The STF2p hydrophilin from Saccharomyces cerevisiae is required for dehydration stress tolerance

The yeast Saccharomyces cerevisiae is able to overcome cell dehydration; cell metabolic activity is arrested during this period but restarts after rehydration. The yeast genes encoding hydrophilin proteins were characterised to determine their roles in the dehydration-resistant phenotype, and STF2p was found to be a hydrophilin that is essential for survival after the desiccation-rehydration process. Deletion of STF2 promotes the production of reactive oxygen species and apoptotic cell death during stress conditions, whereas the overexpression of STF2, whose gene product localises to the cytoplasm, results in a reduction in ROS production upon oxidative stress as the result of the antioxidant capacity of the STF2p protein.     

Myo4p and She3p are required for cortical ER inheritance in Saccharomyces cerevisiae

Myo4p is a nonessential type V myosin required for the bud tip localization of ASH1 and IST2 mRNA. These mRNAs associate with Myo4p via the She2p and She3p proteins. She3p is an adaptor protein that links Myo4p to its cargo. She2p binds to ASH1 and IST2 mRNA, while She3p binds to both She2p and Myo4p. Here we show that Myo4p and She3p, but not She2p, are required for the inheritance of cortical ER in the budding yeast Saccharomyces cerevisiae. Consistent with this observation, we find that cortical ER inheritance is independent of mRNA transport. Cortical ER is a dynamic network that forms cytoplasmic tubular connections to the nuclear envelope. ER tubules failed to grow when actin polymerization was blocked with the drug latrunculin A (Lat-A). Additionally, a reduction in the number of cytoplasmic ER tubules was observed in Lat-A-treated and myo4Delta cells. Our results suggest that Myo4p and She3p facilitate the growth and orientation of ER tubules.     

Saccharomyces cerevisiae pex3p and pex19p are required for proper localization and stability of peroxisomal membrane proteins

The mechanisms by which peroxisomal membrane proteins (PMPs) are targeted to and inserted into membranes are unknown, as are the required components. We show that among a collection of 16 Saccharomyces cerevisiae peroxisome biogenesis (pex) mutants, two mutants, pex3Delta and pex19Delta, completely lack detectable peroxisomal membrane structures and mislocalize their PMPs to the cytosol where they are rapidly degraded. The other pexDelta mutants contain membrane structures that are properly inherited during vegetative growth and that house multiple PMPs. Even Pex15p requires Pex3p and Pex19p for localization to peroxisomal membranes. This PMP was previously hypothesized to travel via the endoplasmic reticulum (ER) to peroxisomes. We provide evidence that ER-accumulated Pex15p is not a sorting intermediate on its way to peroxisomes. Our results show that Pex3p and Pex19p are required for the proper localization of all PMPs tested, including Pex15p, whereas the other Pex proteins might only be required for targeting/import of matrix proteins.     

Msn2p/Msn4p-activation is essential for the recovery from freezing stress in yeast

Recent data suggest that the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptor subtype plays a pivotal role in the pathogenesis of effective disorders and in the action of antidepressant drugs. After chronic treatment with the antidepressants desipramine or paroxetine, we measured by immunoprecipitation and Western blotting, the changes in the interaction of AMPA receptor subunits with proteins involved in trafficking and/or stabilization of the subunits into synaptic membranes of the hippocampus. Both antidepressants increased the interaction of GluR1 subunit with stargazin and of GluR2/3 with NSF. Paroxetine increased the interaction of GluR1 with Rab4A, and desipramine markedly increased the interaction of GluR1 with SAP97. Paroxetine, but not desipramine, also increased membrane levels of CaMKII, autophosphorylated CaMKII and GluR1 phosphorylated at the CaMKII site. Interactions of GluR1 and GluR2/3 with proteins implicated in AMPA receptor trafficking and with scaffolding proteins appear to account for the enhanced membrane expression of AMPA receptors in the hippocampus after antidepressant treatment.     

The ribosome-bound chaperones RAC and Ssb1/2p are required for accurate translation in Saccharomyces cerevisiae

The chaperone homologs RAC (ribosome-associated complex) and Ssb1/2p are anchored to ribosomes; Ssb1/2p directly interacts with nascent polypeptides. The absence of RAC or Ssb1/2p results in a similar set of phenotypes, including hypersensitivity against the aminoglycoside paromomycin, which binds to the small ribosomal subunit and compromises the fidelity of translation. In order to understand this phenomenon we measured the frequency of translation termination and misincorporation in vivo and in vitro with a novel reporter system. Translational fidelity was impaired in the absence of functional RAC or Ssb1/2p, and the effect was further enhanced by paromomycin. The mutant strains suffered primarily from a defect in translation termination, while misincorporation was compromised to a lesser extent. Consistently, a low level of soluble translation termination factor Sup35p enhanced growth defects in the mutant strains. Based on the combined data we conclude that RAC and Ssb1/2p are crucial in maintaining translational fidelity beyond their postulated role as chaperones for nascent polypeptides.     

Saccharomyces cerevisiae Hop1 zinc finger motif is the minimal region required for its function in vitro

Saccharomyces cerevisiae meiosis-specific HOP1, which encodes a core component of synaptonemal complex, plays a key role in proper pairing of homologous chromosomes and processing of meiotic DNA double strand breaks. Isolation and analysis of hop1 mutants indicated that these functions require Cys(371) of Hop1 embedded in a region (residues 343-378) sharing homology to a zinc finger motif (ZnF). However, the precise biochemical function of Hop1, or its putative ZnF, in these processes is poorly understood. Our previous studies revealed that Hop1 is a DNA-binding protein, showed substantially higher binding affinity for G4 DNA, and enhances its formation. We report herein that ZnF appears to be sufficient for both zinc as well as DNA-binding activities. Molecular modeling studies suggested that Hop1 ZnF differs from the previously characterized natural ZnFs. The zinc-binding assay showed that the affinity for zinc is weaker for C371S ZnF mutant compared with the wild type (WT) ZnF. Analysis of CD spectra indicated that zinc and DNA induce substantial conformational changes in WT ZnF, but not in C371S ZnF mutant. The results from a number of different experimental approaches suggested that the DNA-binding properties of ZnF are similar to those of full-length Hop1 and that interaction with DNA rich in G residues is particularly robust. Significantly, WT ZnF by itself, but not C371S mutant, was able to bind duplex DNA and promote interstitial pairing of DNA double helices via the formation of guanine quartets. Together, these results implicate a direct role for Hop1 in pairing of homologous chromosomes during meiosis.