THE JOURNAL OF BI~L~CICAL CHEMISTRY

Communication Vol. 265. No. 26. Issue of September 15, pp. 15357-15360, 1990
Printed in U S.A.

translocated to the grana partition region (12). Turnover of

A Novel Metabolic Form of the the 32kDa-Dl protein is attuned to light intensity (2, 13) and
32kDa-Dl Protein in the Grana- light quality (14-16). Two types of photoreceptors have been
localized Reaction Center of implicated in its degradation: chlorophylls in the visible and
far-red, and plastoquinones in the ultraviolet region of the
Photosystem II* spectrum (15). A scission in the protein backbone between
(Received for publication, June 4, 1990) amino acid residues 238-247, yielding a membrane-bound
Franklin E. Callahan*, Maria L. Ghirardi, product of 23.5 kDa, has been suggested as the primary
Sudhir K. Soporyg, Arkesh M. Mehta, degradation event of 32kDa-Dl protein turnover in Spirodelu
Marvin Edelmanl, and Autar K. Mattool/ thylakoids (13, 16). However, the molecular intermediates
From the Plant Molecular Biology Laboratory, United leading to this event have not yet been deciphered.

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States Department of Agriculture-ARS, Beltsuille We describe here the resolution of Dl (17) into the 32kDa-
Agricultural Research Center (West), Dl form and a novel form designated 32*. The 32* form is
Beltsuille, Maryland 20705 specifically and transiently generated in the grana where it is
a significant component of the reaction center. Its appearance
Two forms of the 32kDa-Dl reaction center protein does not correlate with photosynthetic electron flow.
of photosystem II (PSII), having slightly different mo-
bilities on denaturing polyacrylamide gels, have been EXPERIMENTAL PROCEDURES
resolved in Spirodela oligorrhiza, Glycine max L., Spirodela oligorrhiza plants were grown in sucrose-supplemented
Gossypium hirsutum L., Triticum aestivum L., andZea medium under cool fluorescent lamps (20 pE mm2 s-‘) at 20-25 “C.
mays L. The protein band with faster mobility is iden- Plants were transferred overnight to minus sucrose medium before
tified as the 32kDa-Dl protein, and the less mobile each experiment. Pulse labeling was done with [“‘Slmethionine under
band as a novel form, designated 32*. The two forms the same illumination (12). Specific conditions are described in the
are structurally similar based on immunological and figure legends. For comparison, wheat leaf segments were prepared
partial proteolytic tests. 32* is associated exclusively and suspended in preincubation buffer as described previously (18).
Segments were pulse-labeled with [“‘Slmethionine and chased in the
with the grana and is present in the PSI1 reaction
same medium without the radiolabel at 50 PE mm2 s-‘.
center. Temporally, 32* appears several hours after Thylakoids were isolated and fractionated into grana and stroma
the translocation of newly synthesized and processed lamellae by the dual detergent method (19). PSI1 reaction centers
32kDa-Dl protein from the stroma lamellae to the were isolated from grana preparations according to Marder et al. (5).
grana. Formation of the 32* is strictly light-dependent Proteins of these various isolates were resolved by SDS-PAGE on
under physiological light intensities and correlates lo-20% gradients (20). Electrophoresis was continied until proteins
with a reciprocal loss of the 32-kDa form. Light in- having molecular masses of 20-26 kDa reached the bottom of the gel
duced formation of 32* is inhibited by 3-(3,4-dichlo- as indicated by prestained molecular weight markers. The gels were
rophenyl)- l,l-dimethylurea but is not coupled to linear then either stained with Coomassie R-250 or exposed for fluorogra-
phy. Electroblotting and immunodetection of the 32kDa-Dl protein
electron transport.
were carried out as described previously (20) using alkaline phospha-
tase-linked secondary antibody. An LKB laser densitometer was used
for scans of blots and fluorogiaphs.
Partial proteolytic digestion of isolated 32-kDa and 32* bands was
Light-dependent rapid turnover is a characteristic of the done with either Staphylococcus aureus V8 protease or papain as
Dl or 32-kDa thylakoid protein (l-3). The 32kDa-Dl protein described (11, 21, 22).
is an essential component of the photosystem II (PSII)’
reaction center (4,5) and is also the primary target of triazine RESULTS AND DISCUSSION
and urea-type herbicides (6-9). It is synthesized in the light Radiolabeled 32* Appears in the Grana during a Chase-
on chloroplast ribosomes as a 33.5-34.5-kDa precursor (10, Spatio-temporal aspects of 32kDa-Dl protein metabolism
11). The precursor is integrated into the stroma-exposed were followed using pulse-chase experiments in conjunction
lamellae, processed there to its mature size and subsequently with subfractionation of thylakoids. Spirodela plants were
pulse-labeled in the light for 2 min with [%]methionine, and
* This research was supported in part by a United States-Israel
Binational Agricultural Research and Development Fund grant (to the radioactivity was chased in the light with nonradioactive
M. E. and A. K. M.). The costs of Dublication of this article were methionine for various periods of time. Grana and stroma
defrayed in part by the payment of page charges. This article must lamellae were isolated and equal amounts of membrane pro-
therefore be hereby marked “aduertisement” in accordance with 18 teins analyzed by high resolution SDS-PAGE/fluorography.
U.S.C. Section 1734 solelv to indicate this fact. The distribution of radiolabel in proteins of grana and stroma
$ Permanent address: Crop Science Research Laboratory,
lamellae during the chase is shown in the fluorogram of Fig.
USDA-ARS. P. 0. Box 5367. Mississiuni State. MS 39762-5367.
§ Permanent address: School of Life Scienc&, Jawaharlal Nehru 1A. At 0 time chase most of the radioactivity is still present
University, New Delhi 110067, India. in the 33.5-kDa precursor band in the stroma lamellae (SL).
ll Permanent address: Dept. of Plant Genetics, The Weizmann By 15 min it chases from the precursor into the mature 32-
Institute of Science, Rehovot 76100, Israel. kDa band. Processing of the precursor is complete by 1 h with
11To whom correspondence should be addressed: Plant Molecular concomitant translocation of the mature 32kDa-Dl protein
Biology Lab., Bldg. 006, Rm. 118, USDA-ARS, BARC-W, Beltsville,
MD 20705. from the stroma lamellae to the grana (Fig. lA, see G) as
’ The abbreviations used are: PSII, photosystem II; SDS-PAGE, previously shown (12). The radiolabel in the grana-localized
sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DCMU, 32-kDa protein increases from 15 min to 1 h but thereafter is
3-(3,4-dichlorophenyl)-l,l-dimethylurea. also visible in a novel band with slightly lower mobility,

15357
 Resolution of the 32kDa-Dl Protein into Two Forms

A
STAIN %-MET BLOT
,..-
f= ‘- .’

0 .25 1 3 4 6 12 0 .25 1 3 4 6 12
CHASE TIME, h LHCP- W’

SL G SL G SL G RC

B BLOT

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- w,Jr -
1 2 3 4
0 2 4 6 8 10 12 14
FIG. 2. Immunological reactivity of 32kDa-Dl and 32* pro-
CHASE TIME, h teins, detection of 32* within the PSI1 reaction center and
general occurrence of 32* in higher A, stroma
plant thylakoids.
FIG. 1. Time-dependent appearance of 32* in grana lamel- lamellae (SL) and grana (G) fractions were isolated from radiolabeled
lae. A, Spirodela plants were pulse-labeled in the light (20 PE m-* Spirodela plants as described in the legend to Fig. 1 and resolved by
s-‘) for 2 min with [%]Met (250 &i/ml), washed with fresh medium, SDS-PAGE. The Coomassie-stained gel (STAIN) shows the overall
and then transferred to medium containing 1 mM nonradioactive protein profiles of the fractions used. The highly resolved banding
methionine. At the indicated chase times, a sample of plants was positions of the 33.5-kDa precursor, 32-kDa protein, and 32* are
removed for thylakoid isolation and further fractionated into stroma shown in the fluorograph (35S-MET). The regime for the SL lane was
(SL) and grana (G) lamellae. Proteins associated with these mem- 3-min pulse and no chase, and that for the G lane, 3-min pulse and
brane fractions were resolved by SDS-PAGE/fluorography. The po- 2-h chase. Duplicated lanes of the same gel were electroblotted onto
sitions of the precursor (33.5), 32kDa-Dl protein (32) and modified nitrocellulose and probed with antibody (3) to the 32kDa-Dl protein
32-kDa form (32*) are indicated. B, ratios of 32* to 32-kDa proteins (BLOT). Isolated granal PSI1 reaction centers (RC) were electropho-
were determined by densitometric scans of fluorographs of three resed in parallel and probed in a similar manner. B, thylakoids were
individual experiments such as shown in A. isolated from fully developed leaves excised from greenhouse-grown
6-week-old corn (lane I), 3-week-old wheat (lane 2), 6-week-old
designated 32*. The characteristic turnover process is indi- cotton (lane 3), and 6-week-old soybean (lane 4) plants. Equal
amounts of chlorophyll (2 pg/lane) were run on SDS-PAGE and
cated by the decrease in label in both the 32-kDa and 32* electroblotted onto nitrocellulase paper. The blot was probed with
bands by 12 h of chase. 32-kDa antibody (32) as described in A.
A plot of the ratio of labeled 32* to labeled 32-kDa protein
in the grana showed a progressive increase with time of chase
32 32* 32
(Fig. lB), suggesting conversion of one form of the protein
into the other. Similar time courses of 32* formation were
found for [35S]methionine-labeled wheat leaf segments (data
not shown).
32% Is Immunologically and Structurally Similar to the 32-
kDa Protein-The relationship between the 32* and 32kDa-
Dl protein forms was investigated using immunological and
protein fingerprinting techniques. The results, shown in Fig. 0ao1o.lwu, OJam 0 0.1 u) Lo o.lo
2A (BLOT), indicate that 32* is immunologically related to S. aureus protease, ag Papam, pg
the 32-kDa protein and, moreover, is present in purified FIG. 3. Partial proteolytic digestion patterns of the 32kDa-
granal reaction centers. Thus, the modification of the 32-kDa Dl protein and 32*. Plants
were pulse-labeled for 30 min with
protein resulting in 32* formation occurs within the assembled [35S]Met (150 &i/ml) and chased for 7 h with nonradioactive methi-
PSI1 reaction center. Also shown in Fig. 2A are the banding onine in order to allow significant formation of 32* while also retain-
ing radiolabel in the 32kDa-Dl protein. Thylakoid proteins were
positions of the radiolabeled 33.5kDa precursor, 32* and 32- resolved on preparative gradient SDS gels, dried, and autoradi-
kDa protein forms (35S-ME!?‘), and the overall Coomassie ographed in order to visualize the two bands. The separate bands, as
Blue stained protein profiles of grana and stroma lamellae exemplified in the autoradiogram (A), were excised from the gels by
(STAIN). The steady state ratio of 32*/32-kDa was deter- realignment of the x-ray film and subjected to proteolytic digestion
mined by microdensitometry as an average from several ex- with the indicated concentrations of either S. aureus protease or
papain (B).
periments. It was approximately 1 to 4 in all Spirodelu mem-
brane preparations.
peptide forms are compared in Fig. 3. Digestion of 32* and
Immunoblots of thylakoid preparations from leaves of
the 32-kDa protein with S. aureus V8 protease yielded the
greenhouse-grown corn, cotton, soybean, and wheat plants characteristic (22) 22- and 21-kDa doublet and lo-, 8-, and 6-
reveal the presence of 32* in these plants as well, and further kDa polypeptides in both cases (indicated by arrows in Fig.
indicate that steady state ratios of 32* to 32-kDa protein can 3). Likewise, digestion with papain resulted in the character-
vary from plant to plant, perhaps dependent on the physio- istic 20- and 12-kDa fragments (indicated by arrows) in both
logical age of the plant (Fig. 2B). cases. Thus, 32* is structurally closely related to the 32kDa-
Partial proteolysis maps (23) of the 32* and 32-kDa poly- Dl protein.
 Resolution of the 32kDa-Dl
A L D Fig. 4A demonstrates that formation of 32* is blocked when
y4- -1c
0 CHASE
0.2 1 T& l2
0 0.2 1 4' 14
L scavenger which does not directly interact with the 32kDa-
ru*r*6 valIw&
-!amlmwe
+DCMU
vN4lm
+DINOSEB
L The 32* form is characterized by its decreased mobility vis-
B0.5
FIG. 4. Light dependence of 32* formation and the effect of
PSI1 herbicides. A, plants were pulse-labeled for 6 min with [?S]
Met and then chased as in Fig. 1 in the light (L), in darkness (D),
with DCMU (2.5 PM), or Dinoseb (2.5 PM). At the chase times
indicated, samples were removed and grana were isolated. Grana
uroteins were resolved bv SDS-PAGE/fluoroeranhv. Arrows denote
32*. B, ratios of 32* to 32kDa-Dl proteins were determined by
densitometric scans of the data presented in A. Filled circle, light
control; triangle, light + Dinoseb; square, light + DCMU.
Formation of 32* Does Not Correlate with Electron Flow
through PSZZ-Degradation of the 32kDa-Dl protein in visi-
ble light is inhibited by DCMU (2), a herbicide that displaces
bound quinone from the protein (24) and disrupts linear
electron transport through PSII. Recently, we have shown
that Dinoseb, a herbicide which also interacts with the 32kDa-
Dl protein, markedly inhibits turnover of this protein in
visible light at concentrations where linear electron transport
is only slightly affected (25). These compounds, along with
dark controls, were used to differentiate between association
of 32* with protein degradation or with electron transport.
Spirodelu plants were radiolabeled in the light as in Fig. 1 and
then chased for various times either in the dark or in the light
with and without DCMU or Dinoseb. The results (Fig. 4A)
show that treatments with either DCMU or Dinoseb did not
impair translocation of processed 32-kDa protein into the
grana. Likewise, darkness did not significantly inhibit trans-
location, per se (additionally confirmed by other experiments
not shown, where the 32-kDa protein was weakly labeled in
darkness immediately after removal of plants from light and
then chased in the dark). Thus, these three treatments are
suitable for monitoring effects on 32* formation.
Protein into Two Forms 15359
the chase is performed in the dark (12-h chase, light versus
dark) or in the light in the presence of DCMU (or atrazine,
data not shown). Significantly, Dinoseb also markedly delayed
the appearance of 32* in the light, although some 32* forma-
tion occurred at the longest chase time. The inhibitory effects
of DCMU and Dinoseb on the increase in the ratio of radio-
labeled 32* to 32-kDa protein are quantified in Fig. 4B. We
have recently demonstrated that propylgallate, a free radical
Dl protein, inhibits this protein’s degradation without affect-
ing linear electron transport (26). In the presence of propyl-
gallate, the formation of 32* was markedly inhibited (data not
shown). Finally, ultraviolet (300 nm) irradiation stimulates
32kDa-Dl protein degradation but does not support photo-
synthetic electron transport (15). Yet 32* is readily formed at
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300 nm.’ All of the above suggest a lack of correlation between
photosynthetic electron flow and 32* formation; they are
consistent with (but do not prove) a role for 32* in light-
dependent 32kDa-Dl protein degradation.
a-vis the 32kDa-Dl parent protein on denaturing SDS-PAGE.
Such mobility shifts can arise from single amino acid changes
or modifications (27 and references therein), particularly
those resulting in net charge alteration of a protein (28). The
behavior of 32* on denaturing SDS-polyacrylamide gels would
thus be consistent with some type of protein modification in
a domain that is recalcitrant to complete denaturation by
SDS. Several types of posttranslational modifications of pro-
teins are known in the literature. Examples include glycosyl-
ation, methylation, ADP-ribosylation, adenylation, acylation,
phosphorylation, and oxidative modification. Posttransla-
tional modifications of the 32-kDa protein, such as acylation
(12) and phosphorylation (29, 30), have been reported, with-
out, however, an assignment of a specific function. Similarly,
the 32-kDa protein has been reported to undergo an irrevers-
ible conformational change perhaps due to cross-linking
within the protein molecule under photoinhibitory conditions
(31). Experiments geared at the elucidation of the nature of
the modification that leads to 32* formation have revealed
that neither acylation (12) nor irreversible cross-linking
within the protein (31) lead to formation of the 32* band on
SDS-PAGE.3 Preliminary experiments involving the in vivo
labeling of chloroplast proteins with [32P]orthophosphate
were inconclusive since the presence of any of the phosphoryl-
ated protein bands migrating in the 32-kDa-32* region could
not be rigorously correlated with the 32* specifically. Identi-
fication of the modification that results in 32* formation
should be useful in elucidating the function of this transient
form of the 32kDa-Dl protein of the PSI1 reaction center.
Acknowledgments-F. E. C. thanks Dr. Charles R. Caldwell for
intellectual “happy hour” discussions, distractions that resulted in a
prolonged electrophoresis run and thereby facilitated our initial at-
tempts-at resolution of 32* and 32-kDa proteins within Dl (diffuse
band 1). We thank Drs. James D. Anderson and Tedd Elich for
helpful’discussions, and Drs. Yossi Hirschberg and John Mullet for
gifts of antibodies to the 32kDa-Dl protein.
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