Centrins are a subfamily within the superfamily of Ca2+-modulated proteins that play a fundamental role in centrosome duplication and contraction of centrin-based fiber systems. We examined the individual molecular properties of yeast, green alga, and human centrins. Circular dichroism spectroscopy revealed a divergent influence of Ca2+ binding on the alpha-helical content of these proteins. Ca2+-free centrins were elongated in shape as determined by size exclusion chromatography. The presence of Ca2+ and binding peptide resulted in more spherical shaped centrins. In contrast to yeast calmodulin, centrins formed multimers in the Ca2+-bound state. This oligomerization was significantly reduced in the absence of Ca2+ and in the presence of binding peptide. The Ca2+-dependent polymerization of the green alga Scherffelia dubia centrin (SdCen) resulted in a filamentous network. This molecular property was mainly dependent on the amino-terminal subdomain and the peptide-binding site of SdCen. Finally, we analyzed whether SdCen and Cdc31p-SdCen hybrid proteins functionally substitute for the Saccharomyces cerevisiae centrin Cdc31p. Only hybrid proteins containing the amino-terminal subdomain or the third EF-hand of SdCen and the other subdomains from Cdc31p were functional in vivo.
A novel, large-scale method for the purification of cytochrome-c oxidase from the yeast Saccharomyces cerevisiae is described. The isolation procedure gave highly pure and active enzyme at high yields. The purified enzyme exhibited a heme dprotein ratio of 9.1 nmoVmg and revealed twelve protein bands after Tricine/SDSPAGE. N-terminal sequencing showed that eleven of the corresponding proteins were identical to those recently described by Taanman and Capaldi [Taanman, J.-W. & Capaldi, R. A. (1992) J. Biol. Chem. 267, 22481-2248.51. 15 of the N-terminal residues of the 12th band were identical to subunit VIII indicating that this band represents a dimer of subunit VIII (Mr 5364). We conclude that subunit XI1 postulated by Taanman and Capaldi is the subunit VIII dimer and that cytochrome-c oxidase contains eleven rather than twelve subunits.We obtained the complete sequence of subunit VIa by Edman degradation. The protein contains more than 25 % of charged amino acids and hydropathy analysis predicts one membrane-spanning helix.The purified enzyme had a turnover number of 1500 s-' and the ionic-strength dependence of the K, value for cytochrome-c was similar to that described for other preparations of cytochrome-c oxidase. This was al:io true for the cyanide-binding characteristics of the preparation. When the enzyme was isolated in the presence of chloride, more than 90% of the preparation showed fast cyanide-binding kinetics and was resistant to formate incubation, indicating that chloride was bound to the binuclear center. When the enzyme was isolated in the absence of chloride, approximately 70% of the preparation was in the fast form. This high content of fast enzyme was also reflected in the characteristics of optical and EPR spectra for cytochrome-c oxidase purified with our method.Keywo,rds. Cytochrome-c oxidase ; Saccharomyces cerevisiae ; mitochondria ; subunit composition.Cytochronie-c oxidase is the terminal enzyme of the mitochondrial electron-transfer chain catalyzing reduction of oxygen to water [l], The three largest subunits of the eukaryotic enzyme are encoded by mitochondria1 DNA and form the functional core, as they contain all redox centers and are homologous to the subunits of bacterial oxidases [2]. Two hemes (a and a,) and Cu, are ligated by subunit I and the Cu, center is ligated by subunit I1 [3 -51. In addition mitochondria1 cytochrome-c oxidases consists of up to ten nuclear-coded subunits [6]. Most preparations of Saccharomyces cerevisiae cytochroine-c oxidase contain only six nuclear-coded subunits [7], but recently three more bands were identified by SDSPAGE in a small-scale preparation using dodecyl maltoside as a detergent by Taanman and Capaldi [8]. Two o€ the additional bands could be sequenced and turned out to be the homologs to subunits VIa [9] and VIb [lo] of the bovine enzyme. The third band migrated too close to subunit VI to he sequenced in the 21% polyacrylamide Laemmli gel used by these authors [8]. In this study we describe a large-scale preparation for yeast cytochrome...
Cytochrome-c reductase was isolated from Succhuromyces cerevisiue GM50-3C. A tenth subunit was detected with molecular mass 8.5 kDa on SDSIPAGE.Two yeast mutants selected for resistance to myxothiazol, an inhibitor of the Qo center (Q.ubiquinone) of cytochrome-c reductase, were analysed. The single amino acid substitution in the cytochrome-b subunit, N256Y in the mutant Myx-119 and G137R in the mutant Myx-118, caused a general resistance to all methoxyacrylate inhibitors to about fivefold higher concentrations. The kinetic measurements with the substrate analogue nonylbenzohydroquinone revealed a decrease in the K,,, by fivefold and of the maximal turnover number by fourfold in the N256Y mutant. The K , of the C137R mutant was not affected and the V,,, was 50% higher.Cytochrome-c reductase was isolated from mutants to allow determination of the Kd values of methoxyacrylate-stilbene and myxothiazol by means of fluorescence-quench and red-shift titration. Changes in the structure of the multisubunit complex due to a single amino acid exchange became obvious during the purification procedure. SDSjPAGE of the purified enzyme revealed that the substitution N256Y in cytochrome b led to a loss of the iron-sulfur protein and the fifth small subunit with no change in the pattern of the remaining eight subunits. The subunit pattern of the G137R mutant was identical to the wild type. This is the first report of a single amino acid exchange in the catalytic subunit of cytochrome b, greatly affecting the iron-sulfur protein, the second important catalytic subunit of the Qo center. This is a new approach to obtain structural information about the interaction of cytochrome b with the iron-sulfur subunit.All mitochondrial ubiquinol : cytochrome-c reductases comprise three proteins carrying prosthetic groups. These are cytochrome b, the iron-sulfur protein and cytochrome el. Depending on the species, h -8 additional subunits are present. In yeast, the primary sequence of nine subunits has been determined by cloning and sequencing the respective genes [I -81. Sequence alignments showed that all subunits of the yeast enzyme possess homologous counterparts in bovine heart, but the latter contains two additional polypeptides that have so far not been detected in yeast [9]. The function of the non-redox subunits was analyzed by studying the effect of deletion of the particular gene [6. 8, 101. Most of them seem lo be involved in the assembly of mitochondrial cytochromec reductase [I 11.As has been known for a long time, cytochrome h is the only subunit of mitochondrial origin, but deletion of the gene C.'orrespondence to C. von Jagow. Universitatsklinikum, Institut fur Therapeutische Biochemic, lheodor-Stern-Kai 7 Haus 25B, W-6000 Frankfurt 70, Fcderal Rcpublic of Germany Ahhreviutions. NBH, nonylbenzohydroquinone; Q, ubiquinone; TCso, mcdian inhibitory concentration. also causes defects in thc iron-sulfur protein, and the 14-kDa and 11-kDa subunits [12, 131. A different approach to elucidate the structure and function of cytochro...
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