Mutations in the ubiquitin-binding domain of OPTN/optineurin interfere with autophagy-mediated degradation of misfolded proteins by a dominant-negative mechanism

OPTN (optineurin) is an autophagy receptor and mutations in the OPTN gene result in familial glaucoma (E50K) and amyotrophic lateral sclerosis (ALS) (E478G). However, the mechanisms through which mutant OPTN leads to human diseases remain to be characterized. Here, we demonstrated that OPTN colocalized with inclusion bodies (IBs) formed by mutant HTT/huntingtin protein (mHTT) in R6/2 transgenic mice and IBs formed by 81QNmHTT (nuclear form), 109QmHTT (cytoplasmic form) or the truncated form of TARDBP/TDP-43 (TARDBP^ND251) in Neuro2A cells. This colocalization required the ubiquitin (Ub)-binding domain (UbBD, amino acids 424 to 511) of OPTN. Overexpression of wild-type (WT) OPTN decreased IBs through K63-linked polyubiquitin-mediated autophagy. E50K or 210 to 410Δ (with amino acids 210 to 410 deleted) whose mutation or deletion was outside the UbBD decreased the IBs formed by 109QmHTT or TARDBP^ND251, as was the case with WT OPTN. In contrast, UbBD mutants, including E478G, D474N, UbBDΔ, 411 to 520Δ and 210 to 520Δ, increased accumulation of IBs. UbBD mutants (E478G, UbBDΔ) retained a substantial ability to interact with WT OPTN, and were found to colocalize with polyubiquitinated IBs, which might occur indirectly through their WT partner in a WT-mutant complex. They decreased autophagic flux evidenced by alteration in LC3 level and turnover and in the number of LC3-positive puncta under stresses like starvation or formation of IBs. UbBD mutants exhibited a weakened interaction with MYO6 (myosin VI) and TOM1 (target of myb1 homolog [chicken]), important for autophagosome maturation, in cells or sorted 109QmHtt IBs. Taken together, our data indicated that UbBD mutants acted as dominant-negative traps through the formation of WT-mutant hybrid complexes to compromise the maturation of autophagosomes, which in turn interfered with OPTN-mediated autophagy and clearance of IBs.     

Saccharomyces cerevisiae Mutants Affected in Vacuole Assembly or Vacuolar H+-ATPase are Hypersensitive to Lead (Pb) Toxicity

Lead is an important environmental pollutant. The role of vacuole, in Pb detoxification, was studied using a vacuolar protein sorting mutant strain (vps16Δ), belonging to class C mutants. Cells disrupted in VPS16 gene, did not display a detectable vacuolar-like structure. Based on the loss of cell proliferation capacity, it was found that cells from vps16Δ mutant exhibited a hypersensitivity to Pb-induced toxicity, compared to wild type (WT) strain. The function of vacuolar H⁺-ATPase (V-ATPase), in Pb detoxification, was evaluated using mutants with structurally normal vacuoles but defective in subunits of catalytic (vma1Δ or vma2Δ) or membrane domain (vph1Δ or vma3Δ) of V-ATPase. All mutants tested, lacking a functional V-ATPase, displayed an increased susceptibility to Pb, comparatively to cells from WT strain. Modification of vacuolar morphology, in Pb-exposed cells, was visualized using a Vma2p-GFP strain. The treatment of yeast cells with Pb originated the fusion of the medium size vacuolar lobes into one enlarged vacuole. In conclusion, it was found that vacuole plays an important role in the detoxification of Pb in Saccharomyces cerevisiae; in addition, a functional V-ATPase was required for Pb compartmentalization.     

Ubiquitin-coated nanodiamonds bind to autophagy receptors for entry into the selective autophagy pathway

Selective macroautophagy/autophagy plays a pivotal role in the processing of foreign pathogens and cellular components to maintain homeostasis in human cells. To date, numerous studies have demonstrated the uptake of nanoparticles by cells, but their intracellular processing through selective autophagy remains unclear. Here we show that carbon-based nanodiamonds (NDs) coated with ubiquitin (Ub) bind to autophagy receptors (SQSTM1 [sequestosome 1], OPTN [optineurin], and CALCOCO2/NDP52 [calcium binding and coiled-coil domain 2]) and are then linked to MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) for entry into the selective autophagy pathway. NDs are ultimately delivered to lysosomes. Ectopically expressed SQSTM1-green fluorescence protein (GFP) could bind to the Ub-coated NDs. By contrast, the Ub-associated domain mutant of SQSTM1 (ΔUBA)-GFP did not bind to the Ub-coated NDs. Chloroquine, an autophagy inhibitor, prevented the ND-containing autophagosomes from fusing with lysosomes. Furthermore, autophagy receptors OPTN and CALCOCO2/NDP52, involved in the processing of bacteria, were found to be involved in the selective autophagy of NDs. However, ND particles located in the lysosomes of cells did not induce mitotic blockage, senescence, or cell death. Single ND clusters in the lysosomes of cells were observed in the xenografted human lung tumors of nude mice. This study demonstrated for the first time that Ub-coated nanoparticles bind to autophagy receptors for entry into the selective autophagy pathway, facilitating their delivery to lysosomes.     

Temporal dynamics of PARK2/parkin and OPTN/optineurin recruitment during the mitophagy of damaged mitochondria

Damaged mitochondria are selectively degraded via autophagy in a regulated pathway known as mitophagy. Parkinson disease-linked proteins PINK1 (PTEN induced putative kinase 1) and PARK2 (parkin RBR E3 ubiquitin protein ligase) are recruited to the outer mitochondrial membrane upon mitochondrial damage, leading to the PARK2-mediated ubiquitination of mitochondrial proteins. Here, we discuss our recent work demonstrating that OPTN (optineurin) is recruited to damaged mitochondria, serving as an autophagy receptor for autophagosome formation around mitochondria. Using high-resolution live-cell imaging, we find that OPTN is recruited to ubiquitinated mitochondria downstream of PARK2, and induces autophagosome assembly around mitochondria via its LC3-interacting region. Mutations in OPTN are linked to both glaucoma and ALS (amyotrophic lateral sclerosis), and an ALS-associated E478G mutation in OPTN''s ubiquitin binding domain leads to defective mitophagy and accumulation of damaged mitochondria. Importantly, our results highlight a role for mitophagy defects in ALS pathogenesis, and demonstrate that defects in the same pathway for mitochondrial homeostasis are causal for both familial Parkinson disease and ALS.     

Self-assembly pathways of E22Δ-type amyloid β peptide mutants generated from non-aggregative O-acyl isopeptide precursors

The recently identified E22Δ-type amyloid β peptide (Aβ) mutants are reported to favor oligomerization over fibrillization and to exhibit more-potent synaptotoxicity than does wild-type (WT) Aβ. Aβ(E22Δ) mutants can thus be expected to serve as tools for clarifying the impact of Aβ oligomers in Alzheimer's disease (or Alzheimer's-type dementia). However, the biochemical and biophysical properties of Aβ(E22Δ) have not been conclusively determined. Here, we evaluated the self-assembly pathways of Aβ(E22Δ) mutants generated from water-soluble, non-aggregative O-acyl isopeptide precursors. Circular dichroism spectroscopy, Western blot analysis, and thioflavin-T fluorescence intensity and cellular toxicity assays suggest that the self-assembly pathways of Aβ(E22Δ) differed from those of Aβ(WT). Aβ1-40(E22Δ) underwent a rapid random coil→β-sheet conformational change in its monomeric or low-molecular-weight oligomeric states, whereas Aβ1-40(WT) self-assembled gradually without losing its propensity to form random coil structures. The Aβ1-42(E22Δ) monomer formed β-sheet-rich oligomers more rapidly than did Aβ1-42(WT). Additionally, the Aβ1-42(E22Δ) oligomers appear to differ from Aβ1-42(WT) oligomers in size, shape, or both. These results should provide new insights into the functions of Aβ(E22Δ) mutants.     

The contribution of the S-phase checkpoint genes MEC1 and SGS1 to genome stability maintenance in Candida albicans

Genome rearrangements, a common feature of Candida albicans isolates, are often associated with the acquisition of antifungal drug resistance. In Saccharomyces cerevisiae, perturbations in the S-phase checkpoints result in the same sort of Gross Chromosomal Rearrangements (GCRs) observed in C. albicans. Several proteins are involved in the S. cerevisiae cell cycle checkpoints, including Mec1p, a protein kinase of the PIKK (phosphatidyl inositol 3-kinase-like kinase) family and the central player in the DNA damage checkpoint. Sgs1p, the ortholog of BLM, the Bloom's syndrome gene, is a RecQ-related DNA helicase; cells from BLM patients are characterized by an increase in genome instability. Yeast strains bearing deletions in MEC1 or SGS1 are viable (in contrast to the inviability seen with loss of MEC1 in S. cerevisiae) but the different deletion mutants have significantly different phenotypes. The mec1Δ/Δ colonies have a wild-type colony morphology, while the sgs1Δ/Δ mutants are slow-growing, producing wrinkled colonies with pseudohyphal-like cells. The mec1Δ/Δ mutants are only sensitive to ethylmethane sulfonate (EMS), methylmethane sulfonate (MMS), and hydroxyurea (HU) but the sgs1Δ/Δ mutants exhibit a high sensitivity to all DNA-damaging agents tested. In an assay for chromosome 1 integrity, the mec1Δ/Δ mutants exhibit an increase in genome instability; no change was observed in the sgs1Δ/Δ mutants. Finally, loss of MEC1 does not affect sensitivity to the antifungal drug fluconazole, while loss of SGS1 leads to an increased susceptibility to fluconazole. Neither deletion elevated the level of antifungal drug resistance acquisition.     

PINK1‐mediated mitophagy maintains pluripotency through optineurin

ObjectivesDysfunction of autophagy results in accumulation of depolarized mitochondria and breakdown of self‐renewal and pluripotency in ESCs. However, the regulators that control how mitochondria are degraded by autophagy for pluripotency regulation remains largely unknown. This study aims to dissect the molecular mechanisms that regulate mitochondrial homeostasis for pluripotency regulation in mouse ESCs.Materials and methods Parkin^+/+ and parkin ^−/− ESCs were established from E3.5 blastocysts of parkin^+/− x parkin^+/− mating mice. The pink1 ^−/−, optn ^−/− and ndp52 ^−/− ESCs were generated by CRISPR‐Cas9. shRNAs were used for function loss assay of target genes. Mito‐Keima, ROS and ATP detection were used to investigate the mitophagy and mitochondrial function. Western blot, Q‐PCR, AP staining and teratoma formation assay were performed to evaluate the PSC stemness.ResultsPINK1 or OPTN depletion impairs the degradation of dysfunctional mitochondria during reprogramming, and reduces the reprogramming efficiency and quality. In ESCs, PINK1 or OPTN deficiency leads to accumulation of dysfunctional mitochondria and compromised pluripotency. The defective mitochondrial homeostasis and pluripotency in pink1 ^−/− ESCs can be compensated by gain expression of phosphomimetic Ubiquitin (Ub‐S65D) together with WT or a constitutively active phosphomimetic OPTN mutant (S187D, S476D, S517D), rather than constitutively inactive OPTN (S187A, S476A, S517A) or a Ub‐binding dead OPTN mutant (D477N).ConclusionsThe mitophagy receptor OPTN guards ESC mitochondrial homeostasis and pluripotency by scavenging damaged mitochondria through TBK1‐activated OPTN binding of PINK1‐phosphorylated Ubiquitin.     

Effects of excess succinate and retrograde control of metabolite accumulation in yeast tricarboxylic cycle mutants

Cellular and mitochondrial metabolite levels were measured in yeast TCA cycle mutants (sdh2Δ or fum1Δ) lacking succinate dehydrogenase or fumarase activities. Cellular levels of succinate relative to parental strain levels were found to be elevated ~8-fold in the sdh2Δ mutant and ~4-fold in the fum1Δ mutant, and there was a preferential increase in mitochondrial levels in these mutant strains. The sdh2Δ and fum1Δ strains also exhibited 3-4-fold increases in expression of Cit2, the cytosolic form of citrate synthase that functions in the glyoxylate pathway. Co-disruption of the SFC1 gene encoding the mitochondrial succinate/fumarate transporter resulted in higher relative mitochondrial levels of succinate and in substantial reductions of Cit2 expression in sdh2Δsfc1Δ and fum1Δsfc1Δ strains as compared with sdh2Δ and fum1Δ strains, suggesting that aberrant transport of succinate out of mitochondria mediated by Sfc1 is related to the increased expression of Cit2 in sdh2Δ and fum1Δ strains. A defect (rtg1Δ) in the yeast retrograde response pathway, which controls expression of several mitochondrial proteins and Cit2, eliminated expression of Cit2 and reduced expression of NAD-specific isocitrate dehydrogenase (Idh) and aconitase (Aco1) in parental, sdh2Δ, and fum1Δ strains. Concomitantly, co-disruption of the RTG1 gene reduced the cellular levels of succinate in the sdh2Δ and fum1Δ strains, of fumarate in the fum1Δ strain, and citrate in an idhΔ strain. Thus, the retrograde response is necessary for maintenance of normal flux through the TCA and glyoxylate cycles in the parental strain and for metabolite accumulation in TCA cycle mutants.     

Defects in very long chain fatty acid synthesis enhance alpha-synuclein toxicity in a yeast model of Parkinson's disease

We identified three S. cerevisiae lipid elongase null mutants (elo1Δ, elo2Δ, and elo3Δ) that enhance the toxicity of alpha-synuclein (α-syn). These elongases function in the endoplasmic reticulum (ER) to catalyze the elongation of medium chain fatty acids to very long chain fatty acids, which is a component of sphingolipids. Without α-syn expression, the various elo mutants showed no growth defects, no reactive oxygen species (ROS) accumulation, and a modest decrease in survival of aged cells compared to wild-type cells. With (WT, A53T or E46K) α-syn expression, the various elo mutants exhibited severe growth defects (although A30P had a negligible effect on growth), ROS accumulation, aberrant protein trafficking, and a dramatic decrease in survival of aged cells compared to wild-type cells. Inhibitors of ceramide synthesis, myriocin and FB1, were extremely toxic to wild-type yeast cells expressing (WT, A53T, or E46K) α-syn but much less toxic to cells expressing A30P. The elongase mutants and ceramide synthesis inhibitors enhance the toxicity of WT α-syn, A53T and E46K, which transit through the ER, but have a negligible effect on A30P, which does not transit through the ER. Disruption of ceramide-sphingolipid homeostasis in the ER dramatically enhances the toxicity of α-syn (WT, A53T, and E46K).     

Deletion analysis of regions at the C-terminal part of cycloisomaltooligosaccharide glucanotransferase from Bacillus circulans T-3040

Cycloisomaltooligosaccharide glucanotransferase (CITase) belongs to glycoside hydrolase family 66. According to the sequence alignment of enzymes in the same family, we divided the structure of CITase into five regions from the N terminus to the C terminus: an N-terminal conserved region (Ser1-Gly403), an insertion region (R1; Tyr404-Tyr492), two conserved regions (R2; Glu493-Ser596 and R3; Gly597-Met700), and a C-terminal variable region (R4; Lys701-Ser934). CITase catalyzes the synthesis of cycloisomaltooligosaccharides (CIs) with 7-17 glucose units (CI-7 to CI-17) from dextran. In order to clarify the functions of these C-terminal regions (R1-R4), we constructed 15 deletion mutant enzymes. M123Δ (R4-deleted), MΔ234 (R1-deleted), and MΔ23Δ (R1/R4-deleted) catalyzed CI synthesis, but other mutants were inactive. M123Δ, MΔ234, and MΔ23Δ increased their K(m) values against dextran 40. The wild-type enzyme and M123Δ produced CI-8 predominantly, but MΔ234 and MΔ23Δ lost CI-8 production specificity. The k(cat) values of MΔ234 and MΔ23Δ decreased, and these mutants showed narrowed temperature and pH stability ranges. Our deletion analysis suggests that (i) R2 and R3 are crucial for CITase to generate an active form; (ii) both R1 and R4 contribute to substrate binding; and (iii) R1 also contributes to preference of CI-8 production and enzyme stability.     

Inviability of a DNA2 deletion mutant is due to the DNA damage checkpoint

Dna2 is a dual polarity exo/endonuclease, and 5' to 3' DNA helicase involved in Okazaki Fragment Processing (OFP) and Double-Strand Break (DSB) Repair. In yeast, DNA2 is an essential gene, as expected for a DNA replication protein. Suppression of the lethality of dna2Δ mutants has been found to occur by two mechanisms: overexpression of RAD27^scFEN1, encoding a 5' to 3' exo/endo nuclease that processes Okazaki fragments (OFs) for ligation, or deletion of PIF1, a 5' to 3' helicase involved in mitochondrial recombination, telomerase inhibition and OFP. Mapping of a novel, spontaneously arising suppressor of dna2Δ now reveals that mutation of rad9 and double mutation of rad9 mrc1 can also suppress the lethality of dna2Δ mutants. Interaction of dna2Δ and DNA damage checkpoint mutations provides insight as to why dna2Δ is lethal but rad27Δ is not, even though evidence shows that Rad27^ScFEN1 processes most of the Okazaki fragments, while Dna2 processes only a subset.     

Lipid droplet formation protects against gluco/lipotoxicity in Candida parapsilosis: An essential role of fatty acid desaturase Ole1

Elevated levels of glucose and lipids can result in cellular dysfunction in eukaryotic cells ranging from Saccharomyces cerevisiae yeasts to human cells. Moreover, glucotoxicity and lipotoxicity can cause cell death, although the mechanism(s) for lethality is unclear. In the present study, we utilized Candida parapsilosis fatty acid desaturase (OLE1) and fatty acid synthase (FAS2) gene deletion mutants and wild-type (WT) yeast cells to unravel the relationship to glucose and lipid induced cell death in eukaryotic cells. Incubation of WT yeast cells with glucose led to the rapid accumulation of lipid droplets, whereas lipid droplet formation was severely impaired in yeast cells with deletion of OLE1 (ole1Δ/Δ) or FAS2 (fas2Δ/Δ). Interestingly, ole1Δ/Δ yeast cells died within hours in a 1% glucose medium without fatty acid supplementation, whereas the WT or fas2Δ/Δ yeast cells did not. In glucose medium, ole1Δ/Δ yeast cells accumulated saturated fatty acids, while fas2Δ/Δ did not. Addition of saturated fatty acids (e.g., palmitic acid) enhanced ole1Δ/Δ yeast cell death, whereas the addition of unsaturated fatty acids (e.g., oleic or palmitoleic acid) rescued cell death. Furthermore, palmitic acid and glucose medium induced apopotic cell death in ole1Δ/Δ yeast cells, which was dependent on mitochondrial function. Thus, our results show that glucotoxicity is directly linked to lipotoxicity, which we demonstrate is mediated by mitochondrial function.     

GPR109A alleviate mastitis and enhances the blood milk barrier by activating AMPK/Nrf2 and autophagy

Mastitis causes great psychological and physical pain among women. Our previous studies found that niacin has anti-inflammatory effect, and the realization of this function depends on GPR109A. However, there are no previous reports about the anti-inflammatory function of GPR109A in mastitis. In our study, we observed the effect of niacin on the WT and GPR109A^-/- mice mastitis model. The results showed that administration of niacin to WT mice reduced the damage, proinflammatory mediators and protected the integrity of the blood milk barrier in mammary gland. While in GPR109A^-/- mice, there was no effect on the above indexes. In mammary epithelial cells, GPR109A was able to promote autophagy and Nrf2 nuclear import through AMPK. In LPS-induced mammary epithelial cells, niacin inhibited the LPS-induced inflammatory response and downregulation of tight junction proteins, and these effects were eliminated by knocking down GPR109A, blocking autophagy or inhibiting Nrf2 nuclear import. These results indicate that in mastitis, GPR109A promotes autophagy and Nrf2 nuclear import through AMPK, thereby inhibiting inflammatory damage to the mammary gland and repairing the blood milk barrier. Our results suggested that GPR109A may be a potential target for the treatment of mastitis.     

The type II Ca/calmodulin-dependent protein kinases are involved in the regulation of cell wall integrity and oxidative stress response in Candida albicans

The type II Ca²⁺/calmodulin-dependent protein kinases (CaMKs) are thought to play a vital role in cellular regulation in mammalian cells. Two genes CMK1 and CMK2 in the Candida albicans genome encode homologues of mammalian CaMKs. In this work, we constructed the cmk1Δ/Δ, the cmk2Δ/Δ and the cmk1Δ/Δcmk2Δ/Δ mutants and found that CaMKs function in cell wall integrity (CWI) and cellular redox regulation. Loss of either CMK1 or CMK2, or both resulted in increased expression of CWI-related genes under Calcofluor white (CFW) treatment. Besides, CaMKs are essential for the maintenance of cellular redox balance. Disruption of either CMK1 or CMK2, or both not only led to a significant increase of intracellular ROS levels, but also led to a decrease of the mitochondrial membrane potential (MMP), suggesting the important roles that CaMKs play in the maintenance of the mitochondrial function.     

Autophagy contributes to regulation of nuclear dynamics during vegetative growth and hyphal fusion in Fusarium oxysporum

In the fungal pathogen Fusarium oxysporum, vegetative hyphal fusion triggers nuclear mitotic division in the invading hypha followed by migration of a nucleus into the receptor hypha and degradation of the resident nucleus. Here we examined the role of autophagy in fusion-induced nuclear degradation. A search of the F. oxysporum genome database for autophagy pathway components identified putative orthologs of 16 core autophagy-related (ATG) genes in yeast, including the ubiquitin-like protein Atg8, which is required for the formation of autophagosomal membranes. F. oxysporum Foatg8Δ mutants were generated in a strain harboring H1-cherry fluorescent protein (ChFP)-labeled nuclei to facilitate analysis of nuclear dynamics. The Foatg8Δ mutants did not show MDC-positive staining in contrast to the wild type and the FoATG8-complemented (cFoATG8) strain, suggesting that FoAtg8 is required for autophagy in F. oxysporum. The Foatg8Δ strains displayed reduced rates of hyphal growth, conidiation, and fusion, and were significantly attenuated in virulence on tomato plants and in the nonvertebrate animal host Galleria mellonella. In contrast to wild-type hyphae, which are almost exclusively composed of uninucleated hyphal compartments, the hyphae of the Foatg8Δ mutants contained a significant fraction of hyphal compartments with 2 or more nuclei. The increase in the number of nuclei per hyphal compartment was particularly evident after hyphal fusion events. Time-lapse microscopy analyses revealed abnormal mitotic patterns during vegetative growth in the Foatg8Δ mutants. Our results suggest that autophagy mediates nuclear degradation after hyphal fusion and has a general function in the control of nuclear distribution in F. oxysporum.     

Sustained and rapid chromosome movements are critical for chromosome pairing and meiotic progression in budding yeast

Telomere-led chromosome movements are a conserved feature of meiosis I (MI) prophase. Several roles have been proposed for such chromosome motion, including promoting homolog pairing and removing inappropriate chromosomal interactions. Here, we provide evidence in budding yeast that rapid chromosome movements affect homolog pairing and recombination. We found that csm4Δ strains, which are defective for telomere-led chromosome movements, show defects in homolog pairing as measured in a "one-dot/two-dot tetR-GFP" assay; however, pairing in csm4Δ eventually reaches near wild-type (WT) levels. Charged-to-alanine scanning mutagenesis of CSM4 yielded one allele, csm4-3, that confers a csm4Δ-like delay in meiotic prophase but promotes high spore viability. The meiotic delay in csm4-3 strains is essential for spore viability because a null mutation (rad17Δ) in the Rad17 checkpoint protein suppresses the delay but confers a severe spore viability defect. csm4-3 mutants show a general defect in chromosome motion but an intermediate defect in chromosome pairing. Chromosome velocity analysis in live cells showed that while average chromosome velocity was strongly reduced in csm4-3, chromosomes in this mutant displayed occasional rapid movements. Lastly, we observed that spo11 mutants displaying lower levels of meiosis-induced double-strand breaks showed higher spore viability in the presence of the csm4-3 mutation compared to csm4Δ. On the basis of these observations, we propose that during meiotic prophase the presence of occasional fast moving chromosomes over an extended period of time is sufficient to promote WT levels of recombination and high spore viability; however, sustained and rapid chromosome movements are required to prevent a checkpoint response and promote efficient meiotic progression.     

Requirement of a specific group of sphingolipid-metabolizing enzyme for growth of yeast Saccharomyces cerevisiae under impaired metabolism of glycerophospholipids

Sphingolipids play critical roles in many physiologically important events in yeast Saccharomyces cerevisiae. In this study, we screened for yeast mutants showing high sensitivity to Aureobasidin A, an inhibitor of inositol phosphorylceramide synthase, and found that a lack of SAC1 encoding phosphoinositides phosphatase causes high sensitivity to the inhibitor. Double mutation analysis involving the SAC1 and non-essential sphingolipid-metabolizing enzyme genes revealed that csg1Δ, csg2Δ, ipt1Δ or scs7Δ causes synthetic lethality with deletion of SAC1. As previously reported, SAC1-repressed cells exhibited a reduced cellular phosphatidylserine (PS) level, and overexpression of PSS1 encoding PS synthase complemented the growth defects of scs7Δ, csg1Δ and ipt1Δ cells under SAC1-repressive conditions. Furthermore, repression of PSS1 expression resulted in synthetic growth defect with the deletion of CSG1, IPT1 or SCS7. The growth defects of scs7Δ, csg1Δ and ipt1Δ cells under SAC1- or PSS1-repressive conditions were also complemented by overexpression of Arf-GAP AGE1, which encodes a protein related to membrane trafficking. Under SAC1-repressive conditions, scs7Δ, csg1Δ and ipt1Δ cells showed defects in vacuolar morphology, which were complemented by overexpression of each of PSS1 and AGE1. These results suggested that a specific group of sphingolipid-metabolizing enzyme is required for yeast cell growth under impaired metabolism of glycerophospholipids.     

A vacuolar glucoamylase,Sga1, participates in glycogen autophagy for proper asexual differentiation in Magnaporthe oryzae

Nutrient limitation acts as a trigger for the synthesis of glycogen, which serves as a carbon and energy reserve during starvation. Recently, we reported that an autophagy-deficient mutant (atg8Δ) shows severe reduction in aerial hyphal growth and conidiation in the rice-blast fungus Magnaporthe oryzae, and proposed that autophagy plays an important role in facilitating glycogen homeostasis to ensure proper asexual differentiation in Magnaporthe. Here, we identify and characterize a vacuolar glucoamylase function (Sga1) that hydrolyses glycogen to meet the energy requirements during asexual development in Magnaporthe. Loss of SGA1 resulted in significant reduction in conidiation compared to the wild-type Magnaporthe strain. More importantly, an sga1Δ atg8Δ double deletion mutant showed further reduction in conidiation compared to the atg8Δ mutant in Magnaporthe. Forced localization of GFP-Sga1 to the cytoplasm (through removal of the predicted signal peptide) led to increased conidiation in wild type and the sga1Δ, but more interestingly, significantly restored conidiation in the atg8Δ mutant. Our results indicate that autophagy and Sga1 act cooperatively in vacuolar glycogen breakdown, which is essential for conidia formation but dispensable for pathogenicity in Magnaporthe.     

Role of Sch9 in regulating Ras-cAMP signal pathway in Saccharomyces cerevisiae

In Saccharomyces cerevisiae PKA plays a major role in regulating cell growth, metabolism, and stress resistance. We report that Sch9 regulates PKA directly and SCH9 deletion enhances PKA activity by showing that: (1) Bcy1 predominately localized in the nucleus in glycerol-grown sch9Δ cells; (2) large part of the catalytic subunits of PKA transferred from the nucleus to the cytoplasm in sch9Δ cells; (3) higher protein stability of Tpk2 resulted in higher protein level of Tpk2 in sch9Δ than in wild type cells. Our investigations suggest that Sch9 regulates phosphorylation of Bcy1. We also observed hyper-phosphorylation of Cdc25 in sch9Δ, in contrast to the tpk2Δ and tpk2Δsch9Δ mutants, suggesting that feedback inhibition of PKA on Cdc25 is through Tpk2.