The ceramide-activated protein phosphatase Sit4p controls lifespan,mitochondrial function and cell cycle progression by regulating hexokinase 2 phosphorylation

Sit4p is the catalytic subunit of a ceramide-activated PP2A-like phosphatase that regulates cell cycle, mitochondrial function, oxidative stress resistance and chronological lifespan in yeast. In this study, we show that hexokinase 2 (Hxk2p) is hyperphosphorylated in sit4Δ mutants grown in glucose medium by a Snf1p-independent mechanism and Hxk2p-S15A mutation suppresses phenotypes associated with SIT4 deletion, namely growth arrest at G1 phase, derepression of mitochondrial respiration, H₂O₂ resistance and lifespan extension. Consistently, the activation of Sit4p in isc1Δ mutants, which has been associated with premature aging, leads to Hxk2p hypophosphorylation, and the expression of Hxk2p-S15E increases the lifespan of isc1Δ cells. The overall results suggest that Hxk2p functions downstream of Sit4p in the control of cell cycle, mitochondrial function, oxidative stress resistance and chronological lifespan.     

Vba4p,a vacuolar membrane protein,is involved in the drug resistance and vacuolar morphology of Saccharomyces cerevisiae

In the vacuolar basic amino acid (VBA) transporter family of Saccharomyces cerevisiae, VBA4 encodes a vacuolar membrane protein with 14 putative transmembrane helices. Transport experiments with isolated vacuolar membrane vesicles and estimation of the amino acid contents in vacuoles showed that Vba4p is not likely involved in the transport of amino acids. We found that the vba4Δ cells, as well as vba1Δ and vba2Δ cells, showed increased susceptibility to several drugs, particularly to azoles. Although disruption of the VBA4 gene did not affect the salt tolerance of the cells, vacuolar fragmentation observed under high salt conditions was less prominent in vba4Δ cells than in wild type, vba1Δ, and vba2Δ cells. Vba4p differs from Vba1p and Vba2p as a vacuolar transporter but is important for the drug resistance and vacuolar morphology of S. cerevisiae.     

Swi2/Snf2-like protein Uls1 functions in the Sgs1-dependent pathway of maintenance of rDNA stability and alleviation of replication stress

The Saccharomyces cerevisiae Uls1 belongs to the Swi2/Snf2 family of DNA-dependent ATPases and a new protein family of SUMO-targeted ubiquitin ligases. Here we show that Uls1 is implicated in DNA repair independently of the replication stress response pathways mediated by the endonucleases Mus81 and Yen1 and the helicases Mph1 and Srs2. Uls1 works together with Sgs1 and we demonstrate that the attenuation of replication stress-related defects in sgs1Δ by deletion of ULS1 depends on a functional of Rad51 recombinase and post-replication repair pathway mediated by Rad18 and Rad5, but not on the translesion polymerase, Rev3. The higher resistance of sgs1Δ uls1Δ mutants to genotoxic stress compared to single sgs1Δ cells is not the result of decreased formation or accelerated resolution of recombination-dependent DNA structures. Instead, deletion of ULS1 restores stability of the rDNA region in sgs1Δ cells. Our data suggest that Uls1 may contribute to genomic stability during DNA synthesis and channel the repair of replication lesions into the Sgs1-dependent pathway, with DNA translocase and SUMO binding activities of Uls1 as well as a RING domain being essential for its functions in replication stress response.     

Synthesis of mannosylinositol phosphorylceramides is involved in maintenance of cell integrity of yeast Saccharomyces cerevisiae

Complex sphingolipids play important roles in many physiologically important events in yeast Saccharomyces cerevisiae. In this study, we screened yeast mutant strains showing a synthetic lethal interaction with loss of mannosylinositol phosphorylceramide (MIPC) synthesis and found that a specific group of glycosyltransferases involved in the synthesis of mannan‐type N‐glycans is essential for the growth of cells lacking MIPC synthases (Sur1 and Csh1). The genetic interaction was also confirmed by repression of MNN2, which encodes alpha‐1,2‐mannosyltransferase that synthesizes mannan‐type N‐glycans, by a tetracycline‐regulatable system. MNN2‐repressed sur1Δ csh1Δ cells exhibited high sensitivity to zymolyase treatment, and caffeine and sodium dodecyl sulfate (SDS) strongly inhibited the growth of sur1Δ csh1Δ cells, suggesting impairment of cell integrity due to the loss of MIPC synthesis. The phosphorylated form of Slt2, a mitogen‐activated protein (MAP) kinase activated by impaired cell integrity, increased in sur1Δ csh1Δ cells, and this increase was dramatically enhanced by the repression of Mnn2. Moreover, the growth defect of MNN2‐repressed sur1Δ csh1Δ cells was enhanced by the deletion of SLT2 or RLM1 encoding a downstream target of Slt2. These results indicated that loss of MIPC synthesis causes impairment of cell integrity, and this effect is enhanced by impaired synthesis of mannan‐type N‐glycans.     

Involvement of complex sphingolipids and phosphatidylserine in endosomal trafficking in yeast Saccharomyces cerevisiae

Sphingolipids play critical roles in many physiologically important events in the yeast Saccharomyces cerevisiae. In this study, we found that csg2Δ mutant cells defective in the synthesis of mannosylinositol phosphorylceramide exhibited abnormal intracellular accumulation of an exocytic v‐SNARE, Snc1, under phosphatidylserine synthase gene (PSS1)‐repressive conditions, although in wild‐type cells, Snc1 was known to cycle between plasma membranes and the late Golgi via post‐Golgi endosomes. The mislocalized Snc1 was co‐localized with an endocytic marker dye, FM4‐64, upon labelling for a short time. The abnormal distribution of Snc1 was suppressed by deletion of GYP2 encoding a GTPase‐activating protein that negatively regulates endosomal vesicular trafficking, or expression of GTP‐restricted form of Ypt32 GTPase. Furthermore, an endocytosis‐deficient mutant of Snc1 was localized to plasma membranes in PSS1‐repressed csg2Δ mutant cells as well as wild‐type cells. Thus, the PSS1‐repressed csg2Δ mutant cells were indicated to be defective in the trafficking of Snc1 from post‐Golgi endosomes to the late Golgi. In contrast, the vesicular trafficking pathways via pre‐vacuolar endosomes in the PSS1‐repressed csg2Δ mutant cells seemed to be normal. These results suggested that specific complex sphingolipids and phosphatidylserine are co‐ordinately involved in specific vesicular trafficking pathway.     

Deficiency of the exportomer components Pex1, Pex6, and Pex15 causes enhanced pexophagy in Saccharomyces cerevisiae

Turnover of damaged, dysfunctional, or excess organelles is critical to cellular homeostasis. We screened mutants disturbed in peroxisomal protein import, and found that a deficiency in the exportomer subunits Pex1, Pex6, and Pex15 results in enhanced turnover of peroxisomal membrane structures compared with other mutants. Strikingly, almost all peroxisomal membranes were associated with phagophore assembly sites in pex1Δ atg1Δ cells. Degradation depended on Atg11 and the pexophagy receptor Atg36, which mediates degradation of superfluous peroxisomes. Mutants of PEX1, PEX6, and PEX15 accumulate ubiquitinated receptors at the peroxisomal membrane. This accumulation has been suggested to trigger pexophagy in mammalian cells. We show by genetic analysis that preventing this accumulation does not abolish pexophagy in Saccharomyces cerevisiae. We find Atg36 is modified in pex1Δ cells even when Atg11 binding is prevented, suggesting Atg36 modification is an early event in the degradation of dysfunctional peroxisomal structures in pex1Δ cells via pexophagy.     

PiggyBac-based screening identified BEM4 as a suppressor to rescue growth defects in och1-disrupted yeast cells

Glycoengineered yeast cells, which express human-compatible glycan structures, are particularly attractive host cells to produce therapeutic glycoproteins. Disruption of OCH1 gene, which encodes an α-1,6-mannosyltransferase required for mannan-type N-glycan formation, is essential for the elimination of yeast-specific N-glycan structures. However, the gene disruption causes cell wall defects leading to growth defects. Here, we tried to identify factors to rescue the growth defects of och1Δ cells by in vivo mutagenesis using piggyBac (PB)-based transposon. We isolated a mutant strain, named 121, which could grow faster than parental och1Δ cells. The PB element was introduced into the promoter region of BEM4 gene and upregulated the BEM4 expression. Overexpression of BEM4 suppressed growth defects in och1Δ cells. The slow grow phenotypes were partially rescued by expression of Rho1p, whose function is regulated by Bem4p. Our results indicate that BEM4 would be useful to produce therapeutic proteins in glycoengineered yeast without the growth defects.     

Heme acquisition by Shu1 requires Nbr1 and proteins of the ESCRT complex in Schizosaccharomyces pombe

Assimilation of heme is mediated by the cell surface protein Shu1 in Schizosaccharomyces pombe. Shu1 undergoes internalization from the cell surface to the vacuole in response to high concentrations of hemin. Here, we have identified cellular components that are involved in mediating vacuolar targeting of Shu1. Cells deficient in heme biosynthesis and lacking the polyubiquitin gene ubi4⁺ exhibit poor growth in the presence of exogenous hemin as a sole source of heme. Microscopic analyses of hem1Δ shu1Δ ubi4Δ cells expressing a functional HA₄‐tagged Shu1 show that Shu1 localizes to the cell surface. Ubiquitinated Nbr1 functions as a receptor for the endosomal sorting complexes required for transport (ESCRT) that delivers cargos to the vacuole. Inactivation of nbr1⁺, ESCRT‐0 hse1⁺ or ESCRT‐I sst6⁺ results in hem1Δ cells being unable to use exogenous hemin for the growth. Using lysate preparations from hemin‐treated cells, Shu1‐Nbr1 and Shu1‐Hse1 complexes are detected by coimmunoprecipitation experiments. Further analysis by immunofluorescence microscopy shows that Shu1 is unable to reach vacuoles of hemin‐treated cells harboring a deletion for one of the following genes: ubi4⁺, nbr1⁺, hse1⁺ and sst6⁺. Together, these results reveal that hemin‐mediated vacuolar targeting of Shu1 requires Ubi4‐dependent ubiquitination, the receptor Nbr1 and the ESCRT proteins Hse1 and Sst6.     

Development of callus-like structures and plant regeneration in thallus segments of Grateloupia filiformis Kützing (Rhodophyta)

Callus-like structures (CLS) were observed to develop from sectioned tissues of the thallus segments of Grateloupia filiformis Kützing maintained in Von Stosch enriched-seawater culture medium. Three types were observed: dark CLS originated from pigmented cortical cells, clear CLS and loose clear CLS both originated from colorless medullary cells. Dark and clear CLS developed from segments exposed to light while loose clear CLS developed in darkness. Frequencies of different types of CLS varied according to the origin of the segments (apical, median and basal zones of primary branches). In order to assess the potential for plant regeneration, all CLS were isolated from the original segment and cultured under light. The potential for plant regeneration depends on the type of CLS and on the origin of the segments. The loose clear CLS and the CLS from apical segments produced the largest number of regenerated plantlets. Cultures of CLS might be an effective system for micropropagation in G. filiformis and light seems to be an important factor involved in mechanisms of plant regeneration.     

Mdm35p imports Ups proteins into the mitochondrial intermembrane space by functional complex formation

Ups1p, Ups2p, and Ups3p are three homologous proteins that control phospholipid metabolism in the mitochondrial intermembrane space (IMS). The Ups proteins are atypical IMS proteins in that they lack the two major IMS‐targeting signals, bipartite presequences and cysteine motifs. Here, we show that Ups protein import is mediated by another IMS protein, Mdm35p. In vitro import assays show that import of Ups proteins requires Mdm35p. Loss of Mdm35p led to a decrease in steady state levels of Ups proteins in mitochondria. In addition, mdm35Δ cells displayed a similar phenotype to ups1Δups2Δups3Δ cells. Interestingly, unlike typical import machineries, Mdm35p associated stably with Ups proteins at a steady state after import. Demonstrating that Mdm35p is a functional component of Ups–Mdm35p complexes, restoration of Ups protein levels in mdm35Δ mitochondria failed to restore phospholipid metabolism. These findings provide a novel mechanism in which the formation of functional protein complexes drives mitochondrial protein import.     

Tel1/ATM prevents degradation of replication forks that reverse after topoisomerase poisoning

In both yeast and mammals, the topoisomerase poison camptothecin (CPT) induces fork reversal, which has been proposed to stabilize replication forks, thus providing time for the repair of CPT‐induced lesions and supporting replication restart. We show that Tel1, the Saccharomyces cerevisiae orthologue of human ATM kinase, stabilizes CPT‐induced reversed forks by counteracting their nucleolytic degradation by the MRX complex. Tel1‐lacking cells are hypersensitive to CPT specifically and show less reversed forks in the presence of CPT. The lack of Mre11 nuclease activity restores wild‐type levels of reversed forks in CPT‐treated tel1Δ cells without affecting fork reversal in wild‐type cells. Moreover, Mrc1 inactivation prevents fork reversal in wild‐type, tel1Δ, and mre11 nuclease‐deficient cells and relieves the hypersensitivity of tel1Δ cells to CPT. Altogether, our data indicate that Tel1 counteracts Mre11 nucleolytic activity at replication forks that undergo Mrc1‐mediated reversal in the presence of CPT.     

Absence of superoxide dismutase activity causes nuclear DNA fragmentation during the aging process

Superoxide dismutases (SOD) serve as an important antioxidant defense mechanism in aerobic organisms, and deletion of these genes shortens the replicative life span in the budding yeast Saccharomyces cerevisiae. Even though involvement of superoxide dismutase enzymes in ROS scavenging and the aging process has been studied extensively in different organisms, analyses of DNA damages has not been performed for replicatively old superoxide dismutase deficient cells. In this study, we investigated the roles of SOD1, SOD2 and CCS1 genes in preserving genomic integrity in replicatively old yeast cells using the single cell comet assay. We observed that extend of DNA damage was not significantly different among the young cells of wild type, sod1Δ and sod2Δ strains. However, ccs1Δ mutants showed a 60% higher amount of DNA damage in the young stage compared to that of the wild type cells. The aging process increased the DNA damage rates 3-fold in the wild type and more than 5-fold in sod1Δ, sod2Δ, and ccs1Δ mutant cells. Furthermore, ROS levels of these strains showed a similar pattern to their DNA damage contents. Thus, our results confirm that cells accumulate DNA damages during the aging process and reveal that superoxide dismutase enzymes play a substantial role in preserving the genomic integrity in this process.     

Effect of low pH on organization of the actin cytoskeleton in Saccharomyces cerevisiae

Cell growth in the yeast Saccharomyces cerevisiae depends on polarization of the actin cytoskeleton. In this study, we investigated how the cell regulates the distribution of actin in response to low pH conditions, focusing on the role of mitogen-activated protein kinases, Hog1 and Slt2. Changing the extracellular pH from 6.0 to 3.0 caused a transient depolarization of the actin cytoskeleton. Actin cables were no longer visible, and actin patches appeared randomly distributed after 30 min at pH 3.0. The deletion strain hog1Δ did not show this low-pH phenotype, suggesting that Hog1 is involved in depolarization of the actin cytoskeleton in response to low-pH stress. Yeast cells incubated at pH 3.0 also showed markedly increased endocytosis compared with the control at neutral pH, as indicated by the uptake of Lucifer Yellow (LY). Both the hog1Δ and slt2Δ mutants took up LY into the vacuole to a similar extent as the wild-type strain. In addition, cells grown at pH 3.0 showed a 2-fold increase in phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) levels, as did the hog1Δ or slt2Δ cells. Efficient uptake of LY and actin repolarization at pH 3.0 might therefore require activation of PI(4,5)P2 synthesis.     

The AP‐2 complex is required for proper temporal and spatial dynamics of endocytic patches in fission yeast

In metazoans the AP‐2 complex has a well‐defined role in clathrin‐mediated endocytosis. By contrast, its direct role in endocytosis in unicellular eukaryotes has been questioned. Here, we report co‐ immunoprecipitation between the fission yeast AP‐2 component Apl3p and clathrin, as well as the genetic interactions between apl3Δ and clc1 and sla2Δ/end4Δ mutants. Furthermore, a double clc1 apl3Δ mutant was found to be defective in FM4‐64 uptake. In an otherwise wild‐type strain, apl3Δ cells exhibit altered dynamics of the endocytic sites, with a heterogeneous and extended lifetime of early and late markers at the patches. Additionally, around 50% of the endocytic patches exhibit abnormal spatial dynamics, with immobile patches and patches that bounce backwards to the cell surface, showing a pervasive effect of the absence of AP‐2. These alterations in the endocytic machinery result in abnormal cell wall synthesis and morphogenesis. Our results complement those found in budding yeast and confirm that a direct role of AP‐2 in endocytosis has been conserved throughout evolution.     

The Candida glabrata sterol scavenging mechanism,mediated by the ATP‐binding cassette transporter Aus1p,is regulated by iron limitation

During disseminated infection by the opportunistic pathogen Candida glabrata, uptake of sterols such as serum cholesterol may play a significant role during pathogenesis. The ATP‐binding cassette transporter Aus1p is thought to function as a sterol importer and in this study, we show that uptake of exogenous sterols occurred under anaerobic conditions in wild‐type cells of C. glabrata but not in AUS1‐deleted mutant (aus1Δ) cells. In aerobic cultures, growth inhibition by fluconazole was prevented in the presence of serum, and AUS1 expression was upregulated. Uptake of sterol by azole treated cells required the presence of serum, and sterol alone did not reverse FLC inhibition of growth. However, if iron availability in the growth medium was limited by addition of the iron chelators ferrozine or apo‐transferrin, growth of wild‐type cells, but not aus1Δ cells, was rescued. In a mouse model of disseminated infection, the C. glabrata aus1Δ strain caused a significantly decreased kidney fungal burden than the wild‐type strain or a strain in which AUS1 was restored. We conclude that sterol uptake in C. glabrata can occur in iron poor environment of host tissues and thus may contribute to C. glabrata pathogenesis.