From the Department of Botany, University of Munich, 80638 München, Menzinger Straße 67, Federal Republic of Germany
Upon transfer of lysed chloroplasts from darkness
to light, the accumulation of membrane and stromal chloroplast proteins is strictly regulated at the level of translation elongation. In
darkness, translation elongation is retarded even in the presence of
exogenously added ATP and dithiothreitol. In the light, addition of the
electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethyl urea
inhibits translation elongation even in the presence of ATP. This
inhibition can be overcome by addition of artificial electron donors in
the presence of light, but not in darkness. Electron flow between
photosystem II and I induced by far red light of 730 nm is sufficient
for the activation of translation elongation. This activation can also
be obtained by electron donors to photosystem I, which transport
protons into the thylakoid lumen. Release of the proton gradient by
uncouplers prevents the light-dependent activation of
translation elongation. Also, the induction of translation activation is switched off rapidly upon transfer from light to darkness. Hence, we propose that the formation of a photosynthetic proton gradient across the thylakoid membrane activates translation elongation in chloroplasts.
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INTRODUCTION |
Protein expression in plastids of algae and higher plants is
regulated by light during the light-dependent development
of chloroplasts as well as during the light-dark transitions of fully differentiated chloroplasts. A great proportion of the regulatory mechanisms were found to be mediated at a posttranscriptional level, as
the mRNA levels of many of the concerned genes were little or not
affected (1-4). Most of the posttranscriptional changes of protein
expression were explained by a regulation of translation initiation;
the 5'-untranslated region of the psbA gene encoding the reaction
center protein D1 was shown to direct light-dependent
accumulation of a reporter gene in transformed tobacco plants (5), and
proteins binding to the 5'-regions of several chloroplast genes were
observed in Chlamydomonas reinhardtii (6).
Light-dependent binding of proteins to the 5'-untranslated region of the psbA gene was shown to be responsible for the
light-dependent translation initiation in
Chlamydomonas (7) and in spinach (8).
Other examples of light-regulated protein expression were explained by
translation elongation control; the mRNA encoding the large subunit
of ribulose-1,5-bisphosphate carboxylase was not translated in the dark
but remained bound to polysomes in Amaranthus (9). Accumulation of D1
translation intermediates in the dark was observed in pea chloroplasts
(10, 11) and in spinach (12).
Different explanations were given of how light regulated translation.
Several groups attributed reduced protein expression in the dark to the
reduced level of ATP in the chloroplast in the absence of
photosynthetic activity (13, 14). Conversely, reduced translation
initiation of the psbA gene has been explained by the increased level
of ADP in the dark (15). Binding of an initiation complex to the psbA
mRNA was proposed to be regulated by reduced thioredoxin, which is
generated in photosynthetic electron transport (16). Redox factors were
also suggested to control chloroplast protein expression in general
(17) and translation elongation of the D1 protein (14).
Here, we present a new effect of light on the general efficiency of
translation elongation in barley chloroplasts, which is superimposed on
a regulation of translation by redox factors or ATP. Our results
suggest that the light stimulation of translation elongation is
dependent on the formation of a proton gradient across the thylakoid
membrane, which arises from photosynthetic electron transport. The
effect is manifested rapidly in the light and is also rapidly
suppressed after transfer to darkness.
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EXPERIMENTAL PROCEDURES |
Plant Growth and Plastid Isolation--
Barley (Hordeum
vulgare L. var. Steffi) seeds were planted in moist vermiculite
and grown for 7 days at 23 °C in a light/dark cycle of 12 h
each. In the light phase of the seventh day, the upper half of the
primary leaves was cut, and plastids were isolated as described
(18).
In Organello Translation and Protein Detection--
Isolated
chloroplasts were pulse labeled for 2.5 min as described (18) with
[35S]methionine and in the presence of 0.5 mM
ATP, if not otherwise stated. Additional reagents were added as
indicated in the figure legends. Chloroplasts were lysed osmotically in
the translation reactions because of the absence of sorbitol. Dark
reactions were performed in a dim green safe light (<10
nE/m2 s), and light reactions were performed in a white
light of 50 µE/m2 s. Far red light was applied to the
samples with a Volpi Intralux 150H cold light source provided with an
interference filter of 730 nm, providing a light intensity of about 5 µE/m2 s to the reactions. Reactions were terminated by
freezing the samples in liquid nitrogen. Samples were separated into
stroma and membrane fractions by centrifugation (3000 × g, 3 min). The stromal fraction was centrifuged again
(20,000 × g, 5 min), and the membranes were washed in
a buffer containing 10 mM Tris-HCl, pH 6.8, 10 mM magnesium acetate, and 20 mM potassium
acetate. Membrane-bound polysomes were isolated as described in Ref.
19. Proteins were prepared for SDS-polyacrylamide gel electrophoresis as described (18).
Chemicals--
[35S]Methionine (Redivue) was
purchased from Amersham & Buchler (Braunschweig, FRG). Ascorbate,
carbonyl cyanide-m-chlorophenylhydrazon (CCCP)1,
3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU), duroquinone, gramicidin, lincomycin, and phenazine methosulfate (PMS) were from
Sigma (Munich); dithiothreitol was purchased from Biomol (Hamburg),
2,6-dichlorophenolindophenol (DCPIP) from Merck (Darmstadt), and ATP
and GTP from Boehringer Mannheim. Plastoquinone-1 was a gift from W. Oettmeier (Bochum) and was reduced with NaBH4 and purified
by extraction with ether. Duroquinone was reduced with NaBH4 according to Ref. 20. Hydrophobic reagents were
dissolved in ethanol; the final concentration of ethanol in the
translation reactions and in the control reactions performed in
parallel was kept below 2%.
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RESULTS |
Light Regulates Translation Elongation in Chloroplasts--
The
regulation of translation elongation was examined by radiolabeling of
lysed barley chloroplasts with [35S]methionine in the
presence of the translation initiation inhibitor lincomycin. When
membrane-bound polysomes were isolated from chloroplasts pulse labeled
for 2.5 to 15 min, radiolabel incorporation into defined polysome-bound
translation intermediates was high after 2.5 min in the light but low
in darkness. Thereafter, radiolabel accumulation in translation
intermediates decreased between 2.5 and 15 min in the light, whereas in
darkness radiolabel increased for 7.5 min and only decreased
thereafter. This indicated that translation elongation was increased in
the light by increased ribosome run off (Fig.
1).

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Fig. 1.
Light increases polysome run off in
chloroplasts. Chloroplasts isolated from 7-day-old barley leaves
were lysed during the translation reaction and were pulse labeled in
the presence of lincomycin with [35S]methionine in
darkness or light for 2.5, 7.5, and 15 min at 25 °C. After labeling,
membrane-bound polysomes were isolated, and proteins immunologically
related to the large subunit of ribulose-1,5-bisphosphate carboxylase
(LSU*) and the reaction center protein D1 (D1*)
were separated by 12.5% SDS-polyacrylamide gel electrophoresis
containing 6 M urea as described under "Experimental
Procedures."
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DCMU Inhibits Light Stimulation of Translation Elongation in
Chloroplasts--
In lysed chloroplasts, radiolabeling of soluble and
membrane proteins was strongly enhanced in the presence of light (Fig. 2; light, lanes 3 and
5; dark, lane 1). The main translation product found in the membrane fraction of 7-day-old barley chloroplasts is the
33-kDa precursor of the D1 protein (pD1) (Fig. 2A). During a
10-min chase with nonradioactive methionine, many protein bands with a
lower molecular weight disappeared, whereas pD1 and mature D1
accumulate (Fig. 2A, lanes 7-10). These lower
molecular weight protein bands can be immunoprecipitated with a
N-terminal D1 antibody, indicating that they correspond to translation
intermediates of the D1 protein. However, the light stimulation of
translation elongation was not specific for the D1 protein, as light
also increased radiolabeling of higher molecular weight proteins in the
membrane and in the stromal fraction (Fig. 2B). Also in the stroma, many bands immunoprecipitated in the presence of an antibody against the large subunit of the ribulose-1,5-bisphosphate carboxylase (LSU) and seem to be translation intermediates, as they disappear during a 10-min chase (Fig. 2B, lanes 7-10).
Hereby, a large proportion of the translation products in the stroma is
shown to correspond to LSU, whereas proteins not elongated during the
chase may correspond to other stromal proteins.

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Fig. 2.
DCMU inhibits light stimulation of
translation elongation. Chloroplasts were labeled with
[35S]methionine as described in the legend to Fig. 1 for
2.5 min at 25 °C. After labeling, reactions were separated into
membrane (A) and soluble (B) phases, and the
proteins were separated on 12.5% polyacrylamide gels containing 6 M urea. Reactions were incubated in the presence
(lanes 1, 2, 5, 6-10) or
absence (lanes 3 and 4) of 25 mM
MgATP. Electron transport was inhibited by addition of DCMU
(3-(3,4-dichlorophenyl)-1,1-dimethyl urea) at a concentration of 10 µM (lanes 2, 4, and 6).
In lanes 7-10, accumulation of full-length protein (pD1,
precursor of the D1; D1, processed precursor D1; and LSU, the large
subunit of ribulose-1,5-bisphosphate carboxylase) is followed in
darkness and light by incubation of chloroplast lysates, pulse labeled
for 2.5 min (lanes 7 and 9) in the presence of 8 mM nonradioactive L-methionine, for 10 min
(lanes 8 and 10).
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The light-dependent stimulation of translation elongation
was strongly inhibited by addition of the electron transport inhibitor DCMU (Fig. 2, lanes 3 and 4). DCMU showed no
effect on translation in darkness, indicating that DCMU did not inhibit
translation elongation in general (Fig. 2, lanes 1 and
2). The inhibitory effect of DCMU in the light was also
observed in the presence of exogenously added ATP (Fig. 2, lanes
5 and 6). Hence, the reduced translation efficiency in
the light was not caused by DCMU-dependent inhibition of
ATP synthesis but by inhibition of the photosynthetic electron
transport.
The influence of light on translation elongation was observed in all
experiments for membrane and stromal proteins. Nevertheless, in the
following figures only radiolabeling of membrane proteins will be shown
to focus the data presented.
Electron Donors Overcome the DCMU-dependent Inhibition
of Translation Elongation--
To test which part of the electron
transport chain is responsible for light stimulation of translation
elongation, we restored electron transport in the presence of DCMU by
addition of selected electron donors. As DCMU inhibits reduction of
plastoquinone (PQ) by photosystem II (PS II), we restored the pool of
reduced plastoquinone, and hereby photosynthetic electron transport, by
addition of reduced duroquinone (DQH2) or plastoquinone-1
(PQH2) (20-22). Both substances were able to release the
inhibition of translation by DCMU in the light but did not stimulate
translation in the dark (Fig. 3,
A and B). These data indicated that light is
required in addition to an electron donor component to overcome the
inhibition of electron transport at the site of PQ reduction. Thus, we
concluded that the pool of reduced plastoquinone is not the signal for
light-dependent activation of translation elongation.

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Fig. 3.
Electron donors overcome the
DCMU-dependent inhibition of translation elongation only in
the light. Chloroplast lysates were pulse labeled for 2.5 min as
described in the legend to Fig. 1. In the dark (lane 1) and
light (lane 4), the influence of reduced electron donors for
the photosynthetic electron transport chain (DQH2, 1 mM, reduced according to Ref. 18; PQH2, 5 mM, reduced with NaBH4, and DCPIP, 200 µM, reduced by 5 mM sodium ascorbate;
A, B, and C, respectively) on
translation reactions was compared with the light control (lane
2). In the light, the release of a translation inhibition by 10 µM DCMU (lanes 3 in A and
B) was investigated by addition of the photosynthetic
electron donors (lanes 4 in A, B, and
C). In D, reactions were incubated with 10 µM PMS for 1 min at 25 °C in the light to allow
reduction of PMS before pulse labeling in the absence (lane
1) or presence (lane 2) of DCMU.
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Although reduction of plastocyanin by reduced PQ can take place in the
dark (23), the amount of oxidized plastocyanin most likely limited
electron flow in the dark, whereas in the light constant electron flow
was possible due to PS I action. Hence, after inhibition of electron
transport by DCMU, translation elongation could be reinduced by
restoring electron transport after PS II because of the action of
DQH2 or PQH2 as electron donors.
Electron Flow between PS II and PS I Is Sufficient for Stimulation
of Translation Elongation--
To further localize the activation of
translation elongation to a specific site of the electron transport
chain, we directly reduced PS I by addition of reduced DCPIP (24).
Reduced DCPIP was able to overcome the translation inhibition by DCMU
in the light to the same extent as DQH2 and
PQH2 (Fig. 3C). Also, PMS, which mediates cyclic
electron flow around PS I (24), was able to overcome the inhibition of
DCMU in the light (Fig. 3D). These data indicated that PS I
activity alone was sufficient for light stimulation of translation.
However, PMS and DCPIP are both known to form a proton gradient during
electron transport by PS I (24, 25). We therefore investigated the
formation of a photosynthetic proton gradient by electron transport
between PS II and PS I by illuminating translation reactions with
monochromatic light of 730 nm, which excites PS I but not PS II (26).
Clearly, a stimulation of translation elongation in far red light
occurred only in the presence of an electron donor (DQH2)
(Fig. 4, lane 4 compared with
lanes 1-3). This demonstrated that electron transport
between PS II and PS I was sufficient for induction of translation,
whereas activation of PS I alone was not. To further see whether the
redox state of ferredoxin, thioredoxin, or NADPH, which are reduced by
PS I during photosynthesis, could trigger induction of translation, the
isolated redox compounds were added to the reactions in their reduced
state. However, none of these substances was able to stimulate translation elongation in the dark (data not shown).

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Fig. 4.
Electron flow between PS II and PS I is
sufficient for stimulation of translation elongation. The
induction of translation elongation of the precursor D1 protein (pD1)
was investigated by pulse labeling of chloroplast lysates in the
presence of 0.5 mM MgATP. Translation reactions were
performed in the dark (lane 1) and light of 730 nm
(lane 3) and after addition of 10 µM reduced
duroquinone (DQH2) to lysates in darkness (lanes 2) and 730 nm light (lane 4).
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Release of the Photosynthetic Proton Gradient Prevents
Light-stimulated Translation Elongation--
Our data presented above
suggested that the light-dependent signal for activation of
translation elongation was not triggered by electron transport or a
single redox component of the chain but by the formation of a proton
gradient across the thylakoid membrane. We therefore investigated the
rate of translation elongation during the release of the photosynthetic
proton gradient by addition of uncouplers of photophosphorylation in
the light and in presence of exogenously added ATP. Addition of the
uncouplers Nigericin and CCCP clearly reduced translation elongation to
the level obtained in darkness (Fig. 5,
open bars). The pore-forming protonophore gramicidin,
however, which inhibits photophosphorylation in thylakoid membranes
already at a concentration of 0.1 µM (27), did not block
light-dependent stimulation of translation elongation even in a concentration of 10 µM (Fig. 5). Obviously,
gramicidin only decreases the photosynthetic proton gradient to a level
not high enough for ATP synthesis but does not abolish it even in high concentration (27). We therefore completely abolished the proton gradient by addition of gramicidin in combination with
NH4Cl (27). Hereby, translation elongation in the light was
reduced to the level obtained in darkness. Hence,
light-dependent formation of a weak proton gradient is
sufficient to activate translation elongation. In parallel, we tested
the influence of uncouplers on electron transport by measuring NADP
reduction in the light (Fig. 5, filled bars). Data clearly
revealed that electron transport was not (nigericin, gramicidin,
gramicidin plus NH4+) or only slightly affected
(CCCP) by the uncouplers, indicating that electron transport components
are not involved in regulation of translation elongation.

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Fig. 5.
Uncouplers prevent light stimulation of
translation. Chloroplasts were labeled with
[35S]methionine as described in the legend to Fig. 2.
After separation of the proteins by SDS-polyacrylamide gel
electrophoresis, radioactivity in the pD1 band was quantitated. For
measuring NADP reduction, chloroplast lysates (0.9 × 108) were incubated in 500 µl of translation buffer with
NADP (0.5 mM) for 25 min at 25 °C, and the formation of
NADPH was measured as the difference of absorbance at 338 nm before and
after incubation. Reactions were performed in darkness, in light, or in
light in the presence of nigericin (2 µM), CCCP (10 µM), gramicidin (10 µM), or gramicidin and
NH4Cl (1 mM). Values obtained for
35S incorporation (white bars) or NADP reduction
(shaded bars) from the light reactions were set as 100%
level.
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Light Induction of Translation Elongation Is Rapidly Switched off
upon Transfer of Chloroplasts to Darkness--
The photosynthetic
proton gradient is rapidly released upon transfer of cells from light
to darkness (29). However, a proton gradient-induced signal could
sustain a high rate of translation elongation for some time in
darkness, if it was sufficiently long-lived. We therefore
preilluminated plastids for 1 min on ice to stabilize the putative
light signal and compared the activation of translation with
preillumination at 25 °C. After preillumination,
[35S]methionine and lincomycin were added, and
translation reactions were performed in darkness or light.
Preillumination had no stimulating effect on translation in the dark,
regardless of the temperature in which the preillumination was
performed (Fig. 6). This indicated that
the putative proton gradient-induced signal was not long-lived but was
lost upon transfer of chloroplasts to darkness. We conclude that the
light-induced formation of the photosynthetic proton gradient serves a
dual function for induction of translation elongation. First, a
short-lived signal may be induced to increase the rate of translation
elongation; second, ATP formation is induced after full development of
the proton gradient to sustain the translation elongation process in
general.

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Fig. 6.
Light induction of translation elongation is
rapidly switched off upon transfer of chloroplasts to darkness.
Chloroplast lysates were preilluminated for 1 min on ice (0 °C)
(lanes 1 and 2) or at 25 °C (lanes
3 and 4) before pulse labeling of the precursor form of
the reaction center protein D1 (pD1) was determined in darkness (D) or
light (L) in presence of 0.5 mM MgATP.
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DISCUSSION |
Regulation of Translation Elongation by Light--
Most studies on
posttranscriptional regulation of gene expression in chloroplasts have
concentrated on gene-specific regulation mechanisms (3-5, 7-11,
14-16). Here, we describe a light-dependent mechanism that
is not gene specific but enhances radiolabel accumulation in at least
two proteins localized in the chloroplast stroma and membrane fraction.
Light seems to directly activate radiolabeling by a factor involved in
the enzymatic process of chain elongation, e.g. a ribosomal
component, one of the chloroplast elongation factors, or chloroplast
tRNA-aminoacyl synthases. Alternatively, light-dependent
structural changes at the level of the mRNA or during folding of
the nascent protein chain could indirectly affect the rate of chain
elongation.
It has been observed earlier that inhibitors of photosynthetic electron
flow reduce light-dependent protein expression in chloroplasts (13, 14). Light activation of translation was attributed
merely to the light-dependent increase in the ATP level, and inhibition of translation by inhibitors of photosynthetic electron
flow was explained by inhibition of the light-dependent ATP
synthesis. Our experiments, however, show that the presence of ATP is
only one of the regulating factors, as addition of exogenous ATP was
not sufficient for full activation of translation in the dark or for
restoration of translation in the presence of DCMU or uncouplers.
Kuroda et al. (14) reported an accumulation of translation
intermediates of the D1 protein in the dark, which could not be
overcome by ATP alone, and proposed the involvement of a
light-dependent redox component for activation of
translation, as dithiothreitol partly released the translational block.
However, our experiments were performed in the presence of
dithiothreitol and still revealed a light-dependent
regulation of translation elongation that is superimposed on possible
redox control mechanisms operating on the level of translation
elongation or initiation.
Control mechanisms on initiation level, as proposed for the psbA gene
by several groups (5, 7, 8), may still exist beside the regulation of
translation elongation described here and may be important for the
developmental control of photosystem accumulation, i.e. the
onset of translation after prolonged etiolation or energy depletion in
darkness. The regulation of D1 accumulation by translation elongation
instead of initiation could be necessary to inhibit singlet oxygen
formation by immediate binding of "free" chlorophyll to nascent
apoprotein sites, even in darkness (28).
Regulation of Translation Elongation by the Proton
Gradient--
In the light, translation elongation was only activated
by redox substances that allow the formation of a proton gradient, either by release of protons in the thylakoid lumen (PMS, DCPIP) or by
allowing proton transport via the cytochrome b6/f complex (DQH2, PQH2). In contrast, addition of the same
substances in the dark, or addition of redox substances that do not
promote proton transport, like thioredoxin and ferredoxin, could not
activate translation elongation (Fig.
7A). Furthermore, we show that
a lower proton threshold level is required for the induction of translation elongation than for synthesis of ATP. Hence, the
photosynthetic proton gradient, which is formed and released within
seconds, is used at a very sensitive setting to monitor photosynthetic electron transport and therefore light (29). Such a regulatory mechanism seems useful, as the highly energy-consuming process of
translation will be immediately retarded upon light-dark transfer of
the plant, before the level of ATP is decreased within the organell. A
further indication that the proton gradient in chloroplasts may be used
as a light sensor is its requirement for the
light-dependent translocation of several nuclear-encoded
proteins across the thylakoid membrane (30). Although it is unknown how
the proton gradient signals activation of translation elongation and
membrane translocation, these data indicate that the formation of a
proton gradient is a prerequisite for the assembly of several
chloroplast- and nuclear-encoded proteins in the thylakoid
membrane.

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Fig. 7.
Working model for regulation of translation
elongation by the photosynthetic proton gradient. In the light,
proton gradient formation can be blocked by DCMU
(3-(3,4-dichlorophenyl)-1,1-dimethylurea)-dependent
inhibition of electron transport (black line) between
H2O and NADP+ and can be reactivated by DQ
(duroquinone)-dependent reduction of PQ- or phenazine
methosulfate-dependent (PMS) electron plus
proton transport around PS I (A). Reduced DCPIP (2, 6-dichlorophenolindophenol) serves as a reductant to PS I, and
carbonylcyanide-m-chlorophenylhydrazon (CCCP) releases the
proton gradient over the thylakoid membrane (A). The
translation elongation rate of the psbA mRNA may be regulated by a
proton-sensitive thylakoid membrane component (X), binding the
chloroplast ribosome (R) and the D1 nascent chain (B). In
the light, a high proton concentration in the thylakoid lumen will
structurally alter the configuration of factor X. Hereby, the light
signal will be transmitted from the thylakoid lumen to the
membrane-bound polysomes on the stromal side of the membrane, and the
rate of translation elongation will be increased. In darkness, and upon
proton release in the light, factor X will be reset and translation
elongation will be decreased (B).
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Most interestingly, the main stromal component activated by the
light-induced proton gradient, LSU, was found to be translated on
membrane-bound polysomes (31, 32); this could also be the case for
other stromal proteins. Therefore, we suggest the following model for
the general activation of translation elongation by a proton gradient
(Fig. 7B). The rate of translation elongation is coupled to
the pH gradient by a thylakoid membrane component involved in the
binding of ribosomes or in the enzymatic control of translation
elongation activity. An increase in the luminal pH levels during
illumination alters the conformation of a membrane component leading to
increased chain elongation. The very rapid switch-off observed in our
experiments would favor such a direct mechanism. Also, no indications
for the requirement of signal transduction steps like protein
phosphorylation have been found in our
system.2