Metal ion regulated gene expression: use of a ... - Europe PMC

The EMBO Journal vol.6 no.9 pp.2531-2535, 1987

Metal ion regulated gene expression: use of a plastocyanin-less mutant of Chlamydomonas reinhardtii to study the Cu(L)-dependent expression of cytochrome c-552

Sabeeha Merchant and Lawrence Bogorad The Biological Laboratories, Harvard University, Cambridge, MA 02138, USA Communicated by J.H.Weil

Ac-208, a plastocyanin-deficient strain of Chlamydomonas reinharii, lacks the mRNA for pre-apoplastocyanin although Southern analyses indicate no gross deletion in the single gene for pre-apoplastocyanin. The alternative electron carrier, cytochrome c-552, is expressed only in Cu(II)-deficient cells of the mutant. Thus, the mutant strain can grow photoautotrophically in Cu(I1)-deficient but not in Cu(ll)-sufficientis medium. This indicates that the absence of plastocyanin the only defect (with respect to photosynthesis) in ac-208. Phenotypic revertants of ac-208 are capable of photoautotrophic growth by virtue of constitutive [with respect to Cu(I)] synthesis of cytochrome c-552. The revertant phenotype is attributed to a decreased internal concentration of Cu(IH). This indicates that the Cu(ll)-dependent expression of cytochrome c-552 synthesis is affected directly by the concentration of Cu(II) rather than indirectly by the level of plastocyanin in the cell. Key words: Chlamydomonas reinhardtiilcytochrome c-552 expression Introduction The ability of the unicellular green alga Chlamydomonas reinhardtii to grow heterotrophically by using acetate as a source of reduced carbon (Sager and Granick, 1953) has made it possible to isolate mutant strains with defects in the photosynthetic apparatus (Levine, 1971; Bennoun et al., 1978; Spreitzer and Mets, 1981; Myers et al., 1982). These strains require acetate for growth and are unable to grow photoautotrophically. Strain ac-208, derived from the wild-type (137c, CC-124) by u.v. irradiation, is an acetate-requiring mutant which was originally isolated 1960; on the basis of its inability to fix carbon dioxide (Levine, that concluded Levine and Gorman 1965). Gorman and Levine, photosynthesis to respect with strain this in deficiency only the lies in the absence of plastocyanin (Gorman and Levine, 1965, 1966). This conclusion was based on two experimental observations: plastocyanin could not be detected spectroscopically in extracts from the mutant cells and the addition of purified plastofragcyanin could restore electron transfer activity to chloroplast mapped was mutation The cells. mutant the ments prepared from maintain to linkage group Im and was found to be difficult to phenotypes free of suppressors (Levine, 1971). The suppressed exhibited partially restored photosynthetic activity, but since plastocyanin was not detected in extracts from the phenotypically suppressed cells, the suppressed phenotype was suggested to result from the synthesis of an unstable plastocyanin (Gorman and Levine, 1966). electron Plastocyanin is a type I Cu(II)-protein which catalyzes reaction transfer between the cytochrome b6-f complex and the IRL Press Limited, Oxford, England

center of photosystem I (P700) (Boulter et al., 1977). In some algae and cyanobacteria, a soluble c-type cytochrome can funcand Boger, tionally replace plastocyanin (Wood, 1978;(cytSandmann is related c-52213) 1980). This soluble c-type cytochrome cytochrome and mitochondria, to the soluble cytochromes c from (Dickerson, 1980). The c2 from purple photosynthetic bacteriaor cytochrome c-552 in C. plastocyanin either of accumulation concenCu(ll) the reinhardtii can be regulated by manipulating concentration the When 1978). tration of the medium (Wood, in the medium is < 50 nM, plastocyanin accumulation of is prevented (Wood, 1978; Merchant and Bogorad, 1986a) by the specific degradation of apoplastocyanin in Cu(II)-deficient cells (Merchant and Bogorad, 1986b). Under these conditions, cytochrome c-552 accumulates as a consequence of increased steady state levels of mRNA for pre-apocytochrome c-552 (Merchant and Bogorad, 1986a, 1987). In cultures containing > 200 nM Cu(II), cytochrome c-552 is not detectable. We are interested in identifying the factors which signal the cell to accumulate the message for cytochrome c-552. For example, is the response tois the absence of plastocyanin (e.g. Bohner et al., 1981)? Or, it to the concentration of available Cu(II)? The existence of a plastocyanin-less mutant strain of C. reinhardtii allowed us to address these questions. We have therefore basis characterized this strain in order to determine the molecular study it to used have and function, for the defect in plastocyanin the expression of cytochrome c-552 in the absence of functional plastocyanin. We conclude that the Cu(l)-dependent expression of cytochrome c-552 in C. reinhardtii is affected directly by the concentration of Cu(fl) in the cell.

Cu(ll)

Results Molecular characterization of ac-208 Gorman and Levine (1966) were unable to detect plastocyanin in extracts from cultures of ac-208 by spectroscopic methods. However, visible absorption spectra cannot be used to detect relies apoplastocyanin or pre-apoplastocyanin since the analysis order In in plastocyanin. ligand Cu(ll) the on the absorption of in to plastocyanin precursor any of accumulation the for test to reinhardtii C. purified ac-208, we used antibodies directed against and preplastocyanin (and known to cross-react with apofrom protein total of analyses blot Western in apoplastocyanin) containac-208 C. reinhardtii ac-208. Our results indicated that ed no polypeptide which was immunoreactive with antiC. reinhardtii plastocyanin (not shown). Total RNA isolatedoffrom 17 kd prethe synthesis the direct not could ac-208 is the which 1987) apoplastocyanin (Merchant and Bogorad, shown). not cells (data wild-type in primary translation product This result suggested that the message for pre-apoplastocyanin a was either substantially reduced in cells of ac-208, or that a in resulted for plastocyanin mutation in the coding sequence to distinguish order In product. translation loss of the primary C. between the two possibilities, we used a cDNA cloneoffor RNA analysis Northern in reinhardtii plastocyanin as probe isolated from ac-208. Whereas the RNA isolated from a strain 2531

S.Merchant and L.Bogorad

which exhibits normal plastocyanin activity contains a message 800 bases in length which hybridizes with the cDNA for plastocyanin, RNA isolated from ac-208 contains no sequences which hybridize to the probe (Figure 3, panel A, lanes 1 and 2). Based on normalization to the level of beta-tubulin message in each strain, one can estimate that the level of mRNA for plastocyanin in ac-208 is < 1 % of that in strains containing normal levels of plastocyanin. Southern hybridization of restricted genomic DNA isolated from ac-208 demonstrates that the mutant strair contains sequences which hybridize to the cloned cDNA (Figure 1). This indicates that the mutant phenotype does not result from a gross deletion or re-arrangement of the structural gene for pre-apoplastocyanin. The pattern of bands obtained by digestion of genomic DNA from ac-208 with several restriction enzymes followed by hybridization with plastocyanin cDNA probes is identical to the pattern obtained by the same treatment of wild-type (CC-124, CC-1 173, 2137) DNA (Figure 1). The rFesults of these analyses suggest that either the plastocyanin of

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message is very unstable in ac-208, or that a mutation, which might lie outside the coding sequence for pre-apoplastocyanin, prevents transcription of the gene. Regulation of cytochrome c-552 in ac-208 Cytochrome c-552 can functionally replace plastocyanin in depleted cells of some algae and cyanobacteria (Wood, Cu(IT)1978; Sandmann and Boger, 1980). The level of cytochrome c-552 in C. reinhardtii ac-208 was examined by Western blot as shown in Figure 2. The blot demonstrates that cellsanalysis of ac-208 contain detectable levels of cytochrome c-552 and that the protein accumulates only in Cu(ll)-deficient cells. Thus, the regulation of cytochrome c-552 in ac-208 appears to be normal and independent of the presence or absence of plastocyanin, pre-apoplastoor its

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Fig. 1. Southern analysis of genomic DNA isolated from a plastocyanin-less (CC-1729, ac-208) or from a parental strain which is wild-type for plastocyanin (CC-1 173). Five yg of DNA was digested overnight with the following restriction enzymes: D, HindiJi (50 U); F, Hinfi (50 U); P, PstI (50 U); U, PvuII (50 I); S, SstI (50 U). The DNA was separated in agarose gels containing 89 mM Tris, 89 mM boric acid, 18 mM EDTA, and transferred to GeneScreen. The membrane was probed with a nick-translated plasmid containing a cDNA sequence for plastocyanin (pTZ18RCrPCIA) (Merchant and Bogorad, 1987). The arrows indicate the positions of HindIII fragments of wild-type lambda DNA which were used as mol. wt markers. mutant

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Fig. 2. Western blot analysis of C. reinhardtii proteins from a plastocyaninless mutant (CC-1729, ac-208). C. reinhardtii cells were grown heterotrophically in low light in TAP medium with no added Cu(ll) (-Cu) or with 8 IM added Cu (+Cu). In Cu(II)-free medium, cells could also be grown phototrophically in minimal medium (MM). Total C. reinhardtii proteins were denatured and separated by gel electrophoresis on SDScontaining polyacrylamide gels. The separated proteins were transferred to nitrocellulose and incubated with a solution containing antibodies directed against cytochrome c-552. Bound antibody was detected by use of an enzyme conjugated second antibody and a chromogenic substrate. Details are provided in an earlier publication (Merchant and Bogorad, 1986a). Purified cytochrome c-552 is shown as a marker in the lane on the extreme right.

Cu(l)-dependent expression of cytochrome c-552

ditions where plastocyanin is not required by the cell [i.e. Cu(H)deficient medium], the cell appears to be completely functional with respect to photosynthesis. Thus, plastocyanin appears to be the only deficiency in this mutant strain. Properties of a phenotypically suppressed strain derived from ac-208 Gorman and Levine (1966) had reported that the acetate-requiring phenotype of ac-208 was easily suppressed. Their suppressed strains did not contain any spectroscopically detectable plastocyanin; yet, these strains appeared to have partially restored electron transfer activity. They postulated the existence of an unstable, but partially functional, plastocyanin molecule in the suppressed strains in order to account for the phenotype. Wood (1978), on the other hand, suggested that the suppressed phenotype occurred by virtue of Cu(ll)-independent accumulation of cytochrome c-552. Spectroscopic analyses indicated that cells of the suppressed phenotype contained 10% as much cytochrome c-552 as Cu(II)-depleted wild-type cells (Wood, 1978). Since we were interested in the mechanism of cytochrome c-552 regulation (and Wood's suppressed strains are no longer available), we attempted to identify and characterize phenotypic revertants. Minimal medium containing Cu(fl) was inoculated with heterotrophically grown mutant cells. Cultures which contained cells capable of photoautotrophic growth were spread on agar and allowed to grow photoautotrophically at 300-500 jiEIm2/s. Single colonies were isolated from these agar plates. The properties of all suppressed strains are similar and the characteristics of only one of these (ac-208R2) will be discussed in detail in this report. Whereas ac-208 is capable of photoautotrophic growth only in Cu(II)-free medium, the strain exhibiting the suppressed pheno-

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medium type is able to grow photoautotrophically in minimal - including elements trace of containing the normal complement 8 AM Cu(ll). The strain has been maintained for over two years in the absence of selection for photosynthetic growth [i.e. in low light, on Cu(H)-containing TAP medium (tris-acetatephosphate medium, Gorman and Levine, 1965)]. Under these conditions the suppressed phenotype appears to be stable. The message for plastocyanin is not recovered in the suppressed strain (Figure 3). However, the message for cytochrome at c-552 is detectable at Cu(ll) concentrations in the mediummuplastocyanin-deficient the or (CC-124) wild-type which a tant strain (ac-208, CC-1729) contain no detectable message for cytochrome c-552 (Figure 3). The level of cytochrome c-552 message in ac-208R2 grown in medium containing 8 4iM Cu(ll) is comparable with the level of message detected in Cu(fl)depleted (