Succhuromyces cerevisiae cells contain D and L-lactate : cytochrome c oxidoreductases. The physiological role of these enzymes is investigated by inhibiting the respiratory chain with antimycin A and observing how the vital functions (respiration, protein biosynthesis, growth) are altered. When D or L-lactate are provided as the source of energy, stoichiometric amounts of the antibiotic decrease the growth rate about five-fold, the oxygen uptake two-fold and the energetic efficiency eleven-fold.In the presence of antimycin A, it was often proposed that only a very short chain consisting of one of the lactate dehydrogenases, cytochrome c and cytochrome oxidase could be involved in the oxidation of lactate. Consistent with this scheme are the measurements of lactate and oxygen uptake and the observation that keto acids are released into the external medium, which also allow one to rationalize the growth yield and the amino acid incorporation results.

In spite of their overall evolutionary conservation, the tubulins of ciliates display electrophoretic and structural particularities. We show here that antibodies raised against Paramecium and Tetrahymena ciliary tubulins fail to recognize the cytoplasmic tubulins of all the metazoans tested. Immunoblotting of peptide maps of ciliate tubulins reveals that these antibodies react with one or very few ciliate-specific epitopes, in contrast to polyclonal antibodies against vertebrate tubulins, which are equivalent to autoantibodies and recognize several epitopes in both ciliate and vertebrate tubulins. Furthermore, we show that the anti-ciliate antibodies recognize ciliary and flagellar tubulins of metazoans ranging from sea urchin to mammals (with the exception of humans). The results support the conclusion that although duplication and specialization of tubulin genes in metazoans may have led to distinct types of tubulins, the axonemal one has remained highly conserved.

A method for the estimation of cytochrome concentrations in whole yeast cells was worked out with strains of Saccharornyces cerevisiae obtained in the laboratory. Differences of cytochrome c content were due to mutations affecting the nature or the quantity of synthetized cytochrome c. Respiratory Toutes les souches utilisees (Sacchromyces cerevisiae) ont 6th construites dans notre laboratoire. Les differences de teneur en cytochrome c que l'on peut observer de l'une A l'autre sont dues b des mutations affectant soit la nature, soit la quantitb du -ou des -cytochromes synth6tisbs. En particulier, nous avons utilise les differentes combinai-

The amino acid sequence of Schizosaccharomyces pombe cytochrome c has been established by automatic degradation of the protein and by manual degradation of fragments obtained by cyanogen bromide cleavage and chymotryptic digestion. The chymotryptic peptides were aligned by homology with other known cytochrome c sequences. The protein is 108 residues long, with a four-residue amino-terminal tail. It has only one methionine residue and differs from other fungal cytochromes c in lacking the one-residue deletion at the C-terminal end.After a cyanogen bromide step, an unexpected cleavage of the peptide chain before a cysteine residue was observed. This is ascribed to formation of a dehydroalanyl residue during an incomplete S-carboxymethylation of the apoprotein, and subsequent cleavage under acidic conditions. Experimental evidence is presented in favour of the proposed mechanisms.The preceding paper reports the purification of Schizosaccharomyces pombe cytochrome c [l 1. The spectrum of this protein presents a splitting of the ci band at liquid nitrogen temperature into three components M I , M Z and ~3 .Such a splitting was first described by Estabrook for a number of species (moth, horse, pigeon, wheat germ), but u2 was notably absent from the spectrum of Saccharomyces cerevisiae cytochrome c [2,3]. Subsequently, Claisse extended these observations and showed that, among yeasts, only some species clearly showed an c12 band [1,4,5].We report here the primary structure of the cytochrome c from Schizosaccharomyces pombe. The work was undertaken in order to see whether sequence data would shed light on the origin of the M band splitting. The amino acid composition of the protein has been reported [l]; it shows the presence of only one methionine residue, which must be methionine 80; this fact was used in the sequence strategy. MATERIALS AND METHODSThe cytochrome c was purified as described by Claisse and Simon-Becam [I]. It was free from nontrimethylated species, and gave a single band upon disc gel electrophoresis at acidic pH. Abbreviation. Me3Lys, e-N-trimethyllysine Preparation of S-Carboxymethylated Apocytochrome cApocytochrome c was prepared according to the method of Ambler [6]. To 1 pmol dissolved in 1 ml deionized 8 M urea were added 40 mg HgC12 in 0.1 ml 1 M HCl. The tube was flushed with nitrogen and shaken in the dark at 37 "C for 20 h. The mixture was then filtered on a Sephadex G15 column (2.5 x 30 cm) equilibrated and eluted with 5 acetic acid. The lyophilized protein was carboxymethylated as described by Crestfield [7]. It was taken up in 0.6 ml 0.1 M Tris-HC1 buffer, pH 8.6 into which 720 mg deionized urea were dissolved under nitrogen bubbling; 11 mg dithiothreitol in 0.2 ml 8 M urea, 0.2% EDTA, were then added. After 4 h under nitrogen, carboxymethylation was initiated by addition of 27 mg [2-14C]iodoacetic acid (recrystallized from ether/hexane) dissolved in 0.1 ml 0.5 M NaOH. After 20 min in the dark, the reaction was stopped by addition of 10 pl2-mercaptoethanol. The mixture was then filtered in...

The addition of antimycin A during the logarithmic phase of growth of heterotrophic Euglena gracilis cultures (in lactate or glucose medium) was immediately followed by decreased respiration and a cessation of grwoth. Induced cyanideresistent respiration appeared 5 h after the addition of the inhibitor then the cells started to grow again and could be cultured in the presence of antimycin A. Thus the cells exhibited a cyanide-and antimycin-resistant respiration which was, in addition, sensitive to salicylhydroxamic acid and propylgallate. Antimycin-adapted Euglena and control cells were compared for their biomass production and protein synthesis. The difference in growth yield between control and antimycin-adapted cells was not as high as would be expected if only the first phosphorylation site of the normal respiratory chain was active in the presence of antimycin A. Furthermore, the ability to incorporate labelled valine into proteins, under resting-cell conditions, was not changed. Strong correlations were established between the effects of respiratory effectors on O2 consumption and valine incorporation. These results suggest that sufficient energy for protein synthesis and growth is provided by the operation of the cyanide-resistant respiratory pathway in antimycin-adapted Euglena.