The AFL41 gene (2958 nt) encoding a putative Na+/ H+ antiporter (986 aa) in Saccharomyces cerevisiae was cloned --by selection based on increased NaCl tolerance. The putative protein is highly similar to sodium/proton antiporters from Schizosaccharomyces pombe (gene sod2), and Zygosaccharomyces rouxii (gene Z-SODI). Overexpression of the NHAl gene results in higher and partially pH-dependent tolerance to sodium and lithium; its disruption leads to an increased sensitivity towards these ions.

The R4GI gene of Kluyveromyces lactis encodes a low-affinity glucose/fructose transporter. Its transcription is induced by glucose, fructose, and several other sugars. The RAG4, RAGS, and RAG8 genes are trans-acting genes controlling the expression of the RAG) gene. We report here the characterization of one of these genes, RAG5. The nucleotide sequence of the cloned RAGS gene indicated that it encodes a protein that is homologous to hexokinases of Saccharomyces cerevisiae. ragS mutants showed no detectable hexokinase or glucokinase activity, suggesting that the sugar kinase activity encoded by this gene is the only hexokinase in K. lactis. Both high-and low-affinity transport systems of glucose were affected in ragS mutants. The defect of the low-affinity component was found to be due to a block of transcription of the RAG) gene by the hexokinase mutation. In vivo complementation of the rag5 mutation by the K2 gene of S. cerevisiae and complementation of hxikl hxk2 mutations of S. cerevisiae by the RAGS gene showed that RAGS and HXK2 were equivalent for sugar-phosphorylating activity but that RA4GS could not restore glucose repression in the S. cerevisiae hexokinase mutants.

Some natural isolates and many laboratory strains of the yeast Kluyveromyces lactis cannot grow on glucose when respiration is inhibited by antimycin A. The ability or inability to grow on glucose in the absence of mitochondrial respiration has been called Rag+ or Rag− phenotype (resistance to antimycin on glucose, respectively). Rag− strains, unable to grow on glucose in the presence of the respiratory drug, behave as if they were defective in fermentation. The Rag phenotype was first found to be determined by variant alleles of either of the two nuclear genes, RAG1 and RAG2, which code for a low‐affinity glucose transport protein and for phosphoglucose isomerase, respectively. These findings suggested that the Rag− phenotype can be used to obtain mutations of genes involved in glucose metabolism in K. lactis. We thus looked for other Rag− mutants. Seventy‐four mutants were isolated and genetically characterized. All of the mutations were nuclear recessive alleles, defining 11 new complementation groups, which we designate rag3 through rag13.

The RAG3 gene has been cloned from a Kluyveromyces lactis genomic library by complementation of the rag3 mutation, which shows impaired fermentative growth on glucose in the presence of respiratory inhibitors. From the nucleotide sequence of the cloned DNA, which contained an open reading frame of 765 codons, the predicted protein is 49.5% identical to the Pdc2 protein of Saccharomyces cerevisiae, a regulator of pyruvate decarboxylase in this yeast. Measurement of the pyruvate decarboxylase activity in the original rag3-1 mutant and in the null mutant confirmed that the RAG3 gene is involved in pyruvate decarboxylase synthesis in K. lactis. The effect is exerted at the mRNA level of the pyruvate decarboxylase structural gene KIPDCA. Despite analogies between the RAG3 gene of K. lactis and the PDC2 gene of S. cerevisiae, these genes were unable to reciprocally complement.

A low-affinity glucose transporter gene of Saccharomyces cerevisiae was cloned by complementation of the rag1 mutation in a strain of Kluyveromyces lactis defective in low-affinity glucose transport. Gene sequence and effects of null mutation in S. cerevisiae were described. Data indicated that there are multiple genes for low-affinity glucose transport.