Coenzyme binding capacity of yeast alcohol dehydrogenase.

Commercial lyophilized preparations of yeast alcohol dehydrogenase from Boehringer G.m.b.H. (Mannheim, Germany) bind 2 mols of reduced coenzyme/144000 g of enzyme (1). After the purification by a DEAE-Sephadex column chromatography, the coenzyme binding capacity is raised to 4 mols of NADH/mol of enzyme. Commercial preparations and ionexchange-purified preparations are homogeneous on the ionexchange column chromatography and the disc gel electrophoresis, after reduction with thioglycolic acid. Ionexchange chromatography does not increase the -SH titer, zinc content and the specific activity of enzyme. It is suggested that ionexchange chromatography raises the NADH-binding capacity by removing some impurities present in commercial enzyme preparations.     

Coenzyme A: fatty acid synthetase apoenzyme 4'-phosphopantetheine transferase in yeast

A mixture of two pantetheine-free mutant fatty acid synthetases was dissociated and recombined $\text{in}$$\text{vitro}$ to form a hybrid apoenzyme complex. $\text{In}$$\text{vivo}$ the corresponding $\text{Saccharomyces}$$\text{cerevisiae}$$\text{fas}$-mutants exhibit interallelic complementation when crossed with each other and the enzyme synthesized in the resulting diploid contains pantetheine and exhibits overall fatty acid synthetase activity. Accordingly, the hybrid apoenzyme formed $\text{in}$$\text{vitro}$ could be activated to holo-fatty acid synthetase when incubated with coenzyme A and a partially purified yeast cell extract. The enzyme coenzyme A: fatty acid synthetase apoenzyme 4′-phosphopantetheine transferase has thus been identified in yeast. Further studies on the mechanism of fatty acid synthetase holoenzyme formation will now be possible.     

Functional interaction of yeast elongation factor 3 with yeast ribosomes

Elongation factor 3 (EF-3) is a unique and essential requirement of the fungal translational apparatus. EF-3 is a monomeric protein with a molecular mass of 116,000. EF-3 is required by yeast ribosomes for in vitro translation and for in vivo growth. The protein stimulates the binding of EF-1 alpha :GTP:aa-tRNA ternary complex to the ribosomal A-site by facilitating release of deacylated-tRNA from the E-site. The reaction requires ATP hydrolysis. EF-3 contains two ATP-binding sequence motifs (NBS). NBSI is sufficient for the intrinsic ATPase function. NBSII is essential for ribosome-stimulated activity. By limited proteolysis, EF-3 was divided into two distinct functional domains. The N-terminal domain lacking the highly charged lysine blocks failed to bind ribosomes and was inactive in the ribosome-stimulated ATPase activity. The C-terminally derived lysine-rich fragment showed strong binding to yeast ribosomes. The purported S5 homology region of EF-3 at the N-terminal end has been reported to interact with 18S ribosomal RNA. We postulate that EF-3 contacts rRNA and/or protein(s) through the C-terminal end. Removal of these residues severely weakens its interaction mediated possibly through the N-terminal domain of the protein.     

Induction of yeast killer factor mutations.

Two related killer strains of Saccharomyces cerevisiae were mutagenized and screened for nonkiller variants. About 20% of the mutants derived from one strain lacked all detectable double-straned ribonucleic acid (dsRNA). About 70% of the mutants from the other strain lacked one of the dsRNA species normally associated with the killer factor and had in its place another species of dsRNA with a lower molecular weight.     

Characterization of elongation factor 1 from yeast

Two species of elongation factor 1 (EF-1) differing in molecular weight have been obtained from the postribosomal supernatant fraction of yeast by chromatography on Sephadex G-200. These two forms are present in approximately equal amounts and both appear to be of cytoplasmic origin. Preparations of the higher and lower molecular weight forms of EF-1 catalyze the poly(U)-directed binding of N-acetylphenylalanylt-RNA (AcPhe-tRNA) to yeast ribosomes. The AcPhe-tRNA binding activity of these preparations is consistently lower than the phenylalanyl-tRNA (Phe-tRNA) binding activity and is more sensitive to N-ethylmaleimide. However, the AcPhe-tRNA binding activity co-purifies with EF-1 on phosphocellulose and has the same heat inactivation profile. Several lines of evidence indicate that the AcPhe-tRNA is bound to the acceptor site of the ribosomes. These and other data strongly suggest that yeast EF-1 is capable of catalyzing the binding of both Phe-tRNA and AcPhe-tRNA to ribosomes.     

Coenzyme Q system in the classification of some ascosporogenous yeast genera in the families Saccharomycetaceae and Spermophthoraceae

The Co-Q systems of 11 strains representing the generaSchwanniomyces, Lodderomyces, Lipomyces, Nematospora andMetschnikowia were determined. All the genera were characterized by the Q-9 system except for the genusNematospora with needle-shaped ascospores. The only species,Nem. coryli, was found to have the Q-6 system. These results are discussed from the taxonomic point of view. This constitutes Part VII of a series entitled “Significance of the coenzyme Q system in the classification of yeasts and yeast-like organisms.” The abbreviations used here for coenzyme Q or ubiquinone are: Co-Q, coenzyme Q; Co-Q _n , Q-n or Q _n withn denoting a specified number of isoprene units in a side chain, e.g., Co-Q₆, Q-6, Q₆, etc.     

Coenzyme Q supplementation or over-expression of the yeast Coq8 putative kinase stabilizes multi-subunit Coq polypeptide complexes in yeast coq null mutants

Coenzyme Q biosynthesis in yeast requires a multi-subunit Coq polypeptide complex. Deletion of any one of the COQ genes leads to respiratory deficiency and decreased levels of the Coq4, Coq6, Coq7, and Coq9 polypeptides, suggesting that their association in a high molecular mass complex is required for stability. Over-expression of the putative Coq8 kinase in certain coq null mutants restores steady-state levels of the sensitive Coq polypeptides and promotes the synthesis of late-stage Q-intermediates. Here we show that over-expression of Coq8 in yeast coq null mutants profoundly affects the association of several of the Coq polypeptides in high molecular mass complexes, as assayed by separation of digitonin extracts of mitochondria by two-dimensional blue-native/SDS PAGE. The Coq4 polypeptide persists at high molecular mass with over-expression of Coq8 in coq3, coq5, coq6, coq7, coq9, and coq10 mutants, indicating that Coq4 is a central organizer of the Coq complex. Supplementation with exogenous Q₆ increased the steady-state levels of Coq4, Coq7, and Coq9, and several other mitochondrial polypeptides in select coq null mutants, and also promoted the formation of late-stage Q-intermediates. Q supplementation may stabilize this complex by interacting with one or more of the Coq polypeptides. The stabilizing effects of exogenously added Q₆ or over-expression of Coq8 depend on Coq1 and Coq2 production of a polyisoprenyl intermediate. Based on the observed interdependence of the Coq polypeptides, the effect of exogenous Q₆, and the requirement for an endogenously produced polyisoprenyl intermediate, we propose a new model for the Q-biosynthetic complex, termed the CoQ-synthome.     

Identification of killer factor in the yeast genusMetschnikowia

Summary Out of 260Metschnikowia pulcherrima strains isolated from Sardinian grapes and musts, 6 proved to be killer yeasts. Maximal killing effect occurred between 3.6 and 5.2 pH, and with 48h or 72h cultures.     

Functional characterization of yeast mitochondrial release factor 1

The yeast Saccharomyces cerevisiae mitochondrial release factor was expressed from the cloned MRF1 gene, purified from inclusion bodies, and refolded to give functional activity. The gene encoded a factor with release activity that recognized cognate stop codons in a termination assay with mitochondrial ribosomes and in an assay with Escherichia coli ribosomes. The noncognate stop codon, UGA, encoding tryptophan in mitochondria, was recognized weakly in the heterologous assay. The mitochondrial release factor 1 protein bound to bacterial ribosomes and formed a cross-link with the stop codon within a mRNA bound in a termination complex. The affinity was strongly dependent on the identity of stop signal. Two alleles of MRF1 that contained point mutations in a release factor 1 specific region of the primary structure and that in vivo compensated for mutations in the decoding site rRNA of mitochondrial ribosomes were cloned, and the expressed proteins were purified and refolded. The variant proteins showed impaired binding to the ribosome compared with mitochondrial release factor 1. This structural region in release factors is likely to be involved in codon-dependent specific ribosomal interactions.     

Monoclonal antibody specific for yeast elongation factor 3

Hybridomas have been prepared by fusing mouse myeloma (P3 X 63 Ag8) cells with spleen cells of mice immunized with a yeast fraction enriched with respect to non-ribosomal translational components. Cloned hybridoma lines were grown in the form of ascites tumors, and the monoclonal antibodies produced were purified from the ascites fluid by chromatography on DEAE-Affi-Gel Blue. One of the antibodies, from a hybridoma cell line designated as PSH-1, inhibited the translation of natural mRNA and poly(U) and polysomal chain elongation in a cell-free protein-synthesizing system from yeast. Resolution and partial purification of the elongation factors indicated that the monoclonal antibody from PSH-1 did not interact with EF-1 or EF-2 but reacted with and inactivated EF-3, the 125 000 molecular weight additional elongation factor specifically required with yeast ribosomes. The EF-3 purified from the cytosol by immunoaffinity chromatography was comparable to that prepared by ion-exchange chromatography. Evidence was obtained which indicated that EF-3 was essential for the translation of natural mRNA as well as poly(U), was associated with polysomes but not ribosomal subunits, and was required for every cycle in the elongation phase of protein synthesis.     

Purification and characterization of yeast mitochondrial initiation factor 2

Yeast mitochondrial initiation factor 2 (ymIF2) is encoded by the nuclear IFM1 gene. A His-tagged version of ymIF2, lacking its predicted mitochondrial presequence, was expressed in Escherichia coli and purified. Purified ymIF2 bound both E. coli fMet-tRNA(f)(Met) and Met-tRNA(f)(Met), but binding of formylated initiator tRNA was about four times higher than that of the unformylated species under the same conditions. In addition, the isolated ymIF2 was compared to E. coli IF2 in four other assays commonly used to characterize this initiation factor. Formylated and nonformylated Met-tRNA(f)(Met) were bound to E. coli 30S ribosomal subunits in the presence of ymIF2, GTP, and a short synthetic mRNA. The GTPase activity of ymIF2 was found to be dependent on the presence of E. coli ribosomes. The ymIF2 protected fMet-tRNA(f)(Met) to about the same extent as E. coli IF2 against nonenzymatic deaminoacylation. In contrast to E. coli IF2, the complex formed between ymIF2 and fMet-tRNA(f)(Met) was not stable enough to be analyzed in a gel shift assay. In similarity to other IF2 species isolated from bacteria or bovine mitochondria, the N-terminal domain could be eliminated without loss of initiator tRNA binding activity.