Buthionine sulfoximine-sensitization of C. reinhardtii cells to Cd toxicity. Cells were grown in TAP-agar plates (supplemented as indicated below) and were ...
Plant Physiol. (1992) 98, 127-136
Received for publication May 14, 1991 Accepted August 1, 1991
0032-0889/92/98/0127/1 0/$01 .00/0
Heavy Metal-Activated Synthesis of Peptides in Chiamydomonas reinhardtii' Gregg Howe2 and Sabeeha Merchant* Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90024 ABSTRACT
tionally activated, metal-dependent, enzymatic pathway from precursor GSH (13, 30, 31, 34). This view is supported by genetic evidence showing that yeast cells with defects in the GSH biosynthetic pathway fail to accumulate phytochelatins in response to heavy metal stress (26). Elucidation of the pathway for the biosynthesis of phytochelatins in vascular plants has recently culminated with the identification of a "phytochelatin synthase" activity in suspension cultures of
In this study, we have addressed the capacity of the green alga Chiamydomonas reinhardtii to produce metal-binding peptides in response to stress induced by the heavy metals Cd2 , Hg2+, and Ag+. Cells cultured in the presence of sublethal concentrations of Cd2+ synthesized and accumulated oligopeptides consisting solely of glutamic acid, cysteine, and glycine in an average ratio of 3:3:1. Cadmium-induced peptides were isolated in their native form as higher molecular weight peptide-metal complexes with an apparent molecular weight of approximately 6.5 x 103. The isolated complex bound cadmium (as evidenced by absorption spectroscopy) and sequestered (with a stoichiometry of 0.7 moles of cadmium per mole of cysteine) up to 70% of the total cadmium found in extracts of cadmium-treated cells. In Hg2+-treated cells, the principal thiol-containing compound induced by Hg2 ions was glutathione. It is possible that glutathione functions in plant cells (as it does in animal cells) to detoxify heavy metals. Cells treated with Ag+ ions also synthesized a sulfur-containing component with a charge to mass ratio similar to Cd2+ -induced peptides. But, in contrast to the results obtained using Cd2 as an inducer, these molecules did not accumulate to significant levels in Ag+-treated cells. The presence of physiological concentrations of Cu2+ in the growth medium blocked the synthesis of the Ag+-inducible component(s) and rendered cells resistant to the toxic effects of Ag+, suggesting competition between Cu2+ and Ag+ ions, possibly at the level of metal uptake.
Silene cucubalus (1 1). The isolated enzyme has a heavy metaldependent y-glutamylcysteine dipeptidyl transpeptidase activity that is proposed to be responsible for the metal-regulated biosynthesis of phytochelatins in vivo. The further characterization of phytochelatin synthase will provide a focal point for the study of the biosynthetic regulation and physiological function(s) of these novel peptides. The phytochelatin response pathway in plants presumably constitutes a metal detoxification system analogous to the metallothionein proteins in animals (13, 17, 18). Both animals and plants are capable of providing, upon sensation of elevated concentrations of heavy metals, a quantity of highaffinity, heavy metal-specific ligands (viz. thiols) sufficient to avert the deleterious consequences of heavy metal binding to essential macromolecules. Although current data clearly support the suggestion of a detoxification function for phytochelatins, it has not been possible to define the extent to which these peptides contribute to or are responsible for intracellular metal detoxification, especially in experimental systems in which more than one mechanism for heavy metal homeostasis might be in operation (21, 39). A fundamental yet unanswered question pertaining to phytochelatin-mediated metal detoxification is whether or not the metal specificity of phytochelatin synthase reflects the metal specificity of phytochelatin-mediated metal binding. Although phytochelatin synthesis is activated both in vivo and in cell-free extracts by a broad spectrum of metal ions, including Cd2+, Ag+, Bi3+, Pb2+, Zn2+, Cu2+, Hg2+, and Au'+ (11, 13), only Cd2' and Cu2+ ions have been demonstrated to participate in the formation of peptidemetal complexes in vivo. Furthermore, Delhaize and coworkers (5) have recently found that the heavy metal-induced synthesis of phytochelatin peptides is insufficient in itself to confer cadmium tolerance to cultured cells of Datura innoxia. This work suggested that heavy metal tolerance requires not only synthesis but also the efficient assembly of phytochelatin peptides into higher mol wt peptide-metal complexes. These observations underscore the need to examine the relationship between phytochelatin synthesis and phytochelatin-mediated metal binding in response to a variety of heavy metals for the purpose of establishing the degree to which these peptides
Many organisms respond to the cytotoxic effects of heavy metals by synthesizing metal-chelating proteins or peptides. The predominant class of such molecules in plants, algae, and some fungi are the small, cysteine-rich peptides referred to as phytochelatins (12), Cd2+-binding peptides (25), cadystins (19), or -y-glutamyl peptides (29). These molecules have the general structure (,y-Glu-Cys)n-Gly (12, 17, 20, 34), where n can range from 2 to 11 depending on the species from which the peptides are isolated and the conditions of their induction (10, 13). A wealth of biochemical evidence from experiments conducted in vivo (reviewed in refs. 28, 33) supports a model in which phytochelatins are synthesized via a posttransla-
'This research was supported by a training grant (87-GRAD-90086) from the U.S. Department of Agriculture (G.H.), a U.S. Health and Human Services grant (GM42 143), the Searle Scholars Foundation/Chicago Community Trust (S.M.), and a National Institutes of Health Biomedical Research Support Grant (RR07009-23) to UCLA. 2 Gregg Howe is a graduate student in the Ph.D. program of the Department of Biology, UCLA. 127
HOWE AND MERCHANT
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constitute a cellular strategy for heavy metal tolerance and homeostasis. To further address this issue, we have studied the synthesis and accumulation of sulfur-containing peptides in response to Cd2" as well as in response to Ag+ and Hg2". The latter two metal ions are known to elicit phytochelatin synthesis in some organisms, but the mode of their detoxification (if any) is unknown. A better understanding of the role of these peptides in the complex pathways of heavy metal detoxification and metabolism could be facilitated by an experimental system in which these pathways could be subjected to genetic dissection. Unicellular green algae, some species of which are amenable to genetic analysis, have often been used as experimental models for the study of basic biochemical processes, common to both unicellular and multicellular plants. Several green algal species have been reported to accumulate Cd2+-binding components upon exposure to cadmium (8, 38). Recent characterization of these components from a wide range of algal species has shown them to be phytochelatins (8). In this work, we extend the previous contributions of Gekeler et al. (8) on Cd2+-induced responses in algae by describing the purification and spectral characterization of native metal-peptide complexes in Chiamydomonas reinhardtii and by comparing the distinct cellular responses to different metals. C. reinhardtii is well-suited to the study of the general mechanisms underlying metal tolerance and homeostasis owing to its amenability to biochemical analyses and accessibility to both classical and molecular genetic techniques. MATERIALS AND METHODS
Growth and Radioisotopic Labeling of Cells Cultures of Chiamydomonas reinhardtii wild-type strain CC 124 or CC 102 1, obtained from the Chlamydomonas Culture Collection, Duke University, were grown at 22°C under 125 ,uE/m2/s illumination in TAP3 medium (24) in which the copper concentration was reduced to less than 3 nm. Gibco "Select Grade" agar was included at 1.5% in solid media. The sulfate concentration in the same medium was reduced to 100 AM for [35S]SO4 labeling experiments. Logarithmically growing cultures (50 mL, 1-3 x 106 cells/mL) were typically labeled by the addition of [35S]Na2SO4 (43 Ci/mg S, ICN Biomedicals, Inc.) to a final concentration of 20 ,Ci/mL, 5 min after treatment of the cells with heavy metal ions. Labeling was terminated 20 to 60 min later by the addition of unlabeled Na2SO4 to a final concentration of 10 mm. Incorporation of I5S into soluble protein in pH-neutral extracts (see below) was measured by TCA precipitation as described by Pratt (27) with the exception that Na2SO4 (10 mM), cysteine (0.1%, w/v), and methionine (0.1%, w/v) were added to all TCA solutions at the indicated final concentrations. Preparation of Cell Extracts Cells were collected by centrifugation (7500g for 4 min) and washed once in a solution containing 10 mm phosphate, pH 7.0. Depending on the experiment, either acidified extracts 3Abbreviation: TAP, Tris-acetate-phosphate medium.
Plant Physiol. Vol. 98, 1992
(GSH determination, nonprotein sulfhydryl determination, HPLC analysis) or pH-neutral extracts (G-50 chromatography, gel electrophoresis) were prepared. For the former, the wet weight of the washed cell pellet was measured, and then a volume (in mL equal to the weight in g of the cell paste) of 10% (w/v) sulfosalicylic acid was added. The cell pellet was resuspended by agitation on a Vortex mixer and then stored at -80°C until it was analyzed, at which time the acidsolubilized cells were thawed and the insoluble cell debris was removed by centrifugation (12,000g for 15 min) at 4°C. pHneutral extracts were prepared by resuspending washed cells in 10 mm phosphate, pH 7.0, at a concentration equivalent to 0.5 to 1 mg Chl/mL. Soluble components of the cells were released by freezing the concentrated cell suspensions slowly to -80C, followed by slow-thawing to room temperature (22°C). Quantitative release was achieved by repetition of this freeze-thaw step. After the freeze-thaw cycles, insoluble cell debris was removed by centrifugation (12,000g for 15 min) at 4°C. Nonprotein Sulfhydryl and GSH Assays
Nonprotein sulfhydryl content in acidified extracts was determined essentially as described by Ellman (6). Our standard assay involved combining 10 ,L of extract with 990 ,L of a solution containing 0.1 mm dithionitrobenzoic acid, 100 mM K2HPO4, pH 8.0, followed by measurement of the absorbance at 412 nm. The presence of 1 mol equivalent of AgNO3 or 0.5 mol equivalent of HgCl2 in a standard solution of GSH (25 nmol in 5% sulfosalicylic acid) completely inhibited the reaction of dithionitrobenzoic acid with the GSH sulfhydryl group (owing to the formation of stable GSH mercaptides) and hence prevented their assay in the presence of these metals by the above method. The results of our assay, therefore, are necessarily presented as "reactive" nonprotein sulfhydryl content. CdCl2 had little effect on the sulfhydryl assay when present in a 10-fold excess over GSH. GSH levels in acidified extracts were determined by the GSH recycling assay described by Anderson (1) and are expressed as GSH equivalents. Like the nonprotein sulfhydryl assay, the GSH assay was also sensitive to the presence of Hg2' and Ag+ ions. HPLC Detection of Thiol-Containing Peptides
Thiol-containing compounds were assayed by subjecting acidified extracts (prepared from 50-200 mg cell-pack wet weight) to reverse-phase HPLC on a C- 18 column (Econosphere, 4.6 cm x 250 mm, Alltech Associates, Inc., Deerfield, IL) equilibrated with 0.1% TFA. Peptides were separated on the column with a 0 to 20% gradient of acetonitrile in 0.1% TFA over 40 min at 1 mL/min. Sulfhydryl-containing compounds eluting from the column were detected either by their absorbance at 412 nm after postcolumn derivatization with Ellman's reagent as previously described ( 13) or by determining the 35S content of collected fractions (0.4 mL). Native Gel Electrophoresis
Stacking gels (1.5 mm x 1.5 cm) consisted of a 5% (w/v) acrylamide:bisacrylamide mixture (30:1) in stacking gel buffer
METAL-INDUCED PEPTIDES IN CHLAMYDOMONAS REINHARDTII
(0.062 M Tris, 0.062 N HCl, pH 6.7), and separating gels (1.5 mm x 15 cm) consisted of a 15 to 17% (w/v) acrylamide:bisacrylamide mixture (30:1) in separating gel buffer (0.375 M Tris, 0.06 N HCl, pH 8.9). Both gel layers were polymerized by the addition of 0.03% (w/v) ammonium persulfate and 0.06% (v/v) N,N,N',N-tetramethylethylenediamine. The pH-neutral extract derived from an equivalent of 20,g Chl (approximately 50,ug protein) was mixed with onefourth volume of 5x sample buffer (0.25 M Tris, 0.25 N HCl, pH 6.7, 40% [w/v] sucrose, 0.05% [w/v] bromphenol blue) and electrophoresed in running buffer (0.05 M Tris, 0.38 M glycine, pH 8.3) at room temperature (22°C) for 3 h at 150 V. Gels were fixed in 10% (v/v) acetic acid for 30 min and then prepared for fluorography (3). Purification of the Cd2' -Induced Peptide The pH-neutral extracts prepared from "S-labeled cells were centrifuged at 27,000g for 30 min, and the cleared supernatant was applied to a Sephadex G-50 column (2.5 x 50 cm) equilibrated with 0.1 M KCl, 20 mM Tris-Cl, pH 7.8. The extract was eluted at room temperature (22°C) in the same buffer at a flow rate of 1 mL/min. The peptide-containing fractions were located by liquid scintillation counting, by cadmium analysis, by atomic absorption spectroscopy, and by measurement of the sulfhydryl content (6) of an aliquot of each fraction. The pooled fractions were applied to a 1.5 x 6.5-cm column containing DEAE A25 (Pharmacia) anion exchange resin equilibrated with 0.1 M KCl, 20 mm Tris-Cl, pH 7.0. The peptide was eluted with a linear gradient of 0.1 to 1.0 M KCl in the same buffer at a flow rate of 1.2 mL/min. Fractions containing the peptide were pooled and acidified by the addition of TFA to a final concentration of 0.1 % (v/v). This material was desalted by applying it to a C- 18 Sep-Pak column (Pharmacia) equilibrated with 0.2% TFA (v/v). The column was washed with 10 mL of 0.2% TFA. The metalfree peptide was eluted with 70% acetonitrile, 0.1 % TFA, and concentrated by lyophilization. More than 90% of the radioactivity applied to the Sep-Pak column was recovered in the eluent. The peptide concentration in the desalted DEAE-pure material was determined by quantitative amino acid analysis at the UCLA Protein Microsequencing Laboratory. Samples were hydrolyzed under vacuum in a nitrogen atmosphere for 18 h in 6 N HCl at 1 10°C. Cysteine concentration was measured after oxidation to cysteic acid by treatment with formic acid:hydrogen peroxide (30%) (19:1) for 30 min at 25°C.
Phenylisothiocyanate-derivatized amino acid products were separated by HPLC on a Waters C- 18 Nova-Pak column. Typical yields of the Cd2+-induced peptide ranged from 50 to 250 mg of peptide per L of Cd2t-induced culture, assuming an average peptide structure of (-y-Glu-Cys)3-Gly. The Cd2+binding peptide purified from "S-labeled, cycloheximidetreated cells showed the same amino acid composition and Cd2+-binding stoichiometry as it did when purified from unlabeled cells that were not treated with cycloheximide. Miscellaneous Procedures Chl was estimated from its absorbance at 652 nm (2) after acetone:methanol (80:20, v/v) extraction of whole cells. The
129
absorbance at 652 nm of a 13 ,g/mL standard solution of Chl in acetone:methanol was unaffected by the addition of HgCl2, AgNO3, or CdCl2 to final concentrations of 500, 10, or 500 uM, respectively. Cadmium and silver concentrations were determined by flame atomic absorption spectroscopy and inductively coupled plasma emission spectroscopy, respectively. RESULTS Cell Growth and Intracellular Thiol Content in Response to Heavy Metals The effect of different concentrations of Cd2' and Hg2'
ions on the growth of C. reinhardtii in liquid culture was examined to define metal concentrations that inhibited cell growth but that did not abolish cell viability. Cells exposed to relatively moderate concentrations of heavy metals tended to aggregate into clumps and thus made accurate measurements of cell number and cell viability difficult. Therefore, Chl accumulation was used as a measure of heavy metal-induced toxicity (Fig. lA). During the logarithmic phase of growth, cultures of C. reinhardtii maintained under standard laboratory conditions in the absence of added heavy metals double in cell number and in Chl content in approximately 10 h. The addition of 120 Mm CdCl2 or 5 uM HgCl2 to rapidly growing cultures inhibited the rate of Chl accumulation by 30 and 66%, respectively, during the first 18 h after metal addition. In all cases, the observed inhibition of Chl accumulation was correlated with a proportional increase in cell division time. After 1 to 2 d of continued growth, heavy metal-treated cultures recovered from the deleterious effects of heavy metal toxicity, because their Chl content and cell densities were comparable to those in untreated cultures. Cells cultured in the presence of sublethal concentrations of either Cd2+ or Hg2' accumulated a nonprotein sulfhydryl content significantly greater than that observed in untreated cells (Fig. 1 B). Whereas the Hg2+-induced increase in sulfhydryl content occurred rapidly and tapered off, there was a lag phase for Cd2+-induced sulfhydryl increase. GSH measurements made on the same extracts showed that GSH levels in Cd2+-treated cells do not account for the increase in Cd2+induced, nonprotein thiol content (Fig. lC). In fact, we often observed a slight decrease in the GSH content in Cd2+-treated versus untreated cells. This finding suggests that GSH pools are being depleted as a consequence of Cd2+-induced synthesis of thiol-containing peptides. In remarkable contrast to Cd2+treated cells, the increased nonprotein sulfhydryl content in Hg2`-treated cells was completely accounted for by the increased intracellular concentration of GSH. The GSH levels in Hg2`-treated cells (Fig. IC) probably underestimate the extent to which Hg2' stimulates GSH overproduction, because Hg2+ ions interfere with GSH measurements (see explanation in "Materials and Methods"). We have observed in other experiments that GSH levels in Hg2`-treated cells increase threefold above the levels in untreated cells during a 90-min period after the addition of 2 to 10 Mm HgCl2.
Cd2+-lnduced Peptides To characterize the thiol-containing components that accumulated in response to Cd2" ions, cell-free extracts were
Plant Physiol. Vol. 98, 1992
HOWE AND MERCHANT
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metal analysis and absorption spectra suggest that a specific complex is formed between the sulfur-containing component and Cd2+ ions. The Cd2+-binding material (pooled G-50 fractions 24-33, fig. 2B) was further purified by DEAE chroma-
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prepared from 35SO4-labeled cells and subjected to gel filtration chromatography. The 35S in the extracts prepared from untreated cells typically separated into two peaks, corresponding to "bulk" protein eluting in the void volume (Fig. 2A, peak 1) and material with a lower mol wt of approximately 1 X I03 (Fig. 2A, peak 3). The 35S in extracts from Cd2`-exposed cells, in addition to eluting in peaks 1 and 3, also eluted as a broad peak with an apparent mol wt of 6.5 x 103 (Fig. 2B, peak 2). In some chromatographs, a shoulder (peak 2a) on the lagging edge of peak 2 was observed. Assays of the eluent G-50 fractions for sulfhydryl content revealed the presence of a Cd2+-inducible, thiol-containing component that coeluted with peak 2 (not shown). Electrophoretic analysis of these fractions indicated that the radiolabel was contained in a component (or components) that migrated as a single species on a nondenaturing gel (Fig. 2C). Metal analysis showed that about 70% of the cadmium in the extract applied to the column was also associated with peak 2. The remaining Cd2+
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Figure 2. Gel filtration elution profiles of extracts from cells grown in the absence or presence of CdCI2. Cells grown either in the absence (A) or presence (B) of 50 uM CdCI2 for 24 h were labeled with [35S] Na2SO4 for 1 h as described in "Materials and Methods." pH-neutral cell extracts were prepared and fractionated on a Sephadex G-50 column (2.5 x 50 cm). Collected fractions (4.5 mL) were assayed for protein content (A254), 35S content, or Cd content. Equal volumes (35 AL) of the indicated fractions from the chromatographs shown in panel B or a 5-,AL aliquot of the pH-neutral extract applied to the column were analyzed by nondenaturing PAGE and fluorography (C). The arrow indicates the position of migration in the gel of the 35Scontaining, Cd2+-inducible components (see also Fig. 6).
METAL-INDUCED PEPTIDES IN CHLAMYDOMONAS REINHARDTII
tography and analyzed for Cd content and amino acid composition. These analyses revealed only Glu, Cys, and Gly (95.4% of total amino acids) in a ratio of 3.2:2.7:1.0 and 0.7 mol of Cd/mole of cysteine, suggesting that the Cd2+-binding component characterized in this work is similar to that characterized by Gekeler et al. (8). Reverse-phase HPLC was used to separate the thiol-containing compounds that accumulated in heavy metal-treated cells. Cysteine and GSH constituted the major low mol wt thiol-containing compounds in cells that were not exposed to heavy metals (Fig. 4 A, D, peaks 1 and 2). Cells treated with Cd2" ions, however, accumulated thiol-containing compounds that were not present (or were present at low abundance) in untreated cells (Fig. 4B, C, peaks labeled 3-8). These compounds are inducible within 8 h of treatment with Cd2" ions (Fig. 4B), and their levels remain high 48 h after treatment with Cd2+ ions (Fig. 4C). The inducible compounds account for 75% of the total nonprotein sulfhiydryl in cells treated with 100 zlM CdCl2 for 3 d. The Cd2+-binding complex (Fig. 2, peak 2) was shown by HPLC analysis to consist predominantly of the thiol-containing material in peak 5 (Fig. 4B, C). In contrast with Cd2+-treated cells, cells that were cultured in the presence of 10 /uM HgCl2, a concentration that is known to be toxic (see Fig. 1 for example), did not accumulate comparable amounts of inducible, thiol-containing peptides
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Plant Physiol. Vol. 98, 1992
HOWE AND MERCHANT
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sized GSH and at least two compounds (peaks 1 and 2) during a 90-min labeling period (Fig. 5B). Although GSH does not accumulate over the long term in Cd2+-treated cells (probably owing to its utilization as a substrate for other metal-activated syntheses), its synthesis is stimulated to a significant extent in the short term. Cells exposed to 10 Mm HgCl2 synthesized only GSH like control cells; however, the amount of GSH synthesized in the 90-min period was much greater in Hg2+-treated cells than in control cells.
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fraction number Figure 5. HPLC analysis of thiol-containing peptides synthesized in "pulse"-labeled cells in response to Cd2+ and Hg2+ ions. Cells exposed to either 100 AM CdCI2 (B) or 10 AM HgCI2 (B) for 5 min were then labeled for 90 min in the presence of 20 ACi/mL [35S]Na2SO4. Labeling was terminated by the addition of nonisotopic Na2SO4 to a final concentration of 10 mm. Control cells (A) were not treated with heavy metal but were labeled in an identical manner. Acidified extracts obtained from 200 mg of packed cell wet weight were fractionated by reverse-phase HPLC, and collected fractions (0.4 mL) were measured for 35S content. The 35S-containing compound eluting in fractions 1 1 and 12 is GSH. The area under the GSH peak in extracts obtained from Cd2+- and Hg2+-treated cells increased by 2.7 and 5.3-fold, respectively, as compared with the control shown in panel A. The predominant Cd2+-inducible peaks are denoted as 1 and 2. GSSG indicates the retention time of an oxidized GSH standard and also marks the retention time of an Hg2+-inducible component.
Ag+-Induced Responses
Silver nitrate, at 5 AM in the medium, shows the same inhibition of growth and Chl accumulation as does 5 ,M HgCl2 (data not shown). We have been unable to detect the accumulation of either GSH or thiol-containing peptides in Ag'-treated cells by either HPLC separation or enzymatic assay (in the case of GSH). However, we have noted that Ag+treated cells synthesize an inducible sulfur-containing component that can be resolved by nondenaturing polyacrylamide gel electrophoresis (Fig. 6). (A similar system was used by Wagner [37] as an initial step in the purification of a Cd2+_ binding complex from cabbage leaves.) At low to moderate
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(Fig. 4E, F). However, as observed previously, cells treated with HgCl2 show a transient but striking increase in the levels of assayable GSH. The levels of GSH estimated by the HPLC assay are consistent with the values obtained by enzymatic measurement (Fig. IC), as is the pattern of induction. The decrease in the abundance of assayable GSH and cysteine at the 48-h time point likely results from the titration of cellular GSH and cysteine by Hg, because the cells begin to recover from Hg2' toxicity by this time (Fig. IA). To demonstrate that the inducible increase in thiol-containing components in Cd2+-treated cells and glutathione in Hg2+treated cells results from de novo synthesis, we prepared acidified extracts from metal (Cd2+ or Hg2+)-treated, 35SO4 "pulse"-labeled cells (Fig. 5). The major 35S-containing compound in acidified extracts from untreated cells was GSH (Fig. 5A). Cells cultured in 100 Mm CdCl2, however, synthe-
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Figure 6. Detection by nondenaturing gel electrophoresis of components synthesized in response to heavy metals. A, Cells were treated with the indicated concentrations of metal ions for 15 min and were then labeled for 60 min in the presence of 20 uCi/mL of [35S] Na2SO4. Labeling was terminated by the addition of nonisotopic Na2SO4 to a final concentration of 10 mm. pH-neutral cell extracts derived from an equivalent of 20 MAg Chl were prepared, separated on a 15% nondenaturing gel, and visualized by fluorography. The arrow indicates the migration position of the metal-inducible component and the position at which the sulfur-containing component of the Cd-binding complex migrates (Fig. 2C). B, Relative amounts of 35S incorporated into protein in the extracts shown in panel A.
METAL-INDUCED PEPTIDES IN CHLAMYDOMONAS REINHARDTII
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an apparent mol wt of 1000 versus the Cd2+-binding complex that elutes with an apparent mol wt of 6500. Further, during the preparation of acidified extracts of metal-treated cells, we have noted that it is clearly less soluble in acid (10%, w/v, sulfosalicylic acid) compared with the Cd2+-binding component. It is unlikely that the Ag'-induced component is GSH, because extracts of radiolabeled, HgCl2-treated cells (that we have shown to contain radiolabeled GSH) do not contain this component, and standard, radiolabeled GSH does not survive the gel-fixation procedure used for fluorographic detection of the radiolabeled material.
Competition between Ag+ and Cu2+ During the course of this work, we observed that, whereas cells maintained in Cu2+-deficient media (estimated Cu2" concentration between 1 and 5 nM) readily synthesize the sulfur-containing component in response to Ag+ ions, cells maintained in Cu2+-supplemented media do not (Fig. 8, lanes 1 and 4). Furthermore, the extent to which Ag+-induced pre - treatment
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#*'o _is_ -
cyt
metal concentrations, a positive correlation was found between the amount of metal supplied and the amount of this component synthesized over a fixed labeling period. However, exposure of cultures to high concentrations of metal ions noticeably decreased 35S incorporation into protein (Fig. 6B). This effect results, in part, from a decrease in [35S]SO4 uptake (not shown) and, probably in part, from the inhibition of protein synthesis by heavy metals (16). Synthesis of the Ag+-induced 35S-containing component is activated within 15 min or less after the addition of Ag+ ions (Fig. 7A). Furthermore, treatment of cells with cycloheximide prior to the addition of Ag+ showed that the synthesis of these peptides during short labeling periods (2 ions elicit the "model" detoxification response in C. reinhardtii, we have found that Ag4 and Hg24 ions (at the concentrations tested in this work) do not. Both Ag4 and Hg24 have been reported to induce phytochelatins in plant suspension cultures of Rauvolfia serpentinia (13) as well as in the algae Chlorella fusca and Scenedesmus acutiformis (8). However, metal-phytochelatin interactions were not demonstrated in these reports. And, in the case of the algal work, the concentration of Ag4 or Hg24 used to induce phytochelatins was not stated. It is not physiologically relevant to test the effect of Ag4 and Hg24 ions on C. reinhardtii at higher than 10 gM medium concentration, because the cells do not recover from the toxic effects of these metals at higher concentrations. The experiments in this study show that, whereas Ag4 is indeed an effective activator of the synthesis of an anionic, sulfur-containing component in C. reinhardtii, this component is distinct from the Cd24-induced peptides. Furthermore, we have been unable to establish that the Ag+-induced component(s) bind the inducing metal ions in vivo (as do the Cd24-induced peptides), or that they participate in the formation of higher mol wt metal-peptide complexes. Their induction by very low (micromolar) concentrations of Ag+ ions is, nevertheless, very striking and dramatic, and their characterization warrants further attention. We have observed that two Ag+-elicited responses, namely the synthesis of an inducible sulfur-containing component and Ag4 toxicity, can be blocked by micromolar concentrations of Cu2> ions in the growth medium. The simplest hypothesis for this apparent competition between medium Ag4 and Cu2> ions is that Cu+, because it is an electronic analog of Ag+, prevents Ag+ ion uptake by the cell by competing for a shared receptor site. It is unlikely that this phenomenon is due to a passive process involving an interaction or direct competition between Ag4 and Cu2> salts in the growth medium, because the results shown in Figure 8 indicate that inhibition of Ag4-induced peptide synthesis occurs as a consequence of a process that depends upon the length of exposure to or metabolism of Cu24 ions. Neither is it likely that cuprous or cupric ions mimic Ag4 ions in the pathway for induction of the Ag+-induced component, because medium Cu24 ions at these concentrations are nontoxic (Fig. 6) and do not induce any sulfur-containing compounds (Figs. 6 and 8). Although little is understood about cellular mechanisms of Cu24 ion uptake, one possible model that
136
HOWE AND MERCHANT
could explain the above observation is that Ag+ imported into C. reinhardtii cells by a high affinity system that cannot discriminate between Ag+ and Cu+"2+ and that is induced only in Cu2+-deficient cells. Previous in our laboratory has identified several polypeptides synthesis is induced or repressed under conditions deficiency, some of which are uncharacterized with respect function (15). One or more of these polypeptides involved in Cu+/2+-metabolism or uptake. Alternatively, results could be interpreted as showing that medium Cu>+ ions induce a compound that functions to detoxify ions
are
uptake ions
work
whose
of Cu2+to
may
be
the
Ag+
ions.
ACKNOWLEDGMENTS We thank Jim Roe for providing helpful suggestions of this work and Professor Jeanne Erickson for
course
during
reading
the
manuscript.
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