Mechanisms involved in metalloid transport and tolerance acquisition.

Toxic metalloids such as arsenic and antimony have always been an integral part of the natural environment. To survive in such a hostile habitat, it is crucial to develop strategies to exclude toxic substances from the cell and to acquire tolerance. Cells remove metalloids from the cytosol either by active efflux or by sequestration in an internal organelle. Controlling the influx appears to be another way of maintaining a low intracellular metalloid content. Inside the cell, the metalloid can be reduced to a form that is recognised by the expulsion system(s). In addition, metalloid complexation and compartmentalisation contributes to enhanced cellular tolerance. Finally, the presence of metalloids activates transcription of various cellular defence genes. Metalloid-containing drugs are currently used to treat protozoan infections and promyelocytic leukaemia. Since metalloid resistance hampers efficient treatment, interest in identifying the mechanisms involved in tolerance acquisition has arisen. The possibility of using genetic approaches has made the yeast Saccharomyces cerevisiae a compelling model system to investigate the basis of metalloid tolerance at a molecular level. This review describes the recent progress made in elucidating the mechanisms involved in metalloid transport and tolerance in yeast and other organisms.

The glycerol channel Fps1p mediates the uptake of arsenite and antimonite in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae FPS1 gene encodes a glycerol channel protein involved in osmoregulation. We present evidence that Fps1p mediates influx of the trivalent metalloids arsenite and antimonite in yeast. Deletion of FPS1 improves tolerance to arsenite and potassium antimonyl tartrate. Under high osmolarity conditions, when the Fps1p channel is closed, wild-type cells show the same degree of As(III) and Sb(III) tolerance as the fps1Delta mutant. Additional deletion of FPS1 in mutants defective in arsenite and antimonite detoxification partially suppresses their hypersensitivity to metalloid salts. Cells expressing a constitutively open form of the Fps1p channel are highly sensitive to both arsenite and antimonite. We also show by direct transport assays that arsenite uptake is mediated by Fps1p. Yeast cells appear to control the Fps1p-mediated pathway of metalloid uptake, as expression of the FPS1 gene is repressed upon As(III) and Sb(III) addition. To our knowledge, this is the first report describing a eukaryotic uptake mechanism for arsenite and antimonite and its involvement in metalloid tolerance.

Molecular and physiological characterization of the NAD-dependent glycerol 3-phosphate dehydrogenase in the filamentous fungus Aspergillus nidulans.

In filamentous fungi, glycerol biosynthesis has been proposed to play an important role during conidiospore germination and in response to a hyperosmotic shock, but little is known about the genes involved. Here, we report on the characterization of the major Aspergillus nidulans glycerol 3-phosphate dehydrogenase (G3PDH)-encoding gene, gfdA. G3PDH is responsible for the conversion of dihydroxyacetone phosphate (DHAP) into glycerol 3-phosphate (G3P), which is subsequently converted into glycerol by an as yet uncharacterized phosphatase. Inactivation of gfdA does not abolish glycerol biosynthesis, showing that the other pathway from DHAP, via dihydroxyacetone (DHA), to glycerol is also functional in A. nidulans. The gfdA null mutant displays reduced G3P levels and an osmoremediable growth defect on various carbon sources except glycerol. This growth defect is associated with an abnormal hyphal morphology that is reminiscent of a cell wall defect. Furthermore, the growth defect at low osmolarity is enhanced in the presence of the chitin-interacting agent calcofluor and the membrane-destabilizing agent sodium dodecyl sulphate (SDS). As inactivation of gfdA has no impact on phospholipid biosynthesis or glycolytic intermediates levels, as might be expected from reduced G3P levels, a previously unsuspected link between G3P and cell wall integrity is proposed to occur in filamentous fungi.

Molecular characterization of the plasma membrane H(+)-ATPase, an antifungal target in Cryptococcus neoformans.

The Cryptococcus neoformans PMA1 gene, encoding a plasma membrane H(+)-ATPase, was isolated from a genomic DNA library of serotype A strain ATCC 6352. An open reading frame of 3,380 nucleotides contains six introns and encodes a predicted protein consisting of 998 amino acids with a molecular mass of approximately 108 kDa. Plasma membranes were isolated, and the H(+)-ATPase was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be slightly larger than the S. cerevisiae H(+)-ATPase, consistent with its predicted molecular mass. The plasma membrane-bound enzyme exhibited a pH 6.5 optimum for ATP hydrolysis, K(m) and V(max) values of 0.5 mM and 3.1 micromol mg(-1) min(-1), respectively, and an apparent K(i) for vanadate inhibition of 1.6 microM. ATP hydrolysis in plasma membranes and medium acidification by whole cells were inhibited by ebselen, a nonspecific H(+)-ATPase antagonist which was also fungicidal. The predicted C. neoformans protein is 35% identical to proton pumps of both pathogenic and nonpathogenic fungi but exhibits more than 50% identity to PMA1 genes from plants. Collectively, this study provides the basis for establishing the Cryptococcus H(+)-ATPase as a viable target for antifungal drug discovery.

Stimulation of the yeast high osmolarity glycerol (HOG) pathway: evidence for a signal generated by a change in turgor rather than by water stress.

The Saccharomyces cerevisiae HOG pathway controls responses to osmotic shock such as production of the osmolyte glycerol. Here we show that the HOG pathway can be stimulated by addition of glycerol. This stimulation was strongly diminished in cells expressing an unregulated Fps1p glycerol channel, presumably because glycerol rapidly equilibrated across the plasma membrane. Ethanol, which passes the plasma membrane readily and causes water stress by disturbing the hydration of biomolecules, did not activate the HOG pathway. These observations suggest that stimulation of the HOG pathway is mediated by a turgor change and not by water stress per se.

Fps1p controls the accumulation and release of the compatible solute glycerol in yeast osmoregulation.

The accumulation of compatible solutes, such as glycerol, in the yeast Saccharomyces cerevisiae, is a ubiquitous mechanism in cellular osmoregulation. Here, we demonstrate that yeast cells control glycerol accumulation in part via a regulated, Fps1p-mediated export of glycerol. Fps1p is a member of the MIP family of channel proteins most closely related to the bacterial glycerol facilitators. The protein is localized in the plasma membrane. The physiological role of Fps1p appears to be glycerol export rather than uptake. Fps1 delta mutants are sensitive to hypo-osmotic shock, demonstrating that osmolyte export is required for recovery from a sudden drop in external osmolarity. In wild-type cells, the glycerol transport rate is decreased by hyperosmotic shock and increased by hypo-osmotic shock on a subminute time scale. This regulation seems to be independent of the known yeast osmosensing HOG and PKC signalling pathways. Mutants lacking the unique hydrophilic N-terminal domain of Fps1p, or certain parts thereof, fail to reduce the glycerol transport rate after a hyperosmotic shock. Yeast cells carrying these constructs constitutively release glycerol and show a dominant hyperosmosensitivity, but compensate for glycerol loss after prolonged incubation by glycerol overproduction. Fps1p may be an example of a more widespread class of regulators of osmoadaptation, which control the cellular content and release of compatible solutes.

Probing conserved regions of the cytoplasmic LOOP1 segment linking transmembrane segments 2 and 3 of the Saccharomyces cerevisiae plasma membrane H+-ATPase.

Genetic probing was used to examine conserved amino acid clusters in the first cytoplasmic loop domain (LOOP1) linking transmembrane segments 2 and 3 of the plasma membrane H+-ATPase from Saccharomyces cerevisiae. Deletion of the LOOP1 region in PMA1 resulted in a defective enzyme. Scanning alanine mutagenesis of conserved residues produced lethal cell phenotypes in 14 of 26 amino acids, suggesting major enzyme defects. Most viable mutants showed growth characteristics that were comparable to wild type. Two mutations, I183A and D185A, produced reduced growth rates, hygromycin B resistance, and low pH sensitivity, which are phenotypes associated with defects in the H+-ATPase. However, both mutant enzymes displayed near-normal kinetics for ATP hydrolysis in vitro. Localized random mutagenesis was also performed at sites Glu195, Val196, and Ile210, which all showed lethal phenotypes upon conversion to alanine. Amino acids with polar side groups could substitute for Glu195, while Val196 could not tolerate polar side group moieties. Nine mutations at Ile210 proved lethal, including K, R, E, P, H, N, V, G, and A, while functional enzyme was obtained with S, C, M, and L. Normal rates and extents of pH gradient formation were observed for all mutant enzymes, except I183A and D185A. Detailed analysis of the I183A enzyme indicated that it hydrolyzed ATP like wild type, but it appeared to inefficiently couple ATP hydrolysis to proton transport. In total, these results affirm that conserved amino acids in LOOP1 are important to H+-ATPase function, and purturbations in this region can alter the efficiency of energy coupling.

Normal SPECT thallium-201 bull's-eye display: gender differences.

The bull's-eye technique synthesizes three-dimensional information from single photon emission computed tomographic 201TI images into two dimensions so that a patient's data can be compared quantitatively against a normal file. To characterize the normal database and to clarify differences between males and females, clinical data and exercise electrocardiography were used to identify 50 males and 50 females with less than 5% probability of coronary artery disease. Results show inhomogeneity of the 201TI distributions at stress and delay: septal to lateral wall count ratios are less than 1.0 in both females and males; anterior to inferior wall count ratios are greater than 1.0 in males but are approximately equal to 1.0 in females. Washout rate is faster in females than males at the same peak exercise heart rate and systolic blood pressure, despite lower exercise time. These important differences suggest that quantitative analysis of single photon emission computed tomographic 201TI images requires gender-matched normal files.