(Received for publication, July 13, 1995; and in revised form, August 8, 1995)
From the
In the green alga Chlamydomonas reinhardtii, the copper-dependent accumulation of plastocyanin is effected via the altered stability of the protein in copper-deficient versus copper-sufficient medium (t) < 20 min versus several hours). To understand the mechanism of plastocyanin degradation in vivo, the purified apoprotein was characterized relative to the holoprotein with respect to conformation and protease susceptibility. Circular dichroism spectroscopy revealed that the apoprotein in solution did not display the characteristic secondary structure displayed by the native or reconstituted holoprotein. The apoprotein was also susceptible to digestion in vitro by chymotrypsin whereas the holoprotein was resistant. High ionic conditions, which stabilize the folded structure of apoplastocyanin, also inhibit its degradation by chymotrypsin. These results suggest that one explanation for plastocyanin degradation in copper-deficient cells in vivo might be the increased susceptibility of the apo form to a lumenal protease. Since apoplastocyanin is a normal biosynthetic intermediate for the formation of holoplastocyanin, the increased susceptibility of apoplastocyanin to proteolysis implies that degradative and biosynthetic activities would compete for the same substrate. However, characterization of an apoplastocyanin-accumulating mutant suggests that a plastocyanin-degrading protease is active only in copper-deficient cells. Thus, apoplastocyanin is rapidly degraded in copper-deficient cells, whereas its major fate in copper-supplemented cells is holoplastocyanin formation.
Plastocyanin is a small (97-104 amino acids),
lumen-localized, copper-binding protein that functions in
photosynthesis to catalyze electron transfer from cytochrome f of the membrane-associated cyt ()b
f complex to P700
in Photosystem I, and
in respiration (in cyanobacteria) to catalyze electron transfer from
the cytochrome b
f complex to the
terminal oxidase (reviewed recently by Morand et al., 1994;
Redinbo et al., 1994). It contains a single redox active
copper (E
370 mV) and is referred to
as a ``blue'' copper protein owing to the absorption
properties of the oxidized form of the protein. The catalytic activity
of plastocyanin is, of course, dependent on this copper center.
Accordingly, copper-deficient plants or chelator-treated thylakoid
membranes display loss of plastocyanin activity and impaired
photosynthesis.
In the case of green algae and cyanobacteria,
however, some species have the capacity to survive copper deficiency by
inducing a soluble c-type cytochrome to serve as a functional backup
for plastocyanin (Wood, 1978; Sandmann et al., 1983; reviewed
by Merchant, 1995). In these organisms, if copper is available in
amounts sufficient to satisfy the plastocyanin biosynthetic pathway,
this copper protein accumulates and is used for photosynthetic electron
transfer. Generally, cyt c is not detected in such
cultures. However, under conditions of copper deficiency (which would
limit or prevent the synthesis of a functional form of plastocyanin),
green algae and cyanobacteria remain photosynthetically competent by
inducing the synthesis of heme-containing cyt c
.
These organisms thus serve as excellent model systems for the study of
the copper-dependent synthesis of plastocyanin.
Regulation of plastocyanin accumulation has been examined in Chlamydomonas reinhardtii, Scenedesmus obliquus, and Pediastrum boryanum among the green algae, and Anabaena and Synechocystis spp. among the cyanobacteria (Merchant and Bogorad, 1986a, 1986b; van der Plas et al., 1989; Briggs et al., 1990; Bovy et al., 1992; Li and Merchant, 1992; Nakamura et al., 1992; Zhang et al., 1992; Ghassemian et al., 1994). Examination of mRNA and protein abundance as a function of copper concentration provides evidence for copper-responsive regulation at two stages in plastocyanin biosynthesis: 1) at the level of template accumulation (mRNA abundance) and 2) at the level of accumulation of mature protein. The relative contribution of processes regulating mRNA abundance versus protein abundance differs in various organisms and can depend upon the growth phase of the culture (reviewed by Merchant, 1996). In general, when both levels of control are displayed, lower concentrations of copper are required to induce the accumulation of plastocyanin-encoding mRNAs than are required for the accumulation of the protein. For the latter, the requirement for copper appears to be stoichiometric (Merchant et al., 1991). This suggests that the binding of copper to the polypeptide might be an important factor in determining the steady state abundance of the protein.
The mechanisms underlying differential accumulation of the protein have been investigated further in Chlamydomonas by pulse-radiolabeling experiments. The results indicate that translation of the message as well as import and processing of the translation product occur normally and regardless of whether copper is present, which is consistent with a model in which association of copper with apoplastocyanin occurs after translocation of the protein into the thylakoid lumen (Merchant and Bogorad, 1986b; Li et al., 1990). However, in copper-deficient cells, mature plastocyanin is degraded (t < 20 min), whereas in copper-supplemented cells, the newly synthesized protein is extremely stable (t > several hours). If the apoform of the protein were a better substrate for proteolysis than the holoform, the copper-dependent differential degradation of plastocyanin could be explained. In copper-deficient cells, the newly synthesized protein would remain in the protease-susceptible apoform, whereas in copper-supplemented cells, it would convert to the protease-resistant holoform. Crystallographic studies reveal only minimal structural changes between apoplastocyanin and holoplastocyanin (Garrett et al., 1984); however, in solution, the apoprotein was noted to be much less stable (Koide et al., 1993). Thus, it is reasonable to suggest that it might indeed be more susceptible to one or more chloroplast proteases in vivo compared to the holoprotein, whose conformation is metal-stabilized (McLendon and Radany, 1978; Parsell and Sauer, 1989; Koide et al., 1993).
There are many well-documented examples of specific degradation of proteins in chloroplasts. In Chlamydomonas, the small subunit of Rubisco is selectively degraded if it cannot assemble with large subunits to form the multimeric holoenzyme (Schmidt and Mishkind, 1983). Likewise, in rye, the nuclear-encoded subunits of the chloroplast coupling factor are degraded in the absence of synthesis of the chloroplast-encoded subunits (Biekmann and Feierabend, 1985). In the case of chlorophyll-protein complexes, the apoproteins are degraded under conditions where chlorophyll synthesis is reduced (e.g. Bennett, 1981; Slovin and Tobin, 1982; Kim et al., 1994). And, for the electron transfer complexes, the pleiotropic effects of mutations affecting a single subunit of the photosynthetic complexes have been shown, in many cases, to result from the specific degradation of the remaining subunits of the affected complex while other (unaffected) complexes accumulate normally (Erickson et al., 1986; Kuchka et al., 1989; Pakrasi et al., 1991; Takahashi et al., 1991; Kuras and Wollman, 1994). Distinct protease activities have been localized to chloroplasts, but their relationships to specific degradation processes have not yet been fully described (Liu and Jagendorf, 1984; Kuwabara and Hashimoto, 1990; Hoober and Hughes, 1992; Bushnell et al., 1993). Questions of interest with regard to proteolytic events in the chloroplast relate to location, regulation, substrate recognition, and substrate specificity. In the case of plastocyanin, the degradative activity is presumed to be lumen-localized, or at least thylakoid membrane-associated, since the fully processed form of the protein serves as the substrate for degradation.
We sought to compare the properties of purified apo- and holoplastocyanin with respect to protease susceptibility in vitro and in vivo with a view to increasing our understanding of 1) proteolytic processes in chloroplasts and 2) the mechanism of adaptation of the photosynthetic apparatus to copper-deficiency. The data presented in this work support a model in which apoplastocyanin is relatively unstable in solution. We also show that apoplastocyanin is highly susceptible in vitro to proteases. Nevertheless, the increased susceptibility of apoplastocyanin to proteolysis may not be sufficient to explain the regulation of its degradation in vivo. We propose that a specific mechanism exists for activating an apoplastocyanin-degrading protease in copper-deficient cells and that this additional factor is an important determinant of the rate of apoplastocyanin degradation in vivo.
Figure 1:
Difference absorption
spectra of apo-, holo-, or reconstituted plastocyanin. Purified
plastocyanin (B), apoplastocyanin (A), and
reconstituted plastocyanin (C) at 60 µM in 25
mM Tris-Cl (pH 8.05) were used for the measurements. In order
to estimate the amount of copper-containing plastocyanin in different
samples, 10 µl of 100 mM ascorbic acid was added to 1 ml
of apo-, holo-, or reconstituted plastocyanin (prepared as under
``Experimental Procedures''), and the spectrum was recorded
as the reduced spectrum. A spectrum from the same sample to which was
added 10 µl of 100 mM potassium ferricyanide was measured
and recorded as the oxidized spectrum. The difference spectrum was
obtained by subtraction of the reduced spectrum from the oxidized
spectrum. The measurements were made in a 1-ml quartz cuvette (1-cm
pathlength). The peak at 597 nm is characteristic of copper-containing
plastocyanin. The amount of holoplastocyanin in each sample was
estimated from the A at this wavelength using a
of 49 mM
cm
.
In this particular experiment, the yield of apoplastocyanin was 84%,
and 97% of the apoplastocyanin was reconstituted to holoplastocyanin.
Thus, the total copper-containing plastocyanin in the reconstituted
sample was 97.6%.
Figure 6:
Synthesis of mature plastocyanin in
strain pc235. C. reinhardtii strain pc235 was cultured in
copper-deficient medium (-Cu) or supplemented with
copper chloride (+Cu) prior to radiolabeling with
NaSO
for 15 min. The cells were
sampled after a 100-fold dilution of the radiolabel by the addition of
unlabeled Na
SO
to 10 mM, and after
addition of copper chloride to the copper-deficient culture to 6
µM. Label incorporation into total soluble proteins in
equivalent volumes of cell extract are displayed in the top
panel. Plastocyanin was quantitatively immunoprecipitated from
equal volumes of soluble cell extract and analyzed by electrophoresis
through a (12%) polyacrylamide gel under denaturing conditions (bottom panel).
The structure of the native protein in the crystal form revealed
that it is an eight-stranded, anti-parallel -sandwich (Colman et al., 1978; Guss and Freeman, 1983). Removal of the copper
from the crystal did not affect the structure very significantly, and
it was concluded that copper was not required for the folding of the
polypeptide into its native structure (Garrett et al., 1984).
Nevertheless, in vitro folding studies revealed that although
apoplastocyanin was capable of folding in the absence of copper, the
folded structure was not stable in solution in the absence of high
concentrations of salt (Koide et al., 1993). Our preparation
of apoplastocyanin was therefore characterized by CD spectroscopy for
the diagnostic
-strand spectrum (Draheim et al., 1986) (Fig. 2). The spectra of the native holoprotein and the
reconstituted holoprotein were very similar. In fact, the estimated
secondary structure of the reconstituted holoprotein was not
significantly different from the secondary structure of the native
protein (Table 1). However, the spectrum of the apoprotein is
clearly altered. Specifically, the apoprotein has significantly reduced
-sheet content, which suggests that it does not display
substantial native structure under these conditions. The fact that the
apoprotein could be reconstituted with copper to yield a holoprotein
that displayed the characteristic spectroscopic properties ( Fig. 1and Fig. 2) indicates that our preparation was
indeed capable of adopting the native structure and was not
irreversibly denatured.
Figure 2: Far UV CD spectra of apo-, holo-, and reconstituted plastocyanin. The plastocyanin content of each sample was the same (0.56 mg/ml). Each sample was in a solution containing 25 mM Tris-Cl (pH 8.05) in a cuvette with a pathlength of 0.1 mm. All measurements were made at room temperature. Thin solid line, apoplastocyanin; thick solid line, holoplastocyanin; dashed line, reconstituted plastocyanin.
Analysis of apoplastocyanin preparations by electrophoresis under non-denaturing conditions generally revealed three species. In earlier work, we had identified the three species as apoplastocyanin, a disulfide-linked dimer of apoplastocyanin, and residual holoplastocyanin (Li and Merchant, 1992). Similar analysis of spinach apoplastocyanin revealed the same population of species (Li et al., 1990). Bacterial amicyanin also readily dimerizes once the copper is removed from the holoprotein (Kumar and Davidson, 1992). To demonstrate more convincingly that the species identified as apoplastocyanin indeed contained a reactive accessible thiol group (as opposed to holoplastocyanin where the single cysteine provides a ligand to the copper, or the dimer where the cysteine participates in the disulfide bond), the preparation was treated with a fluorescent maleimide (Fig. 3). As expected, only the band corresponding to apoplastocyanin was found to react with the thiol reagent.
Figure 3: Accessibility of the cysteinyl thiol in apo- versus holoplastocyanin to reaction with maleimidylphenyl methylcoumarin. Apo- and holoplastocyanin were treated with a thiol-reactive reagent, maleimidylphenyl methylcoumarin, for 60 min. Equivalent amounts of apo- and holoplastocyanin were analyzed as follows after separation by electrophoresis through a 15% polyacrylamide gel under either non-denaturing (A) or denaturing (B) conditions: under UV-illumination to detect the fluorescent reagent (lanes 3, 4, 7, and 8) or by staining with Coomassie Blue R-250 to detect protein (lanes 1, 2, 5, and 6). The arrows on the left point to apoplastocyanin (a), disulfide-linked dimer of apoplastocyanin (d), holoplastocyanin (h). The arrows and the numbers on the right indicate the size of the molecular weight markers separated on the SDS-containing denaturing gel.
Figure 4:
Protease susceptibility of apo- versus holoplastocyanin. 10 µg each of apo- or holoplastocyanin was
incubated with 0.02 units of chymotrypsin in 100 mM Tricine
(pH 8.0) in a total volume of 30 µl for the indicated amounts of
time. The reaction was terminated by the addition of
phenylmethylsulfonyl fluoride to >100 µg/ml. Portions of the
products (about 200 ng each lane) were loaded onto alternate lanes of a
12% polyacrylamide gel for electrophoresis under non-denaturing
conditions (Davis, 1964), which separates apo- and holoplastocyanin (Li
and Merchant, 1992). The residual protein was visualized by staining
with a silver reagent (Oakley et al., 1980). In panel
B, 0.5 M (NH)
SO
was
added to the samples before the incubation with protease. The arrows point to the positions of migration of apo- and
holoplastocyanin. The products of digestion are probably too small
(<15 amino acid residues) to be retained on the gel. In the absence
of added protease, apoplastocyanin is not
degraded.
It is not surprising that high salt (which stabilizes the native conformation) only delays rather than prevents degradation. If the kinetic barrier to unfolding is small and the two species (i.e. protease-resistant and protease-susceptible) are in equilibrium (see Koide et al., 1993), the degradation of one form would eventually lead to the degradation of the entire population of molecules. Thus, the fact that the apoprotein can adopt a native structure in the absence of the cofactor does not preclude its recognition as a non-native structure by a protease. The identity of the protease in vivo is not known, but the location of the substrate demands a proteolytic mechanism for plastocyanin degradation in the thylakoid lumen.
The characteristics of a weakly non-photosynthetic mutant strain of C. reinhardtii, pc235 (see ``Experimental Procedures''), permitted us to distinguish between these possibilities. This strain encodes plastocyanin with a wild-type primary sequence but has reduced plastocyanin function owing to decreased abundance of holoplastocyanin relative to a wild-type strain (Fig. 5, compare intensity of bands marked h). In fact, apoplastocyanin accumulates at the expense of holoplastocyanin in copper-supplemented cells of strain pc235. We conclude that holoprotein formation is either inhibited in this strain or that the holoprotein, once formed, is not stable. In either case, the strain provided us with an experimental background where holoprotein formation is decoupled from copper supplementation of the medium. The strain was tested for its ability to synthesize plastocyanin during a brief 15 min labeling period (Fig. 6). Label incorporation into various soluble proteins (top panel) or into plastocyanin (bottom panel) was essentially the same in copper-supplemented or copper-deficient conditions. Thus, we conclude that strain pc235 synthesizes plastocyanin. Further, the immunoprecipitated species co-migrates on SDS-containing polyacrylamide gels with mature plastocyanin synthesized in wild-type cells. The accumulation of apoplastocyanin in copper-supplemented cells of the mutant strain therefore suggests regulated proteolysis of apoplastocyanin in the thylakoid lumen. Apoplastocyanin accumulation in pc235 cannot be attributed to a specific protease defect because apoplastocyanin fails to accumulate in copper-deficient medium (Fig. 5) despite the fact that the mutant is capable of synthesizing plastocyanin under these conditions (Fig. 6). Thus, we conclude that a protease-susceptible conformation may be a prerequisite for apoplastocyanin degradation, but it is not sufficient in vivo.
Figure 5:
Differential accumulation of
apoplastocyanin under copper-supplemented versus copper-deficient conditions. C. reinhardtii strain CC-125 (WT) and plastocyanin-deficient strain (pc235) were cultured in copper-supplemented (+) or
copper-deficient(-) conditions as described under
``Experimental Procedures.'' Cells (usually 100-ml cultures)
were collected at stationary phase (about 1 10
cells/ml for the wild-type strain and 5
10
cells/ml for strain pc235) and resuspended in a minimal volume
(
200 µl) of a solution containing 10 mM sodium
phosphate (pH 7.0). The concentrated cells (equivalent to
1-2 mg chlorophyll/ml) were lysed by two freeze-thaw cycles
(freeze at -80 °C and thaw at room temperature). The
supernatant, collected after centrifugation (15,850
g)
at 4 °C, was identified as the soluble cell extract and separated
by electrophoresis through a 15% polyacrylamide gel under
non-denaturing condition (Li and Merchant, 1992). Total soluble protein
equivalent to 1.0 OD
in a 1-ml protein assay was loaded
onto each lane. After electrophoresis, the proteins were transferred
onto a polyvinylidene difluoride membrane which was subsequently
decorated with plastocyanin-specific antibodies. The arrows indicate apoplastocyanin (a), dimer of apoplastocyanin (d), and holoplastocyanin (h). Differences in the
abundance of various species of plastocyanin do not result from
differential release of the protein from the mutant versus the
wild-type cells, or from +copper versus -copper
cells, during preparation of cell extracts.
At the present time, the identity of this protease is not known. Various types of proteases have been identified in chloroplasts including metalloproteases and ATP-dependent proteases (Liu and Jagendorf, 1986; Gray et al., 1990; Hoober and Hughes, 1992; Bushnell et al., 1993). Although we have noted an ATP-dependent protease activity in chloroplast extracts that recognizes apoplastocyanin preferentially over holoplastocyanin as a substrate (data not shown), the absence of ATP in the thylakoid lumen (see Cline et al., 1992) makes it unlikely that this activity is the putative lumenal protease responsible for apoplastocyanin degradation. Our identification of the substrate for this protease (apoplastocyanin) and our ability to prepare reasonable amounts of radiolabeled substrate now opens the door for the assay and identification of a lumenal protease and the copper-responsive system that might regulate it. Toward this end, we have also characterized preparations of apoplastocyanin by electrophoretic separation under non-denaturing conditions. Independently (e.g. Li et al., 1990), a similar analytical method had indicated that preparations of apoplastocyanin contained multiple species that differed with respect to electrophoretic mobility. In this work, we have identified the species corresponding to apoplastocyanin on the basis of the chemical reactivity of the cysteinyl thiol (Fig. 3), while in previous work we identified the species corresponding to cysteine-linked apoplastocyanin dimers (Li and Merchant, 1992).