Binding of centrins and yeast calmodulin to synthetic peptides corresponding to binding sites in the spindle pole body components Kar1p and Spc110p.

Centrins contain four potential Ca2+ binding sites, known as EF-hands, and have essential functions in centrosome duplication and filament contraction. Here we report that centrins from yeast, green algae, and humans bound with high affinity to a peptide of the yeast centrosomal component Kar1p. Interestingly, centrin binding was regulated by physiological relevant changes in [Ca2+], and this Ca2+ dependence was influenced by acidic amino acids within the Kar1p peptide, which also prevented efficient binding of the related yeast calmodulin. However, a hybrid protein with the third and fourth EF-hands from the yeast centrin Cdc31p and the amino-terminal half from yeast calmodulin behaved more like Cdc31p, indicating that the carboxyl-terminal half of Cdc31p influences binding specificity. Besides Kar1p, centrins bound to a yeast calmodulin binding site, explaining the dosage-dependent suppression of a calmodulin mutant by CDC31. Consistent with an essential role of Ca2+ for centrin functions, mutations in the first or the fourth EF-hands of Cdc31p, impairing the Ca2+-induced conformational change of Cdc31p, resulted in nonfunctional proteins in vivo. Our results suggest that centrins are involved in Ca2+ signaling, likely by influencing the properties of target proteins in response to changes in [Ca2+].

Characterization of green alga, yeast, and human centrins. Specific subdomain features determine functional diversity.

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.

Kinetic properties and ligand binding of the eleven-subunit cytochrome-c oxidase from Saccharomyces cerevisiae isolated with a novel large-scale purification method.

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 a/protein ratio of 9.1 mmol/mg and revealed twelve protein bands after Tricine/SDS/PAGE. 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, 22,481-22,485]. 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 (M(r) 5364). We conclude that subunit XII 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-1 and the ionic-strength dependence of the Km value for cytochrome-c was similar to that described for other preparations of cytochrome-c oxidase. This was also 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.

Analysis of cytochrome-b amino acid residues forming the contact face with the iron-sulfur subunit of ubiquinol:cytochrome-c reductase in Saccharomyces cerevisiae.

Four mutations in the mitochondrial cytochrome b of Saccharomyces cerevisiae have been characterized with respect to catalytic properties, inhibitor resistance and subunit interaction. The respiratory-deficient mutant [G137E]cytochrome b and the pseudo-wild-type revertant [G137E, N256K]cytochrome b were described previously [di Rago, J.-P., Netter, P. & Slonimski, P. P. (1990) J. Biol. Chem. 265, 3332-3339; di Rago, J.-P., Netter, P. & Slonimski, P. P. (1990) J. Biol. Chem. 265, 15750-15757]. Two new mutants [N256K]cytochrome b and [N256I]cytochrome b were isolated by dissociation of the second-site suppressor from the original target mutation. The mutants [G137E]cytochrome b and [G137E, N256K]cytochrome b exhibited a high resistance against methoxyacrylate inhibitors, whereas the suppressors [N256K]cytochrome b and [N256I]cytochrome b showed only a slight resistance. Remarkably, all mutants exhibited stigmatellin cross-resistance. The electron-transfer activity from the substrate nonylubiquinol to cytochrome c of mitochondrial membranes was diminished in all mutants. The substitution G137-->E decreases Vmax/Km by one order of magnitude, indicating a reduced catalytic efficiency for ubiquinol. The amino acid exchange at position 256 to a positively charged lysine residue or to a hydrophobic isoleucine residue resulted mainly in a diminished specific activity. The iron-sulfur subunit and the 8.5-kDa subunit were detectable in all mutants at normal levels in immunoblots of membrane preparations, indicating proper assembly of the complex. However, after purification, the mutant bc1 complex lacked the iron-sulfur subunit and the 8.5-kDa subunit. In contrast, the iron-sulfur subunit can only be dissociated from the parental bc1 complex by harsh treatment. These data suggest that residues 137 and 256 in cytochrome b are crucial for cytochrome-b/iron-sulfur protein interaction.

Point mutation in cytochrome b of yeast ubihydroquinone:cytochrome-c oxidoreductase causing myxothiazol resistance and facilitated dissociation of the iron-sulfur subunit.

Cytochrome-c reductase was isolated from Saccharomyces cerevisiae GM50-3C. A tenth subunit was detected with molecular mass 8.5 kDa on SDS/PAGE. Two yeast mutants selected for resistance to myxothiazol, an inhibitor of the Q0 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 Km by fivefold and of the maximal turnover number by fourfold in the N256Y mutant. The Km of the G137R mutant was not affected and the Vmax 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. SDS/PAGE 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 Q0 center. This is a new approach to obtain structural information about the interaction of cytochrome b with the iron-sulfur subunit.

The effect of mercuric salts on the electro-rotation of yeast cells and comparison with a theoretical model.

The rotational spectrum of yeast cells changed after pre-treatment of the cells with HgCl2 or Hg(NO3)2 and became indistinguishable from that of ultrasonically produced cell walls. The spectrum of the affected cells contained a peak which could only be explained by attributing a conductivity to the cell walls that was higher than that of the medium. Theoretical models of the rotational response are fully in accord with the experimental spectra. It is shown that the rotation method is capable of measuring even the low cell wall conductivity of yeast cells (which was found to be 33 microS/cm at 10 microS/cm medium conductivity). Knowledge of the spectra allowed a field frequency to be selected at which untreated cells showed no rotation, but at which cells affected by treatment with Hg(II) identified themselves by rotating in the same direction as the field. Calculation of the percentage of cells showing this co-field rotation gave an index (termed the co-field rotation value) of the proportion of the cells that were affected. Using this technique, effects of 25 nmol/l Hg(II) could be demonstrated. In media of low conductivity (10 microS/cm) the change in the rotational spectrum was usually 'all-or-none', whereas at 200 microS/cm a graded Hg(II)-mediated change became apparent. The co-field rotation method showed that the action of small quantities of Hg(II) was still increasing after 3 h of incubation and paralleled the Hg(II)-induced K+ release. A rapid reduction of the effects of Hg(II) was seen when 3-30 mM K+ (or Na+) or when 1 mM Ca2+ were present in the incubation medium, or as the pH was increased. At high incubation cell concentrations the toxic effect of Hg(II) was reduced, apparently due to binding by the cells.