HIV-1 Vpu Protein Mediates the Transport of Potassium in Saccharomyces cerevisiae

Human immunodeficiency virus type 1 (HIV-1) Vpu is an integral membrane protein that belongs to the viroporin family. Viroporins interact with cell membranes, triggering membrane permeabilization and promoting release of viral particles. In vitro electrophysiological methods have revealed changes in membrane ion currents when Vpu is present; however, in vivo the molecular mechanism of Vpu at the plasma membrane is still uncertain. We used the yeast Saccharomyces cerevisiae as a genetic model system to analyze how Vpu ion channel impacts cellular homeostasis. Inducible expression of Vpu impaired cell growth, suggesting that this viral protein is toxic to yeast cultures. This toxicity decreased with extracellular acidic pH. Also, Vpu toxicity diminished as the extracellular K(+) concentration was increased. However, expression of the Vpu protein suppresses the growth defect of K(+) uptake-deficient yeast (Δtrk1,2). The phenotype rescue of these highly hyperpolarized cells was almost total when they were grown in medium supplemented with high concentrations of KCl (100 mM) at pH 7.0 but was significantly reduced when the extracellular K(+) concentration or pH was decreased. These results indicate that Vpu has the ability to modify K(+) transport in both yeast strains. Here, we show also that Vpu confers tolerance to the aminoglycoside antibiotic hygromycin B in Δtrk1,2 yeast. Our results suggest that Vpu interferes with cell growth of wild-type yeast but improves proliferation of the hyperpolarized trk1,2 mutant by inducing plasma membrane depolarization. Furthermore, evaluation of the ion channel activity of the Vpu protein in Δtrk1,2 yeast could aid in the development of a high-throughput screening assay for molecules that target the retroviral protein.This study was supported by Grants PI PI05/00013 and PI08/0912 from Fondo de Investigación Sanitaria. L.H. and N.M. were holders of Predoctoral Fellowships from Instituto de Salud Carlos III.S

The Type VI secretion system deploys anti-fungal effectors against microbial competitors

This work was supported by the Wellcome Trust (Senior Research Fellowship in Basic Biomedical Science to S.J.C., 104556; 097377, J.Q.; 101873 & 200208, N.A.R.G.), the MRC (MR/K000111X/1, S.J .C; MC_UU_12016/5, M.T.), and the BBSRC (BB/K016393/1 & BB/P020119/1, J.Q.). We thank Maximilian Fritsch, Mario López Martín and Birte Hollmann for help with strain construction; Gary Eitzen for construction of pGED1; Donna MacCallum for the gift of Candida glabrata ATCC2001; Joachim Morschhäuser for the gift of pNIM1; Gillian Milne (Microscopy and Histology facility, University of Aberdeen) for assistance with TEM; and Peter Taylor, Michael Porter, Laura Monlezun and Colin Rickman for advice and technical assistance.Peer reviewedPostprin

Organization of specific genomic regions of Zygosaccharomyces rouxii and Pichia sorbitophila: comparison with Saccharomyces cerevisiae.

The genomes of Zygosaccharomyces rouxii and Pichia sorbitophila were partially explored. The genome of Z. rouxii CBS 732 consists of seven chromosomes with an approximate size of 1.0-2.75 Mb, 12.8 Mb in total. Five of the chromosomes were labelled with specific probes. Three Z. rouxii genomic DNA fragments were sequenced; all 10 ORFs found were without introns and they have homologues in S. cerevisiae. Gene order comparison revealed that the organization is partially conserved in both species. The genome of P. sorbitophila CBS 7064 consists of seven chromosomes with an approximate size of 1.0-2.9 Mb, 13.9 Mb in total. Three of the chromosomes were labelled with specific probes. The sequencing of a 5.2 kb genomic DNA fragment revealed three ORFs, but no conservation of their organization was found, although all of them have their respective homologues in S. cerevisiae. According to our results, the presence of two overlapping ORFs in S. cerevisiae (YJL107c-YJL108c) could be interpreted as the result of a frameshift mutation.comparative studyjournal articleresearch support, non-u.s. gov't2000 Novimporte

Salt and oxidative stress tolerance in Debaryomyces hansenii and Debaryomyces fabryi

We report the characterization of five strains belonging to the halotolerant highly related Debaryomyces hansenii/fabryi species. The analysis performed consisted in studying tolerance properties, membrane characteristics, and cation incell amounts. We have specifically investigated (1) tolerance to different chemicals, (2) tolerance to osmotic and salt stress, (3) tolerance and response to oxidative stress, (4) reactive oxygen species (ROS) content, (5) relative membrane potential, (6) cell volume, (7) K+ and Na+ ion content, and (8) membrane fluidity. Unexpectedly, no direct relationship was found between one particular strain, Na+ content and its tolerance to NaCl or between its ROS content and its tolerance to H2O2. Results show that, although in general, human origin D.fabryi strains were more resistant to oxidative stress and presented shorter doubling times and smaller cell volume than food isolated D.hansenii ones, strains belonging to the same species can be significantly different. Debaryomyces fabryi CBS1793 strain highlighted for its extremely tolerant behavior when exposed to the diverse stress factors studied

Salt and oxidative stress tolerance in Debaryomyces hansenii and Debaryomyces fabryi

We report the characterization of five strains belonging to the halotolerant highly related Debaryomyces hansenii/fabryi species. The analysis performed consisted in studying tolerance properties, membrane characteristics, and cation incell amounts. We have specifically investigated (1) tolerance to different chemicals, (2) tolerance to osmotic and salt stress, (3) tolerance and response to oxidative stress, (4) reactive oxygen species (ROS) content, (5) relative membrane potential, (6) cell volume, (7) K+ and Na+ ion content, and (8) membrane fluidity. Unexpectedly, no direct relationship was found between one particular strain, Na+ content and its tolerance to NaCl or between its ROS content and its tolerance to H2O2. Results show that, although in general, human origin D.fabryi strains were more resistant to oxidative stress and presented shorter doubling times and smaller cell volume than food isolated D.hansenii ones, strains belonging to the same species can be significantly different. Debaryomyces fabryi CBS1793 strain highlighted for its extremely tolerant behavior when exposed to the diverse stress factors studied

The activity of Saccharomyces cerevisiae Na , K H antiporter Nha1 is negatively regulated by 14 3 3 protein binding at serine 481

Na+/H+ antiporters are involved in ensuring optimal intracellular concentrations of alkali-metal cations and protons in most organisms. In Saccharomyces cerevisiae, the plasma-membrane Na+, K+/H+ antiporter Nha1 mediates Na+ and K+ efflux, which is important for cell growth in the presence of salts. Nha1 belongs among housekeeping proteins and, due to its ability to export K+, it has many physiological functions. The Nha1 transport activity is regulated through its long, hydrophilic and unstructured C-terminus (554 of 985 aa). Although Nha1 has been previously shown to interact with the yeast 14-3-3 isoform (Bmh2), the binding site remains unknown. In this work, we identified the residues through which Nha1 interacts with the 14-3-3 protein. Biophysical characterization of the interaction between the C-terminal polypeptide of Nha1 and Bmh proteins in vitro revealed that the 14-3-3 protein binds to phosphorylated Ser481 of Nha1, and the crystal structure of the phosphopeptide containing Ser481 bound to Bmh1 provided the structural basis of this interaction. Our data indicate that 14-3-3 binding induces a disorder-to-order transition of the C-terminus of Nha1, and in vivo experiments showed that the mutation of Ser481 to Ala significantly increases cation efflux activity via Nha1, which renders cells sensitive to low K+ concentrations. Hence, 14-3-3 binding is apparently essential for the negative regulation of Nha1 activity, which should be low under standard growth conditions, when low amounts of toxic salts are present and yeast cells need to accumulate high amounts of K+