The Na+/Ca2+ exchangers NCX1, NCX2, and NCX3 are vital for the control of cellular Ca2+ homeostasis. Here, we show that a doublet of downstream regulatory element sites in the promoter of the NCX3 gene mediates transcriptional repression of NCX3 by the Ca2+-modulated transcriptional repressor downstream regulatory element antagonist modulator (DREAM). Overexpression of a DREAM EF-hand mutant insensitive to Ca2+ (EFmDREAM) in hippocampus and cerebellum of transgenic mice significantly reduced NCX3 mRNA and protein levels without modifying NCX1 and NCX2 expression. Cerebellar granules from EFmDREAM transgenic mice showed increased levels of cytosolic Ca2+ and were more vulnerable to increased Ca2+ influx after partial opening of voltage-gated plasma membrane Ca2+ channels induced by increasing K+ in the culture medium but survived better in the conditions of reduced Ca2+ influx prevailing in low extracellular K+. Overexpression of NCX3 in EFmDREAM transgenic granules using a lentiviral vector restored the normal survival response to high K+ observed in wild-type granules. Thus, the downregulation of the regulator of Ca2+ homeostasis NCX3 by Ca2+-regulated DREAM is a striking example of the autoregulatory property of the Ca2+ signal in neurons.
A cytochrome b/cl complex which catalyses the reduction of cytochrome c by ubiquinol has been isolated from Rhodopseudomonas sphaeroides GA. It contains two hemes b and substoichiometric amounts of ubiquinone-30 and of the Rieske Fe-S center per cytochrome c1, and is essentially free of reaction center and bacteriochlorophyll. The complex consists of three major polypeptides with apparent molecular masses of 40, 34 and 25 kDa. The 34-kDa polypeptide carries heme. Cytochrome c1 has a midpoint potential of 285 mV. For cytochrome b two midpoint potentials, at 50 and -60 mV, at pH 7.4, can be derived if one assumes two components of equal amount. Ubiquinol -cytochrome c oxidoreductase activity is specific for ubiquinol and bacterial cytochromes c, and is inhibited by antimycin A and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole. The complex shows oxidant-induced reduction of cytochrome b.Respiratory as well as photosynthetic electron transport, in mitochondria [l] (Krinner et al., unpublished). Compared to the mitochondria1 complexes the latter two represent relatively simple polypeptide structures. Preparations from anoxygenic photosynthetic bacteria have not been reported. They would be desirable not only for comparative aspects, but also as a biochemical complimentation to the detailed flash spectrophotometric studies, especially on membranes from Rhodospirillaceae [3 -51.By adotping the procedure for the purification of the cytochrome flb6 complex from spinach chloroplasts [2] we succeeded in the isolation of an active cytochrome b/cl complex from Rhodopseudomonas sphaeroides GA, which is presented here. The procedure is based on the use of octylglucoside/ cholate mixtures to solubilize the complex, which has been also successfully employed to isolate coupling factor ATPases [12,13]. MATERIALS AND METHODS Preparation of Ubiquinol-Cytochrome c2 OxidoreductaseCells of Rhodopseudomonas sphaeroides GA were grown and harvested, and chromatophores were prepared thereDedicated to the memory of Assunta Baccarini-Melandri who initiated this work. Other MaterialsUbiquinols were kindly donated by Hoffmann-LaRoche, Basel. Cytochrome c2 from R. sphaeroides was prepared according to Bartsch [15], and cytochrome c from Saccharomyces cerevisiae was obtained from Sigma. All other sources for materials are given in [2]. AssaysActivity of ubiquinol -cytochrome c2 oxidoreductase was measured in an Aminco DW-2 spectrophotometer as described
The sequence analysis of the human intron 2 from the Na 1 /Ca 21 exchanger 1 (NCX1) gene has revealed a GT repeat of variable length (10±16). The 5 H sequence of intron 2 exhibited significant homology (65±70%) with other minisatellite sequences. DNA segments at the 5 H end of intron 2 were inserted in the NCX1 cDNA (3.7 kb) to reconstruct the exon 2/intron 2 junction. Transient expression of these constructs in HEK293 cells generated shortened mRNAs (< 2.5 kb). RT-PCR and ribonuclease protection analysis of the 3 H end of the short transcripts indicated a splicing event at the intron 2/exon 2 junction (5 H site) and in the vector sequence downstream of the NCX1 insert (3 H site). Molecular dissection of the 5 H -intron 2 sequence showed that the GT repeat was required for splicing activation, whereas the remainder of the 5 H -intron 2 segment was completely inactive. The results indicate that the GT repeat is a strong intronic splicing enhancer that could be involved in the regulation of NCX1 expression, possibly mediating tissue-specific alternative splicing of the mutually exclusive exons 3 and 4, and/or exon-2 circularization.Keywords: human polymorphism; intronic splicing enhancer; Na 1 Ca 21 exchanger.The plasma-membrane Na 1 /Ca 21 exchanger is a reversible transporter that couples the translocation of three Na 1 ions to one Ca 21 [1,2]. The human exchanger is composed of 944 amino acids [3,4], encoded in the 5 H region of a 7-kb mRNA [5], that is assembled by the splicing of 12 exons from the NCX1 gene, located in a 200-kb region of human chromosome 2. In addition, a 5 H -untranslated region is composed of the alternatively spliced exons 1 (a±e) [2,6]. The encoded protein (108 kDa) comprises 11 hydrophobic segments, a cleavable N-terminal signal peptide [5] and a large hydrophilic loop that includes important regulatory domains [7]. About two-thirds of the exchanger protein is encoded in an unusually large exon (exon 2, 1.8 kb), whose structure is conserved in the related NCX3 gene [6]. Tissuespecific variations in the large hydrophilic portion of the NCX1 protein is generated by alternative splicing of exons 3±7, also termed exons A±F [8]. The resulting isoforms are composed of the products of mutually exclusive exons 3 or 4 (A or B), and by different combinations of small cassettetype exons [9±12]. Alternative splicing events at the 3 H end of the canine heart 6-kb cDNA expressed in HK293 cells caused the rearrangement of the C-terminal portion of the protein, i.e. the replacement of the last C-terminal hydrophobic segments, of the last two, or the deletion of the last six hydrophobic segments [13,14]. Two main NCX1 transcripts of 7 kb and 1.8 kb have been identified in heart and in other tissues [3±5,11] corresponding to the full-length mRNA and to a circularized exon 2 form, respectively [15]. Quantitative PCR analysis on the human heart NCX1 cDNA has revealed that the most abundant mRNA was spliced immediately downstream of exon 2, apparently corresponding to the circular exon 2. It has been propos...
The fbc operon from Rhodopseudomonas sphaeroides encodes the three redox carriers of the ubiquinol‐cytochrome‐c reductase (b/c1 complex): FeS protein, cytochrome b and cytochrome c1 [Gabellini, N. et al. (1985) EMBO J. 2, 549‐553]. The nucleotide sequence of 3874 bp of cloned R. sphaeroides chromosomal DNA, including the three structural genes fbcF, fbcB and fbcC has been determined. The reading frames of the fbc genes could be identified readily since the encoded amino acid sequences are highly homologous with the sequences of the corresponding mitochondrial polypeptides. Initiation and termination points for transcription have been investigated by S1 nuclease protection analysis. The transcription of the fbc operon starts approximately 240 base pairs upstream from the start codon of the fbcF gene and terminates 120 base pairs downstream from the stop codon of the fbcC gene. Nucleotide sequences resembling recognition signals for the binding and release of the RNA polymerase were identified. The N‐terminal amino acid sequence of the mature cytochrome c1 was obtained by automated Edman degradation of the isolated subunit, confirming the fbcC reading frame and indicating that the bacterial pre‐apocytochrome c1 has a transient leader sequence including 21 residues. The N‐terminal sequence of one hydrophilic peptide of the FeS protein has been also obtained confirming the fbcF reading frame. The deduced amino acid sequences are discussed in relation to the known primary structures of the homologous proteins from mitochondria and chloroplasts. The primary structures of the polypeptides are evaluated with respect to (a) their topology in the membrane, (b) their biogenesis, (c) the structure of the catalytic sites and (d) subunit interactions.
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