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.

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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 ...

eIF3j/Hcr1p, a protein associated with eIF3, was shown to bind to, and stabilize, the multifactor complex containing eIFs 1, 2, 3, and 5 and Met-tRNA i Met , whose formation is required for an optimal rate of translation initiation. Here we present evidence that eIF3j/Hcr1p is an RNA binding protein that enhances a late step in 40 S ribosome maturation involving cleavage of the 20 S precursor of 18 S rRNA in the cytoplasm. Immunofluorescence staining shows that eIF3j/Hcr1p is localized predominantly in the cytoplasm. The hcr1⌬ mutant exhibits a decreased amount of 40 S subunits, hypersensitivity to paromomycin, and increased levels of 20 S pre-rRNA. Combining the hcr1⌬ mutation with drs2⌬ or rps0a⌬, deletions of two other genes involved in the same step of 40 S subunit biogenesis, produced a synthetic growth defect. p35, the human ortholog of eIF3j/Hcr1p, partially complemented the slow growth phenotype conferred by hcr1⌬ when overexpressed in yeast. heIF3j/p35 was found physically associated with yeast eIF3 and 43 S initiation complexes in vitro and in vivo. Because it did not complement the 40 S biogenesis defect of hcr1⌬, it appears that heIF3j can substitute for eIF3j/Hcr1p only in translation initiation. We conclude that eIF3j/Hcr1p is required for rapid processing of 20 S to 18 S rRNA besides its role in translation initiation, providing an intriguing link between ribosome biogenesis and translation.Once the genetic information is transcribed into mRNA, its final translation into a protein requires charged tRNAs and the translation machinery, consisting of the ribosome and soluble translation factors. The assembly of 40 S and 60 S ribosomal subunits occurs primarily in a specialized subnuclear compartment termed the nucleolus, but the final steps occur in the cytoplasm. A large number of proteins and small nuclear RNAs participate in ribosome biogenesis (for review see Ref. 1). In the process of translation initiation, several translation initiation factors (eIFs) 1 orchestrate the assembly of an 80 S initiation complex containing methionyl initiator tRNA (Met-tRNA i Met ) located in the P site of the ribosome and based paired with the AUG start codon in the mRNA (for review see Ref.2). Correct completion of this process is crucial for further decoding of the genetic information during the elongation phase of translation. In general, proteins acting in the ribosome assembly process do not participate in translation and vice versa. Here we describe a protein in the yeast Saccharomyces cerevisiae that is involved in the maturation of 40 S subunits and also in assembly of the 43 S preinitiation complex, an important intermediate in translation initiation. These findings may indicate that ribosome biogenesis and translation initiation are more closely coordinated processes than was previously thought.The 9.1-kb rDNA unit encoding all four rRNAs occurs in about 100 -200 copies repeated on chromosome XII of S. cerevisiae. Ribosome biogenesis begins by transcription of the rRNA genes by RNA polymerase I to produc...