Two metal-dependent steps in the biosynthesis of Scenedesmus obliques plastocyanin. Differential mRNA accumulation and holoprotein formation.

The accumulation of the interchangeable electron transfer catalysts plastocyanin and cytochrome c6 (cyt c6) in Scenedesmus obliquus is reciprocally regulated by the amount of copper ions in the medium. In copper-deficient cells, plastocyanin levels are severely reduced, whereas cyt c6 levels are high. Western blot analysis indicates that neither pre-apoplastocyanin nor apoplastocyanin accumulate to significant extents in copper-deficient Scenedesmus cells, and time course studies indicate that upon provision of copper salts to copper-deficient cells, the accumulation of plastocyanin to the levels maintained in copper-sufficient cells takes about 12-24 h. By 1) Northern hybridization analysis of Scenedesmus obliquus mRNA and 2) in vitro translation of polyadenylylated mRNA followed by immunoprecipitation of a 19.2-kilodalton precursor to plastocyanin, we demonstrate that the regulation of plastocyanin synthesis by copper must occur primarily at the level of mRNA accumulation. These results suggest that copper-dependent stimulation of holoplastocyanin accumulation requires de novo synthesis of the pre-apoprotein and contradict the conclusion of Bohner, H., Bohme, H., and Boger, P. (1981) FEBS Lett. 131, 386-388 that high levels of apoplastocyanin and a precursor to plastocyanin accumulate in copper-deficient Scenedesmus cells. We note also that although metal ions other than copper (e.g. silver) insert into Scenedesmus obliquus plastocyanin in vitro, synthesis of holoplastocyanin in vivo is specific for copper versus silver or mercury as it is in Chlamydomonas reinhardtii. Finally, the very different electrophoretic mobility and immunoreactivity of apoplastocyanin compared with holoplastocyanin suggests rather significant differences in structure between the copper-protein and the metal-free protein.     

Regulation by copper of the expression of plastocyanin and cytochrome c552 in Chlamydomonas reinhardi.

Plastocyanin and cytochrome c552 are interchangeable electron carriers in the photosynthetic electron transfer chains of some cyanobacteria and green algae (P. M. Wood, Eur. J. Biochem. 87:9-19, 1978; G. Sandmann et al., Arch. Microbiol. 134:23-27, 1983). Chlamydomonas reinhardi cells respond to the availability of copper in the medium and accordingly accumulate either plastocyanin (if copper is available) or cytochrome c552 (if copper is not available). The response occurs in both heterotrophically and phototrophically grown cells. We have studied the molecular level at which this response occurs. No immunoreactive polypeptide is detectable under conditions where the mature protein is not spectroscopically detectable. Both plastocyanin and cytochrome c552 appear to be translated (in vitro) from polyadenylated mRNA as precursors of higher molecular weight. RNA was isolated from cells grown either under conditions favorable for the accumulation of plastocyanin (medium with Cu2+) or for the accumulation of cytochrome c552 (without Cu2+ added to the medium). Translatable mRNA for preapoplastocyanin was detected in both RNA preparations, although mature plastocyanin was detected in C. reinhardi cells only when copper was added to the culture. Translatable mRNA for preapocytochrome, on the other hand, was detected only in cells grown under conditions where cytochrome c552 accumulates (i.e., in the absence of copper). We conclude that copper-mediated regulation of plastocyanin and cytochrome c552 accumulation is effected at different levels, the former at the level of stable protein and the latter at the level of stable mRNA.     

Rapid degradation of apoplastocyanin in Cu(II)-deficient cells of Chlamydomonas reinhardtii

Although plastocyanin is not detected in Cu(II)-deficient cells of Chlamydomonas reinhardtii, accumulation of messenger RNA for pre-apoplastocyanin is independent of the concentration of Cu(II) in the medium (Merchant, S., and Bogorad, L. (1986) Mol. Cell. Biol. 6, 462-469). This work shows that the synthesis, transport, and processing of pre-apoplastocyanin also appear to be unaffected in cells grown in Cu(II)-deficient medium. However, the mature protein, presumably formed after import of the precursor into the chloroplast, is rapidly degraded in Cu(II)-deficient cells. The half-life of the mature protein is estimated to be between 16 and 18 min in cells grown in Cu(II)-deficient medium. In cells grown in medium containing Cu(II), the mature protein is stable. The proteolytic activity thus appears to be specific for apoplastocyanin versus plastocyanin and thereby accounts for the absence of accumulated plastocyanin in Cu(II)-deficient cells. This process may be part of a general mechanism designed to remove chloroplast proteins which cannot be utilized.     

Regulated copper uptake in Chlamydomonas reinhardtii in response to copper availability.

A saturable and temperature-dependent copper uptake pathway has been identified in Chlamydomonas reinhardtii. The uptake system has a high affinity for copper ions (Km approximately 0.2 microM) and is more active in cells that are adapted to copper deficiency than to cells grown in a medium containing physiological (submicromolar to micromolar) copper ion concentrations. The maximum velocity of copper uptake by copper-deficient cells (169 pmol h-1 10(6) cells-1 or 62 ng min-1 mg-1 chlorophyll) is up to 20-fold greater than that of fully copper-supplemented cells, and the Km (approximately 2 x 10(2) nM) is unaffected. Thus, the same uptake system appears to operate in both copper-replete and copper-deficient cells, but its expression or activity must be induced under copper-deficient conditions. A cupric reductase activity is also increased in copper-deficient compared with copper-sufficient cells. The physiological characteristics of the regulation of this cupric reductase are compatible with its involvement in the uptake pathway. Despite the operation of the uptake pathway under both copper-replete and copper-deficient conditions, C. reinhardtii cells maintained in fully copper-supplemented cells do not accumulate copper in excess of their metabolic need. These results provide evidence for a homeostatic mechanism for copper metabolism in C. reinhardtii.     

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

Ac-208, a plastocyanin-deficient strain of Chlamydomonas reinhardtii, 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(II)-deficient but not in Cu(II)-sufficient medium. This indicates that the absence of plastocyanin is 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(II)] synthesis of cytochrome c-552. The revertant phenotype is attributed to a decreased internal concentration of Cu(II). This indicates that the Cu(II)-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.     

Tropoelastin production and tropoelastin messenger RNA activity. Relationship to copper and elastin cross-linking in chick aorta.

The elastin content of the chick thoracic aorta increases 2--3-fold during the first 3 weeks post-hatching. The deposition of elastin requires the covalent cross-linking of tropoelastin by means of lysine-derived cross-links. This process is sensitive to dietary copper intake, since copper serves as cofactor for lysyl oxidase, the enzyme that catalyses the oxidative deamination of the lysine residues involved in cross-link formation. Disruption of cross-linking alters tissue concentrations of both elastin and tropoelastin and results in a net decrease in aortic elastin content. Autoregulation of tropoelastin synthesis by changes in the pool sizes of elastin or tropoelastin has been suggested as a possible mechanism for the diminished aortic elastin content. Consequently, dietary copper deficiency was induced to study the effect of impaired elastin cross-link formation on tropoelastin synthesis. Elastin in aortae from copper-deficient chicks was only two-thirds to one-half the amount measured in copper-supplemented chicks, whereas copper-deficient concentrations of tropoelastin in aorta were at least 5-fold higher than normal. In spite of these changes, however, increased amounts of tropoelastin, copper deficiency and decreased amounts of elastin did not influence the amounts of functional elastin mRNA in aorta. Likewise, the production of tropoelastin in aorta explants was the same whether the explants were taken from copper-sufficient or -deficient birds. The lower accumulation of elastin in aorta from copper-deficient chicks appeared to be due to extracellular proteolysis, rather than to a decrease in the rate of synthesis. Electrophoresis of aorta extracts, followed by immunological detection of tropoelastin-derived products, indicated degradation products in aortae from copper-deficient birds. In extracts of aortae from copper-sufficient chicks, tropoelastin was not degraded and appeared to be incorporated into elastin without further proteolytic processing.     

Coordinate expression of coproporphyrinogen oxidase and cytochrome c6 in the green alga Chlamydomonas reinhardtii in response to changes in copper availability.

To maintain photosynthetic competence under copper-deficient conditions, the green alga Chlamydomonas reinhardtii 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, are 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).     

Cloning and characterization of cDNA of the GPI-anchored purple acid phosphatase and its root tissue distribution in Spirodela oligorrhiza

A cDNA clone of the glycosylphosphatidylinositol (GPI)-anchored purple acid phosphatase (PAP) has been obtained by a combination of cDNA library screening and 5' rapid amplification of cDNA ends from Spirodela oligorrhiza plants grown under phosphate-deficient (−P) conditions. The open reading frame of the S. oligorrhiza PAP cDNA consists of 1 365 bp encoding a 455 amino acid protein. Its deduced amino acid sequence shows 82 and 80% similarity to Arabidopsis thaliana and Phaseolus vulgaris PAP, respectively. The amino acid residue, Ala439, followed by two more small amino acid residues, Asp and Ser, is predicted to be the GPI-anchoring ( ω ) site. The absence of a dibasic motif upstream of the putative ω site suggests that the PAP is a cell wall protein. This presumption is supported by the finding that PAP was released by digestion of the cell wall fraction with cellulase. The GPI anchor is speculated to be a signal for transporting PAP to the cell wall. Immunohistochemical results using −P plant roots demonstrate that PAP is preferentially distributed in the outermost cortical cells of roots but not in the epidermis, suggesting its role in acquiring inorganic phosphate under phosphate-deficient conditions. Northern blot analysis using the S. oligorrhiza PAP cDNA as a probe demonstrates that expression of the PAP gene increased during growth of −P plants and this time-dependent occurrence in mRNA levels of the PAP in −P plants was also observed in their protein and activity levels.     

Complete amino acid sequence of plastocyanin from a green alga, Enteromorpha prolifera

The complete amino acid sequence of the plastocyanin from the green alga Enteromorpha prolifera has been determined by Edman degradation of the intact molecule and fragments produced by enzymatic cleavage of the polypeptide chain with chymotrypsin, Staphylococcus aureus protease, proline-specific endopeptidase, Lys-C endopeptidase and trypsin. The molecule consists of 98 amino acid residues with a calculated relative molecular mass of 10103. The amino acid sequence of E. prolifera plastocyanin shows a high degree of homology with those plastocyanins from other algae and higher plants. In particular, the four residues which are copper ligands in other plastocyanins and in the bacterial electron transport protein azurin (two histidines, one cysteine and one methionine) are conserved. Five out of the six acidic amino acid side-chains which create an 'acidic patch' on the surface of plastocyanin from Populus nigra var. italica [Colman, P. M. et al. (1978) Nature (Lond.) 272, 319-324] are conserved in the amino acid sequence of E. prolifera plastocyanin.     

Identification of the cDNA clone which encodes the 58-kDa protein containing the amino acid sequence of rat liver non-specific lipid-transfer protein (sterol-carrier protein 2). Homology with rat peroxisomal and mitochondrial 3-oxoacyl-CoA thiolases

The relationship between the rat liver non-specific lipid-transfer protein (nsLTP) and the 58-kDa protein cross-reactive with anti-nsLTP antibodies, was investigated by cDNA analysis. A 1945-bp cDNA clone was isolated which encodes a 58.7-kDa protein. This protein is identical to the 58-kDa immunoreactive protein determined by N-terminal sequence analysis of the purified 58-kDa protein. It consists of 546 amino acid residues, of which the 123 C-terminal residues are identical to the sequence of nsLTP. The N-terminal 400 amino acid residues of the 58.7-kDa protein were found to have 23.5% identity with the sequence of both mitochondrial and peroxisomal rat 3-oxoacyl-CoA thiolases, including a hypothetical substrate-binding site. The cDNA insert hybridizes with 1.1-kb, 1.7-kb, 2.4-kb and 3.0-kb mRNA species in RNA isolated from various rat tissues and from Chinese hamster ovary (CHO) cells. Southern blot analysis suggests that these mRNA species are generated from a single gene. Mutant CHO cells, deficient in peroxisomes, lack nsLTP. We have found that the mRNA encoding nsLTP is still present in these cells, which suggests that the absence of this protein is related to the lack of peroxisomes.     

Identification, cloning, and initial characterization of a novel mouse testicular germ cell-specific antigen

A monoclonal antibody, designated TES101, was raised by immunizing BALB/c mice with an allogenic mouse testicular homogenate followed by immunohistochemical selection as the initial screening method. By searching the expressed sequence tag (EST) database with the N-terminal amino acid sequence of TES101 reactive protein, we found that the predicted amino acid sequence encoded by a mouse testicular EST clone matched the TES101 protein sequence. Sequence analysis of the clone revealed no homologous molecule in the DNA/protein database. Based on data obtained from N-terminal amino acid analysis of the TES101 protein, the derived amino acid sequence contained a signal peptide region of 25 amino acids and a mature protein region of 225 amino acids, which translated into a protein with a molecular weight of 24 093. Northern blot analysis showed that mRNA of the TES101 protein was found in testis but not in any other mouse tissues examined. Western blot analysis revealed that TES101 reacted with a 38-kDa band on SDS-PAGE under nonreducing conditions, and this reactivity was abrogated under reducing conditions. Immunoelectron microscopic studies demonstrated that the molecule was predominantly located on the plasma membrane of spermatocytes and spermatids but not in Sertoli cells or interstitial cells, including Leydig cells. Thus, the TES101 protein is a novel molecule present primarily on the surface of developing male germ cells. TES101 protein may play a role in the processes underlying male germ cell formation.     

Rat preprocarboxypeptidase H. Cloning, characterization, and sequence of the cDNA and regulation of the mRNA by corticotropin-releasing factor

Carboxypeptidase H is a putative post-translational processing enzyme which removes basic amino acid residues from intermediates during protein hormone biosynthesis. A 2.2-kilobase pair cDNA was shown to contain the complete amino acid sequence of rat carboxypeptidase H. The deduced amino acid sequence revealed that the enzyme was synthesized as preprocarboxypeptidase H, a precursor form of 476 amino acid residues. Preprocarboxypeptidase H contained a putative hydrophobic signal peptide and a short propeptide which contained 5 adjacent Arg residues at its C terminus. Northern blot analysis identified a single carboxypeptidase H mRNA of approximately 2.3 kilobases in brain, pituitary, and heart, as well as in mouse AtT20 cells. No carboxypeptidase H mRNA was detected in rat liver, spleen, kidney, lung, and mammary gland. Sequence analysis of cDNAs obtained from different rat tissues suggested that a single mRNA encodes an identical carboxypeptidase in several tissues. Treatment of AtT20 cells with dexamethasone decreased the levels of both carboxypeptidase H and preproopiomelanocortin (POMC) mRNAs by approximately 30%. Exposure of the dexamethasone-treated cells to corticotropin-releasing factor effected a 2- to 3-fold increase in the carboxypeptidase H and POMC mRNA levels relative to those of dexamethasone-treated cells exposed to control medium. This suggests that the mRNA levels of POMC and one of its putative post-translational processing enzymes, carboxypeptidase H, are co-regulated by corticotropin-releasing factor and steroid hormones.     

Nutritional copper deficiency does not affect sponge granuloma formation in the rat

Sponge granuloma formation was compared in copper-deficient and copper-sufficient rats following feeding of respective diets for 20, 40, or 60 d. Body weight, total blood hemoglobin, and activities of ceruloplasmin and Cu, Zn-superoxide dismutase in plasma were monitored to ascertain copper deficiency. Mean granuloma weights (mg +/- SEM) in copper-deficient and copper-sufficient groups of rats, respectively, were as follows: 37 +/- 2 and 38 +/- 2 after 20 d, 22 +/- 2 and 23 +/- 2 after 40 d, and 19 +/- 1 and 21 +/- 1 after 60 d on respective diets. Thus, nutritional copper deficiency did not have an effect on sponge granuloma formation in the rat.