Dec 14, 1994 - 4. Synthesis of the 35 kDa protein is inhibited at a copper concentration ..... the GSAT-encoding RNA does increase in response to illumination ...
The EMBO Journal vol.14 no.5 pp.857-865, 1995
Coordinate expression of coproporphyrinogen oxidase and cytochrome c6 in the green alga Chiamydomonas reinhardtii in response to changes
Kent L.Hill and Sabeeha Merchant1 Department of Chemistry and Biochemistry, University of California Los Angeles, 405 Hilgard Avenue, Los Angeles, CA 90024-1569,
'Corresponding author Communicated by D.Meyer
To maintain photosynthetic competence under copperdeficient conditions, the green alga Chlamydomonas reinhardfii substitutes a heme protein (cytochrome c6) for an otherwise essential copper protein, viz. plastocyanin. Here, we report that the gene encoding coproporphyrinogen oxidase, an enzyme in the heme biosynthetic pathway, is coordinately expressed with cytochrome c6 in response to changes in copper availability. We have purified coproporphyrinogen oxidase from copper-deficient C.reinhardtii cells, and have cloned a cDNA fragment which encodes it. Northern hybridization analysis confirmed that the protein is nuclear-encoded and that, like cytochrome c6, its expression is regulated by copper at the level of mRNA accumulation. The copper-responsive expression of coproporphyrinogen oxidase parallels cytochrome c6 expression exactly. Specifically, the copper-sensing range and metal selectivity of the regulatory components, as well as the time course of the responses, identical. Hence, we propose that the expression of these two proteins is controlled by the same metalloregulatory mechanism. Our findings represent a novel metalloregulatory response in which the synthesis of one redox cofactor (heme) is controlled by the availability of another (Cu). Key words: copper/coprogen/cytochrome c6heme/metal are
Introduction Owing to their diverse chemical reactivities, numerous metals are known to have specific biochemical functions and are nutritionally essential for life (Frieden, 1985). However, as a result of their chemical reactivity, many metals are also deleterious to biological systems. Cu and Fe are examples of metals that are both essential for life but toxic at higher concentrations. Their metabolism presents a challenging regulatory problem, since an organism has to maintain appropriate levels of biochemical catalysts (metalloproteins) and a relatively constant intracellular utilization pool, despite variation in the supply of the metal nutrient and the potential for toxicity. The most intensively studied metal-regulated gene systems in eukaryotes include (i) the metallothionein gene family, which functions to protect mammalian and fungal cells against heavy metal toxicity (Hamer, 1986; Bremner and © Oxford University Press
Beattie, 1990; Thiele, 1992), and (ii) the transferrin receptor and ferritin system, which functions to regulate the uptake and storage of iron in vertebrates (Theil, 1990; Klausner et al., 1993; Melefors and Hentze, 1993). Their function in metal homeostasis is to control the availability of essential metals and to provide a mechanism for storage or detoxification when they are in excess. Another important aspect of this problem is metal ion control of the synthesis of catalytic metalloproteins (e.g. redox enzymes/electron transfer proteins). The best documented examples are those involving the metal-dependent synthesis of alternate metalloproteins, where the choice between synthesis and accumulation of one or the other protein depends upon the availability of the metal cofactor. For instance, in some photosynthetic organisms, electron transfer from the cytochrome b - f complex to photosystem I can be catalyzed by either a copper protein (plastocyanin) in copper-replete growth conditions or a heme protein (c-type cytochrome) in copper-deficient conditions (Wood, 1978; Sandmann et al., 1983). Likewise, in some nitrogenfixing bacteria, component I of dinitrogenase may be an Fe-Mo protein if molybdenum is available in the growth environment, a V -Fe protein under molybdenum-deficient growth conditions or a third class of enzyme if neither molybdenum nor vanadium is available (Bishop et al., 1980; Pau, 1989). In cyanobacteria, iron deficiency induces flavodoxins that substitute for ferredoxins in numerous reductive pathways (Laudenbach et al., 1988). In studies of nutritionally induced copper deficiency in fungi it was noted that copper-zinc superoxide dismutase levels were depressed, whereas manganese-containing superoxide dismutase levels rose to compensate (Shatzman and Kosman, 1978). More recently, with the availability of molecular tools (nucleic acid and antibody probes) to study specific gene expression, additional examples of trace elementdependent synthesis of metalloproteins have been noted (Kim and Maier, 1990; Gralla et al., 1991; Privalle and Fridovich, 1993). The regulation of gene expression by catalytic cofactors and the differential cofactor-dependent utilization of alternate 'back up' pathways thus appear to be widespread and common occurrences. Owing to its early discovery (Wood, 1978) and its involvement in an essential biochemical pathway, the copper-dependent reciprocal accumulation of plastocyanin and cytochrome c6 in photosynthesis is a very welldefined example of the above class of metalloregulated systems. Plastocyanin, a type I 'blue' copper protein which catalyzes electron transfer between the cytochrome b-f complex and the reaction center of photosystem I, is the most abundant copper protein in plant leaves and hence a major contributor to a plant's nutritional requirement for copper (Boulter et al., 1977). Terrestrial plants grown in copper-deficient habitats (either naturally or in the laboratory) contain reduced levels of functional plastocyanin 857
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and show symptoms of copper deficiency. Aquatic algae and cyanobacteria, on the other hand, are photosynthetically competent under similar nutrient conditions owing to functional replacement of plastocyanin by a heme containing, soluble c-type cytochrome-cytochrome c6 (Merchant and Bogorad, 1986a, 1987a; Briggs et al., 1990; Nakamura et al., 1992; Zhang et al., 1992). Both plastocyanin and cytochrome c6 are efficient in photosynthetic electron transfer, and whether an organism uses one or the other is determined solely by the availability of copper in the surrounding medium. If the amount of available copper is sufficient to support plastocyanin formation at the stoichiometry required for photosynthesis (about two molecules per reaction center molecule), plastocyanin appears to be the preferred choice. However, if the concentration of copper is too low, cytochrome c6 synthesis is induced. The regulation of plastocyanin accumulation has been examined in various species including Chlamydomonas reinhardtii, Scenedesmus obliquus and Pediastrum boryanum among the green algae, and Anabaena and Synechocystis spp. among the cyanobacteria (Merchant and Bogorad, 1986a,b; 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). The examination of mRNA and protein abundance as a function of copper concentration provides evidence for copper-responsive regulation at two stages in plastocyanin biosynthesis: (i) at the level of template accumulation (mRNA abundance) and (ii) at the level of accumulation of mature protein (reviewed in Merchant, 1995). For cytochrome c6, copper-responsive regulation of its biosynthesis appears to be mediated at the level of mRNA accumulation in all organisms where it has been studied (Merchant and Bogorad, 1986b, 1987a; Bovy et al., 1992; Nakamura et al., 1992; Zhang et al., 1992; Ghassemian et al., 1994). In two cases, Chlamydomonas and Anabaena, further investigation indicates that this is attributable largely to regulation at the level of transcription, with little or no contribution from post-transcriptional processes affecting mRNA stability (Hill et al., 1991; Merchant et al., 1991; Bovy et al., 1992). With respect to the 'signal', the evidence argues most strongly for direct sensation of copper in both prokaryotes and eukaryotes (Merchant and Bogorad, 1987b; Zhang et al., 1994). Specifically, cytochrome c6 synthesis continues to be regulated by copper in plastocyanin-minus mutant variants of responsive strains. In other words, the mere absence of the plastocyanin molecule or the loss of its function does not induce cytochrome c6 synthesis. This suggests that the primary factor involved in cytochrome c6 accumulation is copper ion availability. For most organisms, the response range is in the low nM to sub-,iM (Bohner et al., 1980a,b; Sandmann, 1986; Bovy et al., 1992; Nakamura et al., 1992; Zhang et al., 1992; Ghassemian et al., 1994). This concentration range is consistent with a model in which the regulatory pathway represents an adaptation to copper deficiency. The absolute concentration of medium copper appears to be less meaningful than copper availability measured on a per cell basis (Merchant et al., 1991). In the case of Chlamydomonas, the maintenance of plastocyanin levels requires copper availability in the medium to be -9X 106/cell.
29 kD >
Fig. 1. Synthesis of the 35 kDa protein is inhibited by Cu2+. C.reinhardtii cultures (7X 106 cells/ml), maintained in TAP medium with (+) or without (-) 6 jM Cu2+, were labeled with [35S]Na2SO4 (25 jCi/ml) for 10 min. The radiolabel was diluted with a 100-fold excess of cold Na2SO4, and the cultures were chilled on ice for 10 min prior to the preparation of soluble proteins (Merchant et al., 1991). Extracts containing soluble proteins were analyzed by fluorography following SDS-PAGE. The arrowheads mark the position of migration of molecular weight standards, and the arrow indicates the 35 kDa protein whose synthesis is inhibited by Cu .
In addition to plastocyanin and cytochrome c6, there be other processes that are regulated by copper ion availability and that may be relevant to the adaptation to copper deficiency. For Chlamydomonas, these include differential degradation of plastocyanin (Merchant and Bogorad, 1986a; H.H.Li and S.Merchant, unpublished results), induction of a high-affinity copper uptake pathway (Howe and Merchant, 1992b; Hill, 1994) and the synthesis of a 35 kDa protein (Merchant and Bogorad, 1987a; Hill and Quinn, 1993). Here, we have described the further characterization of the 35 kDa protein and its function during the adaptation of C. reinhardtii to copper deficiency with the long-term goal of providing a more complete description of trace element metabolism under conditions of deficiency. appear to
Results A 35 kDa protein is induced in copper-deficient cells To increase our understanding of copper-responsive gene expression and the mechanism by which organisms adapt to copper deficiency, we sought to identify proteins (in addition to plastocyanin and cytochrome c6) whose abundance is influenced by copper availability. The analysis of soluble proteins of C.reinhardtii after separation by 2-D gel electrophoresis indicates that there are several proteins whose abundance is changed in copper-supplemented versus copper-deficient medium (data not shown). Of these, we chose to pursue the further characterization of the 35 kDa protein, because its response to copper was
Copper-responsive expression of coprogen oxidase
0.1 - +
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43 kD P
Fig. 2. Time course of Cu-dependent inhibition of synthesis of the 35 kDa protein. The experiment was initiated at time 0, when one of two identical cultures (200 ml) received 6 ,M CuS04. The other culture (-) remained Cu-deficient. After the indicated time period, an aliquot (25 ml) was removed from each culture and labeled for 10 min with [35S]Na2SO4, as described in Materials and methods. The extracted proteins were separated by SDS-PAGE and visualized via fluorography. The arrow indicates the position of migration of the 35 kDa protein.
the most pronounced and it appeared to be abundant. The increased abundance of the 35 kDa protein was attributed (in part at least) to de novo synthesis. Pulse radiolabeling experiments demonstrated that more label is incorporated into the 35 kDa protein in -Cu versus +Cu cells (Figure 1). To test whether the expression of the 35 kDa protein might be regulated by the same pathway that regulates cytochrome c6 expression, the physiological characteristics of the expression of the 35 kDa protein were tested.
Coordinate expression of the 35 kDa protein and cytochrome c6 The copper-responsive synthesis of cytochrome c6 displays unique metal selectivity (Cu > Hg >> Ag), high sensitivity and a characteristic response time (Merchant et al., 1991). The synthesis of the 35 kDa protein was therefore examined with respect to these properties (Figures 2-4). A time course analysis indicates that the synthesis of the 35 kDa protein is greatly reduced 2.5 h after the addition of copper, and that the response is complete within 5 h. This is similar to the time course of repression of cytochrome c6 synthesis in response to added copper where 95% of the mRNA is lost in 3 h, and by 4 h the response is 100% complete. The metal selectivity of both responses are likewise similar (Figure 3). In both cases, the response displays selectivity for Cu versus Hg ions, while Ag ions are completely ineffective. At a cell density of 4.5 x 106 cells/ml, -50 nM copper is required to completely repress the synthesis of the 35 kDa polypeptide (Figure 4) and cytochrome c6 (data not shown, see legend to Figure 4). This corresponds to a copper content of -7 x 106/cell, which correlates well with the estimated
cyt c6 Fig. 3. Metal specificity of expression of the 35 kDa protein. C.reinhardtii cells were labeled for 10 min with [35S]Na2SO4 (see Materials and methods) after being supplied with the indicated metal ion for 5 h. Top: soluble proteins were separated by SDS-PAGE and visualized by fluorography. Bottom: cytochrome c6 was immunoprecipitated (Howe and Merchant, 1993) from soluble protein extracts and analyzed by fluorography after electrophoretic separation on SDS-containing polyacrylamide gels. The arrow indicates the position of migration of the 35 kDa protein.
plastocyanin content of cells and the amount of copper required to completely repress the transcription of the cytochrome c6-encoding gene (8-9X 106 copper ions per cell), as determined in our earlier work (Merchant et al., 1991). These features of the expression of the 35 kDa polypeptide suggest that it is likely to be regulated by the same metalloregulatory pathway that is responsible for the copper-responsive synthesis of cytochrome c6; hence, the protein is likely to have a function in this adaptive response. We accordingly chose to purify the 35 kDa polypeptide with a view to determining its function and the mechanism of its regulation. Purification of the 35 kDa polypeptide Since the protein had no known biochemical activity, fractions from the purification procedures were monitored by separation on SDS-containing polyacrylamide gels and analyzed by comparison with marker lanes containing extracts from +Cu versus -Cu cells. The purification of the 35 kDa polypeptide from extracts of soluble proteins from C.reinhardtii is described in Materials and methods. We obtained 65 ,ug of purified protein from 81 mg of total soluble protein, with an estimated 130-fold overall purification (based on enzyme activity; see Table I). Densitometric scanning of lane 4 in the gel shown in Figure 5 indicates that the 35 kDa protein constitutes -90% of the protein in the final fraction. 859
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