Evolutionary conserved N-terminal region of human muscle fructose 1,6-bisphosphatase regulates its activity and the interaction with aldolase.

N-terminal residues of muscle fructose 1,6-bisphosphatase (FBPase) are highly conserved among vertebrates. In this article, we present evidence that the conservation is responsible for the unique properties of the muscle FBPase isozyme: high sensitivity to AMP and Ca(2+) inhibition and the high affinity to muscle aldolase, which is a factor desensitizing muscle FBPase toward AMP and Ca(2+). The first N-terminal residue affecting the affinity of muscle FBPase to aldolase is arginine 3. On the other hand, the first residue significantly influencing the kinetics of muscle FBPase is proline 5. Truncation from 5-7 N-terminal residues of the enzyme not only decreases its affinity to aldolase but also reduces its k-(cat) and activation by Mg(2+), and desensitizes FBPase to inhibition by AMP and calcium ions. Deletion of the first 10 amino acids of muscle FBPase abolishes cooperativity of Mg(2+) activation and results in biphasic inhibition of the enzyme by AMP. Moreover, this truncation lowers affinity of muscle FBPase to aldolase about 14 times, making it resemble the liver isozyme. We suggest that the existence of highly AMP-sensitive muscle-like FBPase, activity of which is regulated by metabolite-dependent interaction with aldolase enables the precise regulation of muscle energy expenditures and might contributed to the evolutionary success of vertebrates.

Glu 69 is essential for the high sensitivity of muscle fructose-1,6-bisphosphatase inhibition by calcium ions.

Muscle fructose-1,6-bisphosphatase (FBPase) is highly sensitive toward inhibition by AMP and calcium ions. In allosteric inhibition by AMP, a loop 52-72 plays a decisive role. This loop is a highly conservative region in muscle and liver FBPases. It is feasible that the same region is involved in the inhibition by calcium ions. To test this hypothesis, chemical modification, limited proteolysis and site directed mutagenesis Glu(69)/Gln were employed. The chemical modification of Lys(71-72) and the proteolytic cleavage of the loop resulted in the significant decrease of the muscle FBPase sensitivity toward inhibition by calcium ions. The mutation of Glu(69)-->Gln resulted in a 500-fold increase of muscle isozyme I(0.5) vs. calcium ions. These results demonstrate the key role that the 52-72 amino acid loop plays in determining the sensitivity of FBPase to inhibition by AMP and calcium ions.

Arsenical resistance genes in Saccharomyces douglasii and other yeast species undergo rapid evolution involving genomic rearrangements and duplications.

We have isolated and characterized three adjacent Saccharomyces douglasii genes that share remarkable structural homology (97% amino acid sequence identity) with Saccharomyces cerevisiae ARR1 (ACR1), ARR2 (ACR2) and ARR3 (ACR3) genes involved in arsenical resistance. The ARR2 and ARR3 genes encoding the cytoplasmic arsenate reductase and the plasma membrane arsenite transporter are functionally interchangeable in both yeast species. In contrast, a single copy of S. douglasii ARR1 gene is not sufficient to complement the arsenic hypersensitivity of a S. cerevisiae mutant lacking the transcriptional activator Arr1p. This inability may be related to a deletion of a 35-bp sequence including the putative Yap-binding element in the ARR1 promoter of S. douglasii. Different mechanisms of regulation of ARR1 genes expression may therefore explain the increased tolerance of S. douglasii to arsenic in comparison with S. cerevisiae. The apparent duplication of the ARR gene cluster in the S. douglasii genome may constitute another factor contributing to the observed differences in arsenic sensitivity. Comparison of ARR genes from the genomes of several yeast species indicates that they are located in subtelomeric regions undergoing rapid evolution involving large-scale genomic rearrangements.

Intragenic suppressors that restore the activity of the maturase encoded by the second intron of the Saccharomyces cerevisiae cyt b gene.

The protein encoded by the second intron (bi2) of the mitochondrial cyt b gene from Saccharomyces cerevisiae functions as a maturase promoting intron splicing. This protein belongs to a large family characterized by the presence of two conserved motifs: LAGLIDADG (or P1 and P2). We have isolated and characterized spontaneous revertants from two mis-sense mutations, G85D and H92P (localized in the P1 motif of the bi2-maturase), that have a detrimental effect on intron splicing. All analyzed revertants are intragenic and resulted from monosubstitutions in the mutated codons. Only true back-mutations that restor the initial glycine 85 and a pseudoreversion that replaces the deleterious aspartic acid 85 by alanine were found in revertants of the mutant G85D. In contrast, all possible monosubstitutions in the mutated codon H92P were identified among the revertants of this mutant. The maturase activity of all novel forms of the protein is similar to the wild-type protein.

Transcriptional activation of metalloid tolerance genes in Saccharomyces cerevisiae requires the AP-1-like proteins Yap1p and Yap8p.

All organisms are equipped with systems for detoxification of the metalloids arsenic and antimony. Here, we show that two parallel pathways involving the AP-1-like proteins Yap1p and Yap8p are required for acquisition of metalloid tolerance in the budding yeast S. cerevisiae. Yap8p is demonstrated to reside in the nucleus where it mediates enhanced expression of the arsenic detoxification genes ACR2 and ACR3. Using chromatin immunoprecipitation assays, we show that Yap8p is associated with the ACR3 promoter in untreated as well as arsenic-exposed cells. Like for Yap1p, specific cysteine residues are critical for Yap8p function. We further show that metalloid exposure triggers nuclear accumulation of Yap1p and stimulates expression of antioxidant genes. Yap1p mutants that are unable to accumulate in the nucleus during H(2)O(2) treatment showed nearly normal nuclear retention in response to metalloid exposure. Thus, our data are the first to demonstrate that Yap1p is being regulated by metalloid stress and to indicate that this activation of Yap1p operates in a manner distinct from stress caused by chemical oxidants. We conclude that Yap1p and Yap8p mediate tolerance by controlling separate subsets of detoxification genes and propose that the two AP-1-like proteins respond to metalloids through distinct mechanisms.

Metalloid tolerance based on phytochelatins is not functionally equivalent to the arsenite transporter Acr3p.

Active transport of metalloids by Acr3p and Ycf1p in Saccharomyces cerevisiae and chelation by phytochelatins in Schizosaccharomyces pombe, nematodes, and plants represent distinct strategies of metalloid detoxification. In this report, we present results of functional comparison of both resistance mechanisms. The S. pombe and wheat phytochelatin synthase (PCS) genes, when expressed in S. cerevisiae, mediate only modest resistance to arsenite and thus cannot functionally compensate for Acr3p. On the other hand, we show for the first time that phytochelatins also contribute to antimony tolerance as PCS fully complement antimonite sensitivity of ycf1Delta mutant. Remarkably, heterologous expression of PCS sensitizes S. cerevisiae to arsenate, while ACR3 confers much higher arsenic resistance in pcsDelta than in wild-type S. pombe. The analysis of PCS and ACR3 homologues distribution in various organisms and our experimental data suggest that separation of ACR3 and PCS genes may lead to the optimal tolerance status of the cell.