A DNA fragment containing the Saccharomyces cerevisiae CYS3 (CYII) gene was cloned. The clone had a single open reading frame of 1,182 bp (394 amino acid residues). By comparison of the deduced amino acid sequence with the N-terminal amino acid sequence of cystathionine 'y-lyase, CYS3 (CYI1) was concluded to be the structural gene for this enzyme. In addition, the deduced sequence showed homology with the following enzymes: rat cystathionine y-lyase (41%), Escherichia coli cystathionine -y-synthase (36%), and cystathionine 13-lyase (25%). The N-terminal half of it was homologous (39%o) with the N-terminal half of S. cerevisiae O-acetylserine and O-acetylhomoserine sulfhydrylase. The cloned CYS3 (CYIJ) gene marginally complemented the E. coli metB mutation (cystathionine 'y-synthase deficiency) and conferred cystathionine 'y-synthase activity as well as cystathionine y-lyase activity to E. coli; cystathionine 'y-synthase activity was detected when O-succinylhomoserine but not O-acetylhomoserine was used as substrate. We therefore conclude that S. cerevisiae cystathionine 'y-lyase and E. coli cystathionine -y-synthase are homologous in both structure and in vitro function and propose that their different in vivo functions are due to the unavailability of O-succinylhomoserine in S. cerevisiae and the scarceness of cystathionine in E. coli.Saccharomyces cerevisiae biosynthetic pathways of the sulfur-containing amino acids cysteine and methionine are shown in Fig. 1. Cysteine is synthesized via two pathways. One consists of serine O-acetyltransferase (SATase) (EC 2.3.1.30) and O-acetylserine sulfhydrylase (OAS SHLase) (EC 4.2.99.8). This is the autotrophic pathway for sulfur utilization, because inorganic sulfur is converted to organic sulfur. It is similar to the enteric bacterial and plant cysteine biosynthetic pathways (for reviews, see references 9 and 37). In enteric bacteria and plants, cysteine sulfur is converted to homocysteine sulfur via transsulfuration consisting of cystathionine y-synthase (-y-CTSase) (EC 4.2.99.9) and cystathionine P-lyase (1-CTLase) (EC 4.4.1.8), and the resultant homocysteine is used for methionine biosynthesis. The same takes place in S. cerevisiae. However, it should be stressed that while S. cerevisiae uses O-acetylhomoserine (OAH) for cystathionine biosynthesis, enteric bacteria and plants use O-succinylhomoserine (OSH) and O-phosphohomoserine, respectively.The other S. cerevisiae cysteine biosynthetic pathway is reverse transsulfuration, which consists of cystathionine ,B-synthase (P-CTSase) (EC 4.2.1.22) and cystathionine y-lyase (-y-CTLase) (EC 4.4.1.1). In this pathway, homocysteine sulfur is converted to cysteine sulfur. This pathway is present in mammals but in neither enteric bacteria nor plants (for a review, see reference 10). Mammals synthesize homocysteine exclusively by demethylation of methionine and convert homocysteine sulfur to cysteine sulfur. In this respect, reverse transsulfuration is a part of the heterotrophic pathway for sulfur utilization. S. cerevisiae syn...

Regulation of the two enzymes in reverse trans-sulfuration was investigated in Saccharomyces cerevisiae. In wild-type strains, cystathionine gamma-lyase, but not cystathionine beta-synthase, was depressed nearly 15-fold if cells were starved for both inorganic and organic sulfur compounds. In a met17 strain which is defective of O-acetylserine and O-acetylhomoserine sulfhydrylase, the same enzyme was derepressed if organic sulfur compounds were limited; the repressive effect was in the order of glutathione greater than methionine greater than cysteine. The repressive effect of methionine was not observed, however, in a cys2 cys4 strain which is deficient of serine O-acetyltransferase and cystathionine beta-synthase, indicating that methionine itself is not the effector. The weak repressive effect of cysteine was attributed to inefficient uptake of this amino acid. Our observations indicate that cystathionine gamma-lyase is the target of regulation in reverse trans-sulfuration and that cysteine is very likely to be the effector of this regulation.

A DNA fragment containing the CYS4 gene of Saccharomyces cerevisiae was isolated from a genomic library. The cloned fragment hybridized to the transverse-alternating-field-electrophoresis band corresponding to chromosomes VII and XV. According to the 2 microns DNA chromosome-loss procedure, the cys2 and cys4 mutations, which are linked together and co-operatively confer cysteine dependence, were assigned to chromosome VII. By further mapping involving tetrad analysis, the cys2-cys4 pair was localized between SUP77 (SUP166) and ade3 on the right arm of chromosome VII.

This study suggests that our programme in a university setting seems to be effective mainly because of peer-supports among the participants through regular face-to-face meetings and their own mailing supports.

Although Saccharomyces cerevisiae strains had different cysteine uptake activities, they revealed monophasic uptake kinetics and had the same KT (83.3 microM). The optimal pH of cysteine uptake was between 4.5 and 5.0, but the activity was quickly lost if cells were kept in buffer. When the activity was measured in the growth medium, it increased in the presence of EDTA and greatly decreased in the presence of mercuric chloride. Thioglycol as well as metabolic inhibitors such as dinitrophenol and azide were inhibitory. Homocysteine and methionine were competitive and non-competitive inhibitors, respectively. Cysteamine and cysteic acid were not inhibitory. From these observations, we conclude that the system mediating uptake of cysteine is specific (we thus name it the cysteine transport system) and that the cysteine transport system recognizes not only the SH-group but also amino- and carboxyl-groups. In wild-type strains the cysteine transport system was derepressed only when the cells were incubated without any sulfur source. On the other hand, in cysteine-dependent mutants, cysteine uptake activity increased with increase of exogenous supply of cysteine, glutathione or methionine. From this result, we suspect that the cellular cysteine level is the limiting factor for biosynthesis of the cysteine transport system in cysteine-dependent strains.