Environmental stresses inducing translation arrest are accompanied by the deposition of translational components into stress granules (SGs) serving as mRNA triage sites. It has recently been reported that, in Saccharomyces cerevisiae, formation of SGs occurs as a result of a prolonged glucose starvation. However, these SGs did not contain eIF3, one of hallmarks of mammalian SGs. We have analyzed the effect of robust heat shock on distribution of eIF3a/Tif32p/Rpg1p and showed that it results in the formation of eIF3a accumulations containing other eIF3 subunits, known yeast SG components and small but not large ribosomal subunits and eIF2α/Sui2p. Interestingly, under these conditions, Dcp2p and Dhh1p P-body markers also colocalized with eIF3a. Microscopic analyses of the edc3Δlsm4ΔC mutant demonstrated that different scaffolding proteins are required to induce SGs upon robust heat shock as opposed to glucose deprivation. Even though eIF2α became phosphorylated under these stress conditions, the decrease in polysomes and formation of SGs occurred independently of phosphorylation of eIF2α. We conclude that under specific stress conditions, such as robust heat shock, yeast SGs do contain eIF3 and 40S ribosomes and utilize alternative routes for their assembly.

The large protein superfamily of NADPH oxidases (NOX enzymes) is found in members of all eukaryotic kingdoms: animals, plants, fungi, and protists. The physiological functions of these NOX enzymes range from defense to specialized oxidative biosynthesis and to signaling. In filamentous fungi, NOX enzymes are involved in signaling cell differentiation, in particular in the formation of fruiting bodies. On the basis of bioinformatics analysis, until now it was believed that the genomes of unicellular fungi like Saccharomyces cerevisiae and Schizosaccharomyces pombe do not harbor genes coding for NOX enzymes. Nevertheless, the genome of S. cerevisiae contains nine ORFs showing sequence similarity to the catalytic subunits of mammalian NOX enzymes, only some of which have been functionally assigned as ferric reductases involved in iron ion transport. Here we show that one of the nine ORFs (YGL160W, AIM14) encodes a genuine NADPH oxidase, which is located in the endoplasmic reticulum (ER) and produces superoxide in a NADPH-dependent fashion. We renamed this ORF YNO1 (yeast NADPH oxidase 1). Overexpression of YNO1 causes YCA1-dependent apoptosis, whereas deletion of the gene makes cells less sensitive to apoptotic stimuli. Several independent lines of evidence point to regulation of the actin cytoskeleton by reactive oxygen species (ROS) produced by Yno1p.cell cycle | integral membrane reductase | wiskostatin | latrunculin R eactive oxygen species (ROS) have multiple roles in physiology and pathophysiology, in particular during aging and induction of programmed cell death. This includes also nonmitochondrial sources, besides the long-studied mitochondrially generated ROS. These findings can be viewed as important additions to the classical "free radical theory of aging" (1) and theories developed thereafter (2, 3).In higher organisms, among others, at least two major sources of superoxide other than mitochondria are known. On the one hand, xanthine oxidase, an enzyme in the catabolism of purines, which catalyses the oxidation of hypoxanthine to xanthine and to uric acid, produces superoxide (4). On the other hand, NADPH oxidases (NOX) catalyze the production of superoxide from oxygen and NADPH (5).The NADPH oxidase superfamily of membrane-located enzymes of higher cells has been known for a decade (for review, ref. 5). Whereas the human NOX2 was discovered early on, other NOX (Nox1/3/4/5) as well as dual oxidase (DUOX) (Duox1/2) enzymes (displaying two domains: a NADPH oxidase domain and a peroxidase domain) have been found relatively recently in human cells. The human NOX2 was discovered as a defense enzyme of neutrophils and macrophages, which produce a burst of superoxide (O 2 · − ) as a first line of defense against invading microorganisms. Although X-ray or NMR structure determinations are not available, we know from indirect evidence and bioinformatics that the catalytic subunit of the macrophage enzyme contains six transmembrane helices, is located in the plasma membrane, and produces superoxide in a vectorial ...

In response to severe environmental stresses eukaryotic cells shut down translation and accumulate components of the translational machinery in stress granules (SGs). Since they contain mainly mRNA, translation initiation factors and 40S ribosomal subunits, they have been referred to as dominant accumulations of stalled translation preinitiation complexes. Here we present evidence that the robust heat shock-induced SGs of S. cerevisiae also contain translation elongation factors eEF3 (Yef3p) and eEF1Bγ2 (Tef4p) as well as translation termination factors eRF1 (Sup45p) and eRF3 (Sup35p). Despite the presence of the yeast prion protein Sup35 in heat shock-induced SGs, we found out that its prion-like domain is not involved in the SGs assembly. Factors eEF3, eEF1Bγ2 and eRF1 were accumulated and co-localized with Dcp2 foci even upon a milder heat shock at 42°C independently of P-bodies scaffolding proteins. We also show that eEF3 accumulations at 42°C determine sites of the genuine SGs assembly at 46°C. We suggest that identification of translation elongation and termination factors in SGs might help to understand the mechanism of the eIF2α factor phosphorylation-independent repression of translation and SGs assembly.

Fröhlich postulated coherent polar oscillations as a fundamental biophysical property of biological systems. Recently, Pelling et al. (2004, 2005) detected mechanical vibrations of yeast cell membrane with atomic force microscope (AFM) and analyzed by Fourier analysis in the frequency range 0.5-2 kHz with amplitudes of the order of 1 nm. This article describes the measurement of electric activity of yeast cells in the acoustic frequency range and of mechanical vibrations of cell membrane. Spectrum analyzer and electrically and electromagnetically screened box with point sensor and amplifiers fed by batteries were used for measurement of synchronized and non synchronized tubulin mutants of yeast cells. We show that the electric activity of synchronized cells in the M phase is greater that of non synchronized cells. That corresponds to the findings of Pohl et al. (1981). Obtained results of measurement of cell electric activity are in good agreement with AFM findings.