indicated. At the indicated
times, reactions were quenched by addi-
tion of SDS sample buffer and the re-
sulting samples analyzedby SDS-PAGE,
immunoblotting,andautoradiography.
B
100
A, immunoblotand its autoradiogram
with the positions of the Dl parent pro-
tein and its phosphorylated form (DI- 9E
P) marked. B, densitometric quantifica- .rl
tion of the bandslabeled Dl-P in the
immunoblot (open square) andautora-
diogram (solid square).
3dp
0 20
Time (min)
3525
to 20 h. Phosphorylated D l exhibited a time-dependent loss
of radiolabel which was partially inhibited by NaF (Fig. 3, A,
Autorad, and B), indicative of protein phosphatase activity
(Bennett, 1980). Since this dephosphorylation was paralleled
by the concomitant reversion of the slower migrating Dl-P
form back to the parentD l form (Fig. 3, A,Blot, and B ) , we
conclude that phosphorylation itself was the cause of the
electrophoretic mobility shift.
The in vitro dephosphorylation of D l followed apparent
first order kinetics and exhibited a rather long half-life of
approximately 10 h (Fig. 3B). In contrast, the phosphorylated
bands that co-migrate with the light harvesting chlorophyll
proteins of PSII (LHCII), the most studied thylakoid phos-
phoproteins, were dephosphorylated very quickly (Fig. 3A),
Autorad), as expected (Michel et al., 1987), indicating that
phosphorylated D l is a relatively poor substrate for the pro-
tein phosphatase activity contained in Spirodela thylakoids.
In Vivo Phosphorylation of Dl-Several in vivo phosphoryl-
ation experiments were performed to determine whether in
vitro phosphorylated D l and in vivo derived 32*, which CO-
electrophorese, are identical. Phosphate-depleted Spirodela
plants were incubated in the presence of carrier-free [”PI
orthophosphate and the pattern of radiolabeled thylakoid
proteins compared to those from thylakoids phosphorylated
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in vitro with [T-~*P]ATP(Fig. 4). While qualitative and
quantitative differences were apparent between the in vivo
and in vitro phosphorylated thylakoid protein patterns, sev-
eral proteins,including Dl, appeared to be phosphorylated in
both cases (Fig. 4,see position of Dl-P). Furthermore, DCMU
inhibited the in vitro phosphorylation of D l (Fig. 5), as
expected (Allen et al., 1981),as well as thein vivo phosphoryl-
ation of the co-migrating band (Fig. 5). Interestingly, the
effect of DCMU in vivo was less pronounced than that in
vitro, and observable only on those proteins which appeared
to co-migrate with in vitro phosphorylated polypeptides with
the exception of the approximately 9-kDa band (Fig. 5).
In vitro phosphorylated D l and the in vivo 32P-labeled
protein that co-electrophoresed with it were then subjected to
phosphoamino acid analysis. The results demonstrated that
Dark Light Dark
t
”
- t t
”
- +
- t -
”
+
“*r“ ’ 131c-””’
0 5 10 20 4 0 60 60 60 60
Immunoblot Autoradiogram
0 Blot
Autorad
40 60
3526 Phosphorylation of D l in Vivo
A
-Ne +Ne
Blot
2a 2.- Dl-P 7
Dl

200 -
91.4 -
69 -
46 - laCII{

0 5 10 20 5 10 20
Time (h)

14.3 -
. - .-
C

m
kDa
0 P (tNaF)
FIG.2. Immunoprecipitation of phosphorylated D l . Isolated 8 Blot (-NaF)
thylakoids were phosphorylated in the light with [Y-~~PIATP as 10 ! Blot (tNaF)

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described in the legend to Fig. 1, washed to remove excess ATP, and 0
20 10
resuspended to a chlorophyll concentration of 0.2 mg/ml. Immuno-
precipitation with anti-Dl antibodies was performed as described Time (h)
under “Experimental Procedures.” Samples from the starting thyla- FIG.3. In vitro dephosphorylation of D l by a thylakoid-
koids and immunoprecipitate were denatured in SDS sample buffer, bound phosphatase. Thylakoids were phosphorylated in the dark
resolved ona 10-20% linear gradient SDS gel, and analyzed by by addition of [ Y - ~ ~ P I A T P ferredoxin/NADPH, washed to re-
and
autoradiography (phos thylakoids) or fluorography (imm p p t ) . The move excess ATP and reductant, and further incubated in the dark
positions of molecular weight markers and phosphorylated D l ( D l - in the presence or absence of 10 m M NaF. At the indicated times,
P) are indicated. reactions were quenched by the addition of SDS sample buffer and
the resulting samples analyzed by SDS-PAGE, immunoblotting, and
autoradiography. A, immunoblot (upper panel; Blot) and its autora-
both contained exclusivelyphosphothreonine (Fig. 6). Finally, diogram (lower panel; Autorad) labeled as in Fig. 1except that LHCII
two-dimensional thin layer electrophoresis/chromatography marks the position of the light harvesting chlorophyll proteins of
(TLE/TLC) phosphopeptide mapping of terminal trypsin di- photosystem 11. B, quantification of the phosphorylated D l bands
gests of the two proteins was performed. Two tryptic phos- (Dl-P) on the immunoblot by densitometry (solid symbols) or 3!,
counting (open symbols).
phopeptides, with similar mobilities and ratios of radiolabel,
were resolved from both in vitro phosphorylated D l (Fig. 7,
panel A ) and the co-electrophoresing in vivo phosphorylated
protein (Fig. 7,panel B ) . Additionally, only two phosphopep-
tides were observed whena pH 6.5 buffer system was used in
the first dimension TLE (data not shown). Mixing experi-
ments (Fig. 7, panel C) confirmed that the phosphopeptides . .
from both proteins co-migrated providing strong evidence that .e .E
they were identical. Thus, we conclude that the previously
detected in vivo modification which alters theelectrophoretic
mobility of D l (Callahan et al., 1990) is a protein phosphoryl-
ation.
Mapping the D l PhosphoTylutwn Site-To localize the D l
phosphorylation site(s), partial proteolytic mapping experi-
ments were performed with gel-excised D l derived from in
vivo [%]Met labeling, in vivo 32Piphosphorylation, or in vitro
[T-~*P]ATP phosphorylation experiments. Digestion of 35S-
labeled D l with S. aureus V8 protease (Fig. 8) yielded the 14.3 -
characteristic 21-kDa fragment derived from the N terminus
as well as 10- and 8-kDa fragments which are mainly C-
6.2 ””II
terminal (Marder et al., 1984). The 3zPlabel from the phos-
phorylated proteins was associated primarily with the large
N terminal 21-kDa region (Fig. 8). Papain digestion of 35S- FIG.4. Comparison of thylakoid proteins phosphorylated in
labeled Dl yielded 20- and 12-kDa fragments (Fig. 8) derived vitro and in vivo. Thylakoids were phosphorylated in vitro with
from the N and C termini, respectively (Marder et al., 1984). [ Y - ~ ~ P I A T P described
as in the legend to Fig. 1 or isolated from
The 32Plabel from phosphorylated D l was associated exclu- Spirodela plants thatwere radiolabeled in uiuo by incubation with 0.5
mCi/ml of carrier-free [32P]orthophosphatefor 3 h under 50 p E m-2
sively with the N-terminal 20-kDa papain fragment (Fig. 8). s” white light. The samples were then resolved on 10-20% linear
Additionally, in both protease-derived phosphopeptides, the gradient SDS gels and analyzed by autoradiography. The positions of
mobility shift associated with phosphorylation was apparently the in vitro phosphorylated form of D l ( D l - P )and molecular weight
retained. These results map the phosphorylation site(s) tothe standards are indicated.
 Phosphorylation of Dl in Vivo
in vitro in vivo
DCMU: - +
-200 -
-"
+
-91.4 -
- 69 -
D"P-B -330--'
- FIG.7. Two-dimensional phoephopeptide mapping of in vi-
? phosphorylated in vitro with [y?'P]ATP or in vivo with [32P]ortho-
-14.3 -"
m to nitrocellulose membrane. Blot bands corresponding to phosphoryl-
kDi' ma
FIG.5. Effect of DCMU on thylakoid protein phosphoryla-
tion in vivo and in vitro.Thylakoids were phosphorylated in vitro
with [y3'P]ATP as described in the legend to Fig. 1 or in vivo with
["P]orthophosphate as described in the legend to Fig. 4, in the
presence or absence of 5 p~ DCMU. The samples were then resolved
on 10-20% linear gradientSDS gels and analyzed by autoradiography.
The position of the in vitro phosphorylated form of D l ( D l - P ) is
indicated.
.-e .-g
Y
I x x - ori
0
FIG.6. Phosphoamino acid analysis of in vitro and in vivo
phosphorylated D l . Thylakoid proteins, either phosphorylated in
vitro with [r-"]ATP or in vivo with [32P]orthophosphate,were sub-
jected to SDS-PAGE followed by electrotransfer to PVDFmembrane.
Blot bands corresponding to phosphorylated D l were excised and
hydrolyzed in pH 5.7 constant boiling HCl a t 110 "C for 1.5 h. The
resulting hydrolysates were dried and resuspended in a smallvolume
of H 2 0containing phosphoserine ( P S ) ,phosphothreonine (PT),
phosphotyrosine ( P Y )standards. The samples were subjected to thin
layer electrophoresis a t pH 3.5 for 30 min a t 1000 V and the plates
analyzed by fluorography. The positions of the ninhydrin-stained
phosphoamino acid standards (PS, PT, and P Y ) , orthophosphate
( P i ) ,and sample origin (ori) are indicated. The radioactive spot below
P Y is presumably due to a product of incomplete hydrolysis.
N-terminal two-thirds of the protein.
Fine mapping of the phosphorylation site(s) was accom-
plished by in vitro trypsinization of thylakoids. I n vivo and in
vitro phosphorylated thylakoids were incubated in the dark
a t room temperature in the presence or absence of trypsin.
The reactions were quenched by addition of SDS sample
buffer and the resulting samples subjected to SDS-PAGE,
immunoblotting, and autoradiography. Immunoblot analysis
indicated that the majority of the Dl present in the in vitro
labeled samples was in the phosphorylated form (Fig. 9A,
compare lanes 1 and 2) while the nonphosphorylated form
predominated in vivo (Fig. 9B,lane 1 ).Incubation with trypsin
resulted in a significant conversion of both forms in both
samples to a 31-kDa fragment (Fig. 9, A, lane 3, and B, lane
2) in agreement with previous studies (Marder et aL, 1984).
Autoradiography of the immunoblots demonstrated that the
32Plabel was not associated with the 31-kDa fragments de-
rived from either sample (Fig. 9, A, compare lanes 3 and 3a,
and B, compare lanes 2 and 2a). The original description of
this trypsin cleavage assigned it to the C terminus (Marderet
3527
vivo in vitro in mix
0
A
-.
ii
"
tro and in vivo phosphorylated D l . Thylakoid proteins, either
phosphate, were subjected to SDS-PAGE followed by electrotransfer
ated D l were excised and trypsinized in situfor 18h a t 37 "C. Released
peptides were dried, resuspended in a small volume of 20% pyridine,
and subjected to thin layer electrophoresis (TLE)at pH 3.5 for 30
min a t 1000 V in the horizontal dimension followed by ascending
chromatography (TLC) in the vertical dimension. The plates were
dried and analyzed by fluorography. A, in vitro phosphorylated D l
digest; B, in vivo phosphorylated D l digest; and C, mixture of equal
counts of A and B. Positions of sample application (X), anode (+I,
and cathode (-) are indicated.
Downloaded from www.jbc.org by on March 16, 2008
46
30 -e.
21.5 0.0
14.2 *
6.5
kD2 .-
con
S.a. V8 papain
FIG.8. Partial proteolytic mapping of in vivo and in vitro
phosphorylated D l , and [35S]methionine-labeledD l . Thyla-
koid proteins, either phosphorylated in vitro with [y3'P]ATP or in
vivo with [32P]orthophosphate, or radiolabeled in vivo with
methionine, were resolved by SDS-PAGE, after which the gels were
dried and autoradiographed to visualize the radiolabeled D l bands.
The D l bands were excised and applied on a new15-20% linear
gradient SDS gel in the presence of no protease (con),0.1 pglwell of
S. aureus V8 protease (S. a. V8),or 0.1 pg/well of papain. After
and stacking, electrophoresis was halted for 30 min to ensure proteolysis.
Electrophoresis was then completed and the resulting gel dried and
fluorographed. The positions of molecular weight standards are in-
dicated on the left.
al., 1984), whilea subsequent report concluded that cleavage
at the N terminus also occurred (Marder et al., 1988). Our
combined partial proteolysis results confirm the latter conclu-
sion and therefore map the phosphorylation site to within 1
kDa of the N terminus. Consistent with this conclusion is the
observation that threonine residues are absent from the de-
duced C-terminal28 amino acid residues of mature Spirodela
D l (Avni et al., 1991).
Steady-state Distribution of Phosphorylated D l -The ex-
tent to which PSII-associated proteins are phosphorylated
either in vitro or in vivo has not been previously established.
Exploiting the differential electrophoretic mobility of phos-
phorylated Dl, immunoblot analysis was usedto address this
question for this reaction center protein. As demonstrated in
Fig. lA, virtually all of the D l present in thylakoids can be
phosphorylated in vitro. It is also apparent from this figure
that the in vivo level of phosphorylated Dl, assumed to be
that present in freshly isolated thylakoids, is quite low under
our normal growth conditions (Fig. IA, time 0 lane). We
further tested the effect of light intensity on the in vivo steady-
state levels of phosphorylated Dl. Thylakoids were isolated
from Spirodela plants incubated for 1day under various light
3528 Phosphorylation of Dl in Vivo
A. In Vitro Phosphorylated kDa from the N terminusof this PSII reaction center protein.

trypln:
Dl-P
Rlot

-
Dl> r*
1-31
1 2
-
Auto

- us
3
- +

z a 3 a
T o our knowledge, this is the first rigorous characterization
of an i n vivo phosphorylated form of a PSII-associated pro-
tein. The Dl phosphorylation site mapping reported here can
be further interpreted in light of previous literature. Michel
et al. (1988) isolated terminal tryptic phosphopeptides from
spinach PSII core particles phosphorylated in oitro and iden-
tified the sequences of two of the peptides as TAILER and
R. In Vivo Phosphorylated TAILERR by tandemmassspectrometry.Thesepeptides
Blot Auto
start with an N-acetyl-0-phosphothreonineresidue and cor-

--
trypsin: - + - +
respond to the N terminus of the deduced Dl sequence (minus
1jl.l'
the initiating methionine residue) (Michel et 01.. 1988). Given
Dl> that: 1) terminal trypsinization of phosphorylated Spirodda
1-31 D l yielded two phosphopeptides which were both localized to
I 2 la 2a within 1 kDafrom the N terminus;2)phosphorylatedDl
FIG.9. I n situ trypsinization of i n vi t r o and in vivo phos- from Spirodela contains exclusively phosphothreonine; 3 ) D l
phorylated thylakoids.Thylakoid preparations, either phosphoryl- from Spirodela is N-terminally blocked (Marder et al., 1984);
ated in vitro with [y-'"P]ATP or in oioo with ['*PJorthophosphate, and 4) the deduced Dl amino acid sequences of spinach and
were dilutedto 0.2 mg/ml of chlorophyll and incubated in the presence Spirodela (Avni et al., 1991) are identical at the N terminus,
or absence of 0.01 pg/ml of trypsin for 1 h a t room temperature. The
reactions were stopped by addition of SDS sample buffer and the it seems almost certain that the i n oivo and in vitro Spirodda
resulting samplessubjected to SDS-PAGE, immunoblot(Blot)analy- D l phosphorylation sites characterized here are identical to
sis, and autoradiography( A u t o ) .A, isolated thylakoids before in oitro that reported for the spinach protein phosphorylatedin oitro.
phosphorylation ( h e I ) , and in uitro phosphorylatedthylakoids A similar conclusion was also reached from studies on the in
incubated in the ahsence (lanes 2 and 2a), or presence (lams 3 and oitro phosphorylation of pea Dl (Marderet al., 1988).

Downloaded from www.jbc.org by on March 16, 2008

3 a ) of trypsin. H, in oioo phosphorylated thylakoids incubated in the
absence (lanes 1 and l a ) or presence ( l a n e s 2 and Pa) of trypsin.
In addition to D l , five other PSII-associated proteinshave
Positions of phosphorylated ( D l - P ) andnonphosphorylated ( I l l ) been unambiguously demonstrated to be phosphorylated in
D l , and their major 31-kDa tryptic fragments ( T - 3 / ) are , indicated. vitro: D2, CP47, the psbH gene product, and t-ype I and I1
Note thatwhile D l and D l- P a r enot well-resolved in lane I ofpanel LHCII polypeptides (Bennett, 1991). The in vitrophosphoryl-
H,the less mobile radiolabeled band visible in lane l a corresponds ation sitesof these proteins have been determined andshown
specifically with the D l - P region of lane I . Also note that in both to be on or near the N terminus in all cases (see Bennett,
panel A, lane 2a, and panel R,lane l a , the faster migrating radiolabeled
bands that are lost upon trypsinization are from a thylakoid phos- 1991). However, it has never been conclusively shown that
phoprotein unrelated to D l . identicalphosphorylationsexist in viuo. This is important
since it is apparent from data presented here that there are
differences between i n vitro and in vivo phosphorylated thy-
D
, I -1' lakoid proteins. Thus, the demonstration that Dl is phos-
-0"-
I
phorylated identically in oivo and in vitro validates the use-
fulness of in vitro model systems for some purposes.
0 s so 1.50 300 While not proven, it is generally thought that there aretwo
liplll intensily ( p m-2 ~ ,-I)
kinases involved inphosphorylating PSII-associated proteins,
one acting on the LHCII polypeptides and the other on the
FIG. 10. Steady levels of phosphorylated D l in vivo. Spiro- PSII core proteins (Bennett, 1991). If there are two kinases,
dela plants were incubated under white light (cool white fluorescent they are similar to the extent that both are membrane-bound,
bulbs) at the intensitiesindicated ( P P F D ) for1 dayafter which
thylakoids were isolated and analyzed by SDS-PAGE and immuno- immunologically related, and
redox-regulated (Bennett,
blotting with anti-Dl antibodies. Positionsof the nonphosphorylated 1991). Results from in vitro studies have implicated plasto-
( I l l ) and phosphorylated ( D l - P )forms of Dl are indicated. quinone as the redox-effector of bothactivities; however,
LHCII phosphorylationrequiresanactive cytochrome b,J
intensities and analyzed by immunoblotting (Fig. 10). The complex while phosphorylation of the PSI1core proteins does
total amount of D l protein remained relatively constant in not (Bennett, 1991). In the present study we have demon-
all of the samples indicating that Dl synthesis is tightly strated that DCMU inhibits the phosphorylation of Dl both
coupled to the rapid light-dependent degradationof this pro- in vitro and in viuo. Furthermore, we have shown that the in
tein (Mattoo et al., 1984).However, dark incubated plants vivo level of phosphorylated D l increases with increasing light
contained virtually no phosphorylated D l while increasing intensity. Under subsaturating light, the plastoquinone pool
levels of this form were observed with increasing light inten- would be expected to become more reduced with increasing
sity to a maximum of about 20% of the total D l under 300 light intensity. Thus, our results are consistent with PSI1 core
MEm" s" white light (Fig. 10). In other experiments, 1- and protein phosphorylation being regulated similarly in ~ ~ ani w
%day incubations were found to result in similar levels of has been demonstrated in vitro.
phosphorylated D l indicating thatsteady-state
a was The steady-statelevel of phosphorylated D l will depend on
achieved within a 1-day incubation. The percentage of D l in its formation as well as its turnover. In this case, turnover
the phosphorylated form a t a given light intensity showed could either be due to dephosphorylation, protein degradation,
some variation in different experiments, perhaps depending or a combination of the two. Thylakoid-bound phosphatase
on the age or developmentalstage of the plants, but was activity has been reported to be insensitive to light or redox
always observed to increase with increasing light intensity. regulation in vitro (Bennett, 1980; Michel et al., 1987) while
degradation of D l i n vivo is known to be light-dependent and
DISCUSSION
proportionaltolightintensity(Mattoo et al., 1984). The
In this studywe have identified thein vivo phosphorylated observation that the steady-state level of phosphorylated Dl
form of Dl and mapped the phosphorylation site to within1 increases with increasing light intensity suggests that kinase
 Phosphorylation of Dl in Vivo
activation is more sensitive to light intensity than are the
processes responsible for phosphorylated D l turnover. Fur-
thermore, these results directly demonstrate that dynamic
heterogeneity in the phosphorylation state of Dl, and there-
fore the PSII reaction center, exists under physiological con-
ditions as has been recently suggested (Giardi et al., 1991).
LHCII polypeptide phosphorylation has received consider-
able attention since, at least in vitro, this modification is
known to mediate changes in functional antennae size which
compensate for imbalances between PSI and PSII. Under
conditions that favor PSII excitation, the plastoquinone pool
becomesmore reduced which resultsinactivation of the
thylakoidkinase(s).Phosphorylation of so called “mobile
LHCII” results in the dissociation of this antennae subpopu-
lation from PSII centers located in the grana, thus effectively
decreasing the optical cross-section of PSII. Furthermore, it
is thought that dissociated mobile LHCII can translocate to
the stroma lamellae where it may act to transfer energy to
PSI (for reviews, see Haworth et al., 1982; Williams and Allen,
1987;Bennett, 1991). In contrast, the role of PSII core protein
phosphorylation is not well established. Although lacking
experimental evidence, it hasbeen speculated that these phos-
phorylations may facilitate dissociation of mobile LHCII due
to charge repulsion (Allen and Holmes, 1986), or that the N-
terminal phosphoryl groups may be important for buffering
the partition gap between stackedmembranes (Bennett,
1991). In addition, a variety of effects on PSII functional
properties have been reported to occur following in vitro
phosphorylation of thylakoids, including alterations of: her-
bicide binding (Shochat et al., 1982; Vermaas et al., 1984;
Habash and Baker, 1990); QB stability (Jursinic and Kyle,
1983; Hodges et al., 1987), light-saturated photosynthetic
electron transport (Horton andLee, 1984; Hodges et al., 1985;
Packham, 1987; Habash and Baker, 1990), and photoinhibi-
tion (Horton andLee, 1985). It should be noted that in none
of these cases were the observed effects correlated with phos-
phorylation of any specific polypeptide(s) nor was the extent
of protein phosphorylation known. Furthermore, many of the
measured differences between phosphorylated and nonphos-
phorylated thylakoids were relatively small. Assuming that:
1)the noted effects are proportionalto the extent of thylakoid
protein phosphorylation; and 2) thylakoid proteins are phos-
phorylated to a significantly greater extent i n vitro than i n
vivo as has been shown here for Dl, the significance of these
observations under physiological conditionsremains to be
determined.
A novel hypothesis concerning the function of D l phos-
phorylation is that this modification may somehow be in-
volved in the light-dependent degradation of this protein
(Callahan et al., 1990). This hypothesis is speculative and
based only on circumstantial evidence; the spatio-temporal
correlation between D l phosphorylation and degradation, as
well as thelight dependence and herbicide inhibition of both
processes (Callahan et al., 1990 andthis study). Present
studies are aimed at testing this hypothesis using a variety of
approaches. First, the ability to specifically resolve the two
forms of D l is being exploited to examine the metabolism of
the phosphorylated form in vivo under various conditions to
see if this modification correlates with D l degradation. Sec-
ond, we are attempting toidentify kinase inhibitors that can
effectively block D l phosphorylation i n vivo so that we can
test their effect on D l degradation. Finally, we are testing
whether D l is phosphorylated in organisms such as green
algae and cyanobacteria, where rapid light-dependent degra-
dation of this protein is known to occur.
3529
REFERENCES
Aebersold, R. H., Leavitt, J., Saavedra, R. A., Hood, L. E., and Kent,
S. B. H. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 6970-6974
Allen, J. F., Bennett, J., Steinback, K. E., and Arntzen, C. J. (1981)
Nature 291, 25-29
Allen, J. F., and Holmes, N.G. (1986) FEBS Lett. 2 0 2 , 175-181
Arnon, D. I. (1949) Plant Physiol. 24, 1-15
Avni, A., Mehta, R. A,, Mattoo, A. K., Greenberg, B. M., Chattoo, B.
B., Heller, D., and Edelman, M. (1991) Plant Mol. Biol. 1 7 , 919-
921
Bennett, J. (1977) Nature 2 6 9 , 344-346
Bennett, J. (1980) Eur. J. Biochem. 104, 85-89
Bennett, J. (1991) Annu. Reu. Plant Physiol. Plant Mol. Biol. 42,
281-311
Callahan, F. E., Wergin, W. P., Nelson, N., Edelman, M., and Mattoo,
A. K. (1989) Plant Physiol. 91, 629-635
Callahan, F. E., Ghirardi, M. L., Sopory, S. K., Mehta, A.M.,
Edelman, M., and Mattoo, A. K. (1990) J Biol. Chem. 2 6 5 , 15357-
15360
Cooper, J. A., Sefton, B. M., and Hunter,T. (1983) Methods Enzymol.
99,387-402
Coughlan, S. J., and Hind, G. (1987) Biochemistry 2 6 , 6515-6521
Edelman, M., and Reisfeld, A. (1978) in Chloroplast Deuelopment,
Elsevier/North-Holland, (Akoyunoglou, G., and Argyroudi-Ako-
yunoglou, J. H., eds) pp. 641-652, New York
Farchaus, J., Dilley, R. A,, and Cramer, W. A. (1985) Biochim.
Biophys. Acta 809, 17-26
Giardi, M. T., Rigoni, F., Barbato, R., and Giacometti, G. M. (1991)
Downloaded from www.jbc.org by on March 16, 2008
Biochem. Biophys. Res. Commun. 176,1298-1305
Grebanier, A. E., Coen, D. M., Rich, A., and Bogorad, L. (1978) J.
Cell Biol. 78, 734-746
Habash, D. Z., and Baker, N. R. (1990) J. Exp. Bot. 4 1 , 761-767
Haworth,P., Kyle, D. J., Horton, P., and Arntzen, C. J. (1982)
Photochem. Photobiol. 3 6 , 743-748
Hodges, M., Boussac, A., and Briantais, J.-M. (1987) Biochim. Bio-
phys. Acta 8 9 4 , 138-145
Hodges, M., Packham, N. K., and Barber, J. (1985) FEBS Lett. 1 8 1 ,
83-87
Horton, P., and Lee, P. (1984) Biochim. Biophys. Acta 767,563-567
Horton, P., and Lee, P. (1985) Planta (Basel) 165,37-42
Jursinic, P. A., and Kyle, D. J. (1983) Biochim. Biophys. Acta 723,
37-44
Kamps, M. P., and Sefton, B. M. (1989) Anal. B i o c h m . 176,22-27
Marder, J. B., Goloubinoff, P., and Edelman, M. (1984) J Biol. Chem.
259,3900-3908
Marder, J. B., Mattoo, A. K., and Edelman, M. (1987) Methods
Enzymol. 1 1 8 , 384-396
Marder, J. B., Telfer, A,, and Barber, J. (1988) Biochim. Biophys.
Acta 932,362-365
Mattoo, A. K., Hoffman-falk, H., Marder, J. B., and Edelman, M.
(1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1380-1384
Mattoo, A. K., and Edelman, M. (1987) Proc. Natl. Acad. Sci. U. S. A.
84, 1497-1501
Mattoo, A. K., Marder, J. B., and Edelman, M. (1989) Cell 5 6 , 241-
246
Michel, H., Hunt, D. F., Shabanowitz, J., and Bennett, J. (1988) J.
Biol. Chem. 263, 1123-1130
Michel, H., Shaw, E. K., and Bennett, J. (1987) in Plant Membranes:
Structure, Function, Biogenesis (Leaver, C., and Sze, H., eds) pp.
85-102, Alan R. Liss, New York
Mullet, J. E., Klein, P. G., and Klein, R. R. (1990) Proc. Natl. Acad.
sci. U. S. A. 87,4038-4042
Ohad, I., Adir, N., Koike, H., Kyle, D. J., and Inoue, Y. (1990) J Biol.
Chem. 165, 1972-1979
Packham, N. K. (1987) Biochim. Biophys. Acta 8 9 3 , 259-266
Peter, M., Nakagawa, J., Doree, M., Labbe, J. C., and Nigg, E. A.
(1990) Cell 6 1 , 591-602
Posner, H.B. (1967) in (Witt, F. A., and Wessels, N. K., eds) Methods
in Developmental Biology pp. 301-317, Crowell, New York
Reisfeld, A., Mattoo, A. K., and Edelman, M. (1982) Eur. J . Biochem.
1 2 4 , 125-129
Rutherford, A. W. (1989) Trends Biochem. Sci. 14, 227-232
Shochat, S., Owens, G. C., Hubert, P., and Ohad, I. (1982) Biochim.
Biophys. Acta 6 8 1 , 21-31
Vermaas, W. F. J., Steinback, K. E., and Arntzen, C. J. (1984) Arch.
Biochem. Biophys. 2 3 1 , 226-232
Williams, W. P., and Allen, J. F. (1987) Photosynth. Res. 13, 19-45