Stanislaw Ulaszewski

Isolation of three contiguous genes, ACR1, ACR2 and ACR3, involved in resistance to arsenic compounds in the yeast Saccharomyces cerevisiae.

A 4·2 kb region from Saccharomyces cerevisiae chromosome XVI was isolated as a yeast fragment conferring resistance to 7 mM‐sodium arsenite (NaAsO2), when put on a multicopy plasmid. Homology searches revealed a cluster of three new open reading frames named ACR1, ACR2 and ACR3. The hypothetical product of the ACR1 gene is similar to the transcriptional regulatory proteins, encoded by YAP1, and YAP2 genes from S. cerevisiae. Disruption of the ACR1 gene conduces to an arsenite and arsenate hypersensitivity phenotype. The ACR2 gene is indispensable for arsenate but not for arsenite resistance. The hypothetical product of the ACR3 gene shows high similarity to the hypothetical membrane protein encoded by Bacillus subtilis ORF1 of the skin element and weak similarity to the ArsB membrane protein of the Staphylococcus aureus arsenical‐resistance operon. Overexpression of the ACR3 gene confers an arsenite‐ but not an arsenate‐resistance phenotype. The presence of ACR3 together with ACR2 on a multicopy plasmid expands the resistance phenotype into arsenate. These findings suggest that all three novel genes: ACR1, ACR2 and ACR3 are involved in the arsenical‐resistance phenomenon in S. cerevisiae.

Genetic mapping of nuclear mucidin resistance mutations in Saccharomyces cerevisiae

SummaryIn the yeast Saccharomyces cerevisiae, two nuclear pleiotropic drug resistance mutations pdr3-1 (former designation mucPR) and pdr3-2 (former designation DRI9/T7) have been selected as resistant to mucidin and as resistant to chloramphenicol plus cycloheximide, respectively. The pdr3 mutations were found not to affect the plasma membrane ATPase activity measured in a crude membrane fraction. Meiotic mapping using strains with standard genetic markers revealed that mutation pdr3-1 is centromere linked on the left arm of chromosome II at a distance of 5.9 ± 3.3 cM from its centromere and 11.6 ± 3.1 cM from the marker pet9. The centromere linked pdr3-2 mutation exhibited also genetic linkage to pet9 with a map distance of 9.8 ± 3.2 cM. These results indicate that pdr3-1 and pdr3-2 are alleles of the same pleiotropic drug resistance locus PDR3 which is involved in the control of the plasma membrane permeability in yeast.

Genetic and molecular mapping of the pma1 mutation conferring vanadate resistance to the plasma membrane ATPase from Saccharomyces cerevisiae

SummaryIn the yeast Saccharomyces cerevisiae, the pma1 mutations confers vanadate-resistance to H+-ATPase activity when measured in isolated plasma membranes. In vivo, the growth of pma1 mutants is resistant to Dio-9, ethidium bromide and guanidine derivatives. This phenotype was used to man the pma1 mutation adjacent to LEU1 gene on chromosome VII. From a cosmid library of a wild-type Saccharomyces cerevisiae genome, a large 30 kb DNA fragment was isolated by complementation of a leu1-pma1 double mutant. A 5 kb HindIII fragment was subcloned and it restored both Leu+ and Pma+ phenotypes after integrative transformation. The restriction map of the 5 kb HindIII fragment and Southern blot analysis reveal that the cloned fragment contains the entire structural gene for the plasma membrane ATPase and the 5′ end of the adjacent LEU1 gene. The pma1 mutation conferring vanadate-resistance is thus located in the structural gene for the plasma membrane ATPase.

Cyclic AMP controls the plasma membrane H+-ATPase activity from Saccharomyces cerevisiae.

The thermosensitive G1‐arrested cdc35‐10 mutant from Saccharomyces cerevisiae, defective in adenylate cyclase activity, was shifted to restrictive temperature. After 1 h incubation at this temperature, the plasma membrane H+‐ATPase activity of cdc35‐10 was reduced to 50%, whereas that in mitochondria doubled. Similar data were obtained with cdc25, another thermosensitive G1‐arrested mutant modified in the cAMP pathway. In contrast, the ATPase activities of the G1‐arrested mutant cdc19, defective in pyruvate kinase, were not affected after 2 h incubation at restrictive temperature. In the double mutants cdc35‐10 cas1 and cdc25 cas1, addition of extracellular cAMP prevented the modifications of ATPase activities observed in the single mutants cdc35‐10 and cdc25. These data indicate that cAMP acts as a positive effector on the H+‐ATPase activity of plasma membranes and as a negative effector on that of mitochondria.

Different sensitivities of mutants and chimeric forms of human muscle and liver fructose-1,6-bisphosphatases towards AMP

Abstract AMP is an allosteric inhibitor of human muscle and liver fructose-1,6-bisphosphatase (FBPase). Despite strong similarity of the nucleotide binding domains, the muscle enzyme is inhibited by AMP approximately 35 times stronger than liver FBPase: I0.5 for muscle and for liver FBPase are 0.14 uM and 4.8 uM, respectively. Chimeric human muscle (L50M288) and chimeric human liver enzymes (M50L288), in which the N-terminal residues (1-50) were derived from the human liver and human muscle FBPases, respectively, were inhibited by AMP 2-3 times stronger than the wild-type liver enzyme. An amino acid exchange within the Nterminal region of the muscle enzyme towards liver FBPase (Lys20→Glu) resulted in 13-fold increased I0.5 values compared to the wild-type muscle enzyme. However, the opposite exchanges in the liver enzyme (Glu20→Lys and double mutation Glu19→Asp/Glu20→Lys) did not change the sensitivity for AMP inhibition of the liver mutant (I0.5 value of 4.9 uM). The decrease of sensitivity for AMP of the muscle mutant Lys20→Glu, as well as the lack of changes in the inhibition by AMP of liver mutants Glu20→Lys and Glu19→Asp/Glu20→Lys, suggest a different mechanism of AMP binding to the muscle and liver enzyme.

Vmr 1p is a novel vacuolar multidrug resistance ABC transporter in Saccharomyces cerevisiae

The Saccharomyces cerevisiae Yhl035p/Vmr1p is an ABC transporter of the MRP subfamily that is conserved in all post Whole Genome Duplication species. The deletion of the YHL035 gene caused growth sensitivity to several amphiphilic drugs such as cycloheximide, 2,4-dichlorophenoxyacetic acid, 2,4-dinitrophenol as well as to cadmium and other toxic metals. Vmr1p-GFP was located in the vacuolar membrane. The ATP-dependent transport of a DNP-S-glutathione conjugate was reduced in a vesicular fraction from the VMR1 deletant. The energy-dependent efflux of rhodamine 6G was increased by VMR1 deletion. Growth sensitivity to cadmium of the VMR1-deleted strain was more pronounced in glycerol/ethanol than in glucose-grown cells. The VMR1 promoter had higher activity when grown in glycerol/ethanol compared with glucose. In glucose, the VMR1 promoter was activated by the deletion of the glucose-dependent repressor ADR1.

A new mutation for multiple drug resistance and modified plasma membrane ATPase activity in Schizosaccharomyces pombe

SummaryThe mutant JV66 was selected from the wild type strain of S. pombe 972h−ade7-413 by its ability to grow on solid rich medium containing 200 μg Dio-9/ml. The single nuclear mutation, designated pma1 gives resistance towards diguanidines and several other positively charged compounds. The pma1 mutation also decreases plasma membrane ATPase activity and confers resistance of ATPase to vanadate. The pma1 locus is localized on chromose I at 5.3 map units from cyh1-C7 and at about 20.7 map units from the centromere. This new mutation is genetically and phenotypically different from the mutation cyh3 and cyh4 previously described (Johnston and Coddington 1983).

The origin of the high sensitivity of muscle fructose 1,6-bisphosphatase towards AMP

Adenosine 5′‐monophosphate (AMP) inhibits muscle fructose 1,6‐bisphosphatase (FBPase) about 44 times stronger than the liver isozyme. The key role in strong AMP binding to muscle isozyme play K20, T177 and Q179. Muscle FBPase which has been mutated towards the liver enzyme (K20E/T177M/Q179C) is inhibited by AMP about 26 times weaker than the wild‐type muscle enzyme, but it binds the fluorescent AMP analogue, 2′,3′‐O‐(2,4,6‐trinitrophenyl)adenosine 5′‐monophosphate (TNP‐AMP), similarly to the wild‐type liver enzyme. The reverse mutation of liver FBPase towards the muscle isozyme significantly increases the affinity of the mutant to TNP‐AMP. High affinity to the inhibitor but low sensitivity to AMP of the liver triple mutant suggest differences between the isozymes in the mechanism of allosteric signal transmission.

Functional analysis of three adjacent open reading frames from the right arm of yeast: Chromosome XVI

A 7·24 kb genomic DNA fragment from the yeast Saccharomyces cerevisiae chromosome XVI was isolated by complementation of a new temperature‐sensitive mutation tsa1. We determined the nucleotide sequence of this fragment located on the right arm of chromosome XVI. Among the three, complete open reading frames: YPR041w, YPR042c and YPR043w contained within this fragment, the gene YPR041w was shown to complement the tsa1 mutation and to correspond to the TIF5 gene encoding an essential protein synthesis initiation translation factor. The YPR042c gene encodes a hypothetical protein of 1075 amino acids containing four putative transmembrane segments and is non‐essential for growth. The gene YPR043c encoding the 10 kDa product, highly similar to the human protein L37a from the 60S ribosomal subunit, was found to be essential and a dominant lethal. We conclude that three tightly linked yeast genes are involved in the translation process.

Genetic modification of cytochrome b deficient mutants in Saccharomyces cerevisiae

SummaryGenetic investigation of the interaction between two respiratory deficient mutants pet29 and op1 led to discovery of a new dominant gene Mod2 which modifies the phenotype of mutant carrying pet29 gene, to full respiratory proficiency.