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Lipid droplets (LD) are storage sites for neutral lipids that can be used as a source of energy during nutrient starvation, but also function as hubs for fatty acid (FA) trafficking between organelles. In the yeast Saccharomyces cerevisiae, the absence of LD causes a severe disorganization of the endomembrane network during starvation. Here we show that cells devoid of LD respond to amino acid (AA) starvation by activating the serine/threonine phosphatase calcineurin and the nuclear translocation of its target protein Crz1. This activation was inhibited by treatments that restore a normal endomembrane organization, i.e. inhibition of FA synthesis with cerulenin or deletion of the inhibitory transcription factor Opi1. Activation of calcineurin increased the lifespan of LD-deficient cells during AA starvation. Indeed, deletion of its regulatory or catalytic subunits accelerated cell death. Surprisingly, calcineurin activation appeared to be calcium-independent. An increase in intracellular calcium was observed in LD-deficient cells during AA starvation, but its inhibition by genetic deletion of MID1 or YVC1 did not affect calcineurin activity. In contrast, calcineurin activation required the direct regulator of calcineurin Rcn1 and its activating (GSK-3)-related protein kinase Mck1., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Lipid droplets are ubiquitous organelles in eukaryotes that act as storage sites for neutral lipids. Under normal growth conditions, they are not required in the yeast Saccharomyces cerevisiae. However, recent works have shown that lipid droplets are required for autophagy to proceed in response to nitrogen starvation and that they play an essential role in maintaining ER homeostasis. Autophagy is a major catabolic pathway that helps degradation and recycling of potentially harmful proteins and organelles. It can be pharmacologically induced by rapamycin even in the absence of lipid droplets. Here, we show that amino acid starvation is responsible for autophagy failure in lipid droplet-deficient yeast. It not only fails to induce autophagy but also inhibits rapamycin-induced autophagy. The general amino acid control pathway is not involved in this paradoxical effect of amino acid shortage. We correlate the autophagy failure with mitochondria aggregation and we show that amino acid starvation-induced autophagy is restored in lipid droplet-deficient yeast by increasing mitochondrial biomass physiologically (respiration) or genetically (REG1 deletion). Our results establish a new functional link between lipid droplets, ER and mitochondria during nitrogen starvation-induced autophagy., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Plants live in association with complex populations of microorganisms, including Plant Growth-Promoting Rhizobacteria (PGPR) that confer to plants an improved growth and enhanced stress tolerance. This large and diverse group includes endophytic bacteria that are able to colonize the internal tissues of plants. In the present study, we have isolated a nonrhizobial species from surface sterilized root nodules of Retama monosperma , a perennial leguminous species growing in poor and high salinity soils. Sequencing of its genome reveals this endophytic bacterium is a Bacillus megaterium strain (RmBm31) that possesses a wide range of genomic features linked to plant growth promotion. Furthermore, we show that RmBm31 is able to increase the biomass and positively modify the root architecture of seedlings of the model plant species Arabidopsis thaliana both in physical contact with its roots and via the production of volatile organic compounds. Lastly, we investigated the molecular mechanisms implicated in RmBm31 plant beneficial effects by carrying out a transcriptional analysis on a comprehensive set of phytohormone-responsive marker genes. Altogether, our results demonstrate that RmBm31 displays plant growth-promoting traits of potential interest for agricultural applications., (Copyright © 2020 Dahmani, Desrut, Moumen, Verdon, Mermouri, Kacem, Coutos-Thévenot, Kaid-Harche, Bergès and Vriet.)

Autophagy is a eukaryotic process responsible for the degradation of intracellular content such as damaged organelles. Several putative autophagy-related genes have been identified within the annotated genome of the free-living amoeba Acanthamoeba castellanii. However, the involvement of the corresponding proteins in the autophagy pathway had not been formerly established. Here, we report that AcAtg8 cDNA can complement ATG8-deficient Saccharomyces cerevisiae., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Macroautophagy is a degradative pathway whereby cells encapsulate and degrade cytoplasmic material within endogenously-built membranes. Previous studies have suggested that autophagosome membranes originate from lipid droplets. However, it was recently shown that rapamycin could induce autophagy in cells lacking these organelles. Here we show that lipid droplet-deprived cells are unable to perform autophagy in response to nitrogen-starvation because of an accelerated lipid synthesis that is not observed with rapamycin. Using cerulenin, a potent inhibitor of fatty acid synthase, and exogenous addition of palmitic acid we could restore nitrogen-starvation induced autophagy in the absence of lipid droplets., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Canthinones are natural substances with a wide range of biological activities, including antipyretic, antiparasitic, and antimicrobial. Antiproliferative and/or cytotoxic effects of canthinones on cancer cells have also been described, although their mechanism of action remains ill defined. To gain better insight into this mechanism, the antiproliferative effect of a commercially available canthin-6-one (1) was examined dose-dependently on six cancer cell lines (human prostate, PC-3; human colon, HT-29; human lymphocyte, Jurkat; human cervix, HeLa; rat glioma, C6; and mouse embryonic fibroblasts, NIH-3T3). Cytotoxic effects of 1 were investigated on the same cancer cell lines by procaspase-3 cleavage and on normal human skin fibroblasts. Strong antiproliferative effects of the compound were observed in all cell lines, whereas cytotoxic effects were very dependent on cell type. A better definition of the mechanism of action of 1 was obtained on PC-3 cells, by showing that it decreases BrdU incorporation into DNA by 60% to 80% and mitotic spindle formation by 70% and that it causes a 2-fold accumulation of cells in the G2/M phase of the cell cycle. Together, the data suggest that the primary effect of canthin-6-one (1) is antiproliferative, possibly by interfering with the G2/M transition. Proapoptotic effects might result from this disturbance of the cell cycle.

In crustaceans, the androgenic gland (AG), thanks to the synthesis of the androgenic gland hormone (AGH), controls the differentiation of the primary and secondary male sexual characters. In this study, we amplified 12 new AGH cDNAs in species belonging to five different families of the infra-order Ligiamorpha of terrestrial isopods. Putative essential amino acids for the production of a functional AGH protein exhibit signatures of negative selection and are strictly conserved including typical proteolytic cleavage motifs, a putative N-linked glycosylation motif on the A chains and the eight Cys positions. An insulin-like growth factor motif was also identified in Armadillidium AGH sequences. The phylogenetic relationships of AGH sequences allowed one to distinguish two main clades, corresponding to members of the Armadillidiidae and the Porcellionidae families which are congruent with the narrow specificity of AG heterospecific grafting. An in-depth understanding of the regulation of AGH expression would help deciphering the interaction between Wolbachia, widespread feminizing endosymbiotic bacteria in isopods, and the sex differentiation of their hosts., (Copyright © 2014 Elsevier B.V. All rights reserved.)

This study reports that the spontaneous 50-fold activation of rhodopsin gene transcription, observed in cultured retinal precursors from 13-day chicken embryo, relies on a Ca(2+)-dependent mechanism. Activation of a transiently transfected rhodopsin promoter (luciferase reporter) in these cells was inhibited (60%) by cotransfection of a dominant-negative form of the cAMP-responsive element-binding protein. Both rhodopsin promoter activity and rhodopsin mRNA accumulation were blocked by Ca(2+)/calmodulin-dependent kinase II inhibitors, but not by protein kinase A inhibitors, suggesting a role of Ca(2+) rather than cAMP. This was confirmed by the inhibitory effect of general and T-type selective Ca(2+) channel blockers. Oscillations in Ca(2+) fluorescence (Fluo8) could be observed in 1/10 cells that activated the rhodopsin promoter (DsRed reporter). A robust and reversible inhibition of rhodopsin gene transcription by ZD7288 indicated a role of hyperpolarization-activated channels (HCN). Cellular localization and developmental expression of HCN1 were compatible with a role in the onset of rhodopsin gene transcription. Together, the data suggest that the spontaneous activation of rhodopsin gene transcription in cultured retinal precursors results from a signaling cascade that involves the pacemaker activity of HCN channels, the opening of voltage-gated Ca(2+)-channels, activation of Ca(2+)/calmodulin-dependent kinase II and phosphorylation of cAMP-responsive element-binding protein. Rhodopsin gene expression in cultured retinal precursors from chicken embryo relies on a Ca2+-dependent mechanism whereby hyperpolarization-activated cyclic nucleotide-gated channels (HCN) activate T-type voltage-dependent Ca2+ channels (VDCC) through membrane depolarization, causing calmodulin-dependent kinase II (CaMKII) to phosphorylate the cAMP-responsive element-binding protein (CREB) and leading to activation of rhodopsin gene transcription. Photoreceptor localization and development of HCN1 channels suggest similar role in vivo., (© 2013 International Society for Neurochemistry.)

Screening for suppressors of canthin-6-one toxicity in yeast identified Yap1, a transcription factor involved in cell response to a broad range of injuries. Although canthin-6-one did not promote a significant oxidative stress, overexpression of YAP1 gene clearly increased resistance to this drug. We demonstrated that Yap1-mediated resistance involves the plasma membrane major-facilitator-superfamily efflux pump Flr1 but not the vacuolar ATP-binding-cassette transporter Ycf1. FLR1 overexpression was sufficient to reduce sensitivity to the drug, but strictly dependent on a functional YAP1 gene., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Petite mutations have been described in Saccharomyces cerevisiae and pathogenic yeasts. However, previous studies of the phenotypic traits of these petite mutants reported that they express azole resistance. We describe a clinical isolate of Candida glabrata with a striking association between increased susceptibility to azoles and respiratory deficiency. This isolate was obtained from a urine sample together with a respiration-competent C. glabrata isolate which exhibited azole resistance. The respiratory status of the two isolates was confirmed by cultivation on glycerol-containing agar and oxygraphy. Flow cytometry revealed the normal incorporation of rhodamine 123, and mitochondrial sections with typical cristae were seen by transmission electron microscopy for both isolates. Together, these results suggested a nuclear origin for the reduced respiratory capacity of the hypersusceptible isolate. The sterol contents of these isolates were similar to the sterol content of a reference strain. Sequencing of the ERG11 and PDR1 genes revealed that the sequences were identical in the two isolates, demonstrating their close relatedness. In addition to silent mutations, they carried a nonsense mutation in PDR1 that led to the truncation of transcription factor Pdr1p. They also overexpressed both PDR1 and one of its targets, CDR1, providing a possible explanation for the azole resistance of the respiration-competent isolate. In conclusion, in addition to azole resistance, which is a common feature of C. glabrata mitochondrial petite mutants, the mutation of a nuclear gene affecting aerobic growth may lead to azole hypersusceptibility; however, the mechanisms underlying this phenotype remain to be determined.

Stress within the endoplasmic reticulum (ER) induces a coordinated response, namely the unfolded protein response (UPR), devoted to helping the ER cope with the accumulation of misfolded proteins. Failure of the UPR plays an important role in several human diseases. Recent studies report that intracellular accumulation of saturated fatty acids (SFAs) and cholesterol, seen in diseases of high incidence, such as obesity or atherosclerosis, results in ER stress. In the present study, we evaluated the effects of perturbations to lipid homeostasis on ER stress/UPR induction in the model eukaryote Saccharomyces cerevisiae. We show that SFA originating from either endogenous(preclusion of fatty acid desaturation) or exogenous (feeding with extracellular SFA) sources trigger ER stress and that ergosterol, the major sterol in yeast, acts synergistically with SFA in this process. This latter effect is connected to ergosterol accumulation within microsomal fractions from SFA-accumulating cells, which display highly saturated phospholipid content. Moreover, treating the cells with the molecular chaperone 4-phenyl butyrate abolishes UPR induction, suggesting that lipid-induced ER stress leads to an overload of misfolded protein that acts, in turn, as the molecular signal for induction of the UPR. The present data are discussed in the context of human diseases that involve lipid deregulation.

Unlike the molecular mechanisms that lead to azole drug resistance, the molecular mechanisms that lead to polyene resistance are poorly documented, especially in pathogenic yeasts. We investigated the molecular mechanisms responsible for the reduced susceptibility to polyenes of a clinical isolate of Candida glabrata. Sterol content was analyzed by gas-phase chromatography, and we determined the sequences and levels of expression of several genes involved in ergosterol biosynthesis. We also investigated the effects of the mutation harbored by this isolate on the morphology and ultrastructure of the cell, cell viability, and vitality and susceptibility to cell wall-perturbing agents. The isolate had a lower ergosterol content in its membranes than the wild type, and the lower ergosterol content was found to be associated with a nonsense mutation in the ERG6 gene and induction of the ergosterol biosynthesis pathway. Modifications of the cell wall were also seen, accompanied by increased susceptibility to cell wall-perturbing agents. Finally, this mutation, which resulted in a marked fitness cost, was associated with a higher rate of cell mortality. Wild-type properties were restored by complementation of the isolate with a centromeric plasmid containing a wild-type copy of the ERG6 gene. In conclusion, we have identified the molecular event responsible for decreased susceptibility to polyenes in a clinical isolate of C. glabrata. The nonsense mutation detected in the ERG6 gene of this isolate led to a decrease in ergosterol content. This isolate may constitute a useful tool for analysis of the relevance of protein trafficking in the phenomena of azole resistance and pseudohyphal growth.

The antibiofilm activity of 10 terpenes was tested in vitro against three Candida species by 24-h treatment of biofilms aged 1-5 days. Treatment of 24-h-old Candida albicans biofilms with carvacrol, geraniol or thymol (0.06%) resulted in >80% inhibition. Carvacrol (0.03%) inhibition was > or =75% independent of the age of the C. albicans biofilm. Carvacrol (0.125%) inhibition was >75% against Candida glabrata and Candida parapsilosis biofilms. Geraniol (> or =0.125%) and thymol (0.06% or 0.125%) inhibition was >75% against C. parapsilosis biofilms whatever their age. This study demonstrates the antibiofilm activity of terpenes and points out the exceptional efficiency of carvacrol, geraniol and thymol, which could represent candidates in the treatment of candidiasis associated with medical devices.

When heme biosynthesis is disrupted, the yeast Saccharomyces cerevisiae becomes unable to synthesize its major sterol, ergosterol, and desaturate fatty acids. We took advantage of this physiological peculiarity to evaluate the consequences of ergosterol and/or unsaturated fatty acid (UFA) depletions on the biogenesis of a model polytopic plasma membrane protein, the uracil permease Fur4p. We show that under UFA shortage, which results in low amounts of diunsaturated phospholipid species, and under ergosterol depletion, Fur4p is prematurely routed from the Golgi apparatus to the vacuolar lumen in a process that requires the ubiquitin ligase Rsp5p. Interestingly, this diversion is not correlated to Fur4p exclusion from detergent-resistant membranes. In an independent set of experiments, we show that Fur4p targeting to the plasma membrane depends on phosphatidylethanolamine amounts and more specifically on the propensity of this phospholipid to form a hexagonal phase. In light of recent literature, we propose a model in which ergosterol and diunsaturated phospholipid species maintain optimal membrane curvature for Fur4p to evade the Golgi quality control process and to be properly delivered to its normal destination.

The yeast Saccharomyces cerevisiae is a facultative anaerobic organism. Under anaerobiosis, sustained growth relies on the presence of exogenously supplied unsaturated fatty acids and ergosterol that yeast is unable to synthesize in the absence of oxygen or upon haem depletion. In the absence of exogenous supplementation with unsaturated fatty acid, a net accumulation of SFA (saturated fatty acid) is observed that induces significant modification of phospholipid profile [Ferreira, Régnacq, Alimardani, Moreau-Vauzelle and Bergès (2004) Biochem. J. 378, 899-908]. In the present paper, we focus on the role of SFH2/CSR1, a hypoxic gene related to SEC14 and its involvement in lipid metabolism upon haem depletion in the absence of oleic acid supplementation. We observed that inactivation of SFH2 results in enhanced accumulation of SFA and phospholipid metabolism alterations. It results in premature growth arrest and leads to an exacerbated sensitivity to exogenous SFA. This phenotype is suppressed in the presence of exogenous oleic acid, or by a controlled expression of FAS1, one of the two genes encoding FAS. We present several lines of evidence to suggest that Sfh2p and oleic acid regulate SFA synthase in yeast at different levels: whereas oleic acid acts on FAS2 at the transcriptional level, we show that Sfh2p inhibits fatty acid synthase activity in response to haem depletion.

Little information is available about the molecular mechanisms responsible for polyene resistance in pathogenic yeasts. A clinical isolate of Candida glabrata with a poor susceptibility to polyenes, as determined by disk diffusion method and confirmed by determination of MIC, was recovered from a patient treated with amphotericin B. Quantitative analysis of sterols revealed a lack of ergosterol and an accumulation of late sterol intermediates, suggesting a defect in the final steps of the ergosterol pathway. Sequencing of CgERG11, CgERG6, CgERG5, and CgERG4 genes revealed exclusively a unique missense mutation in CgERG6 leading to the substitution of a cysteine by a phenylalanine in the corresponding protein. In addition, real-time reverse transcription-PCR demonstrated an overexpression of genes encoding enzymes involved in late steps of the ergosterol pathway. Moreover, this isolate exhibited a pseudohyphal growth whatever the culture medium used, and ultrastructural changes of the cell wall of blastoconidia were seen consisting in a thinner inner layer. Cell wall alterations were also suggested by the higher susceptibility of growing cells to Calcofluor white. Additionally, complementation of this isolate with a wild-type copy of the CgERG6 gene restored susceptibility to polyenes and a classical morphology. Together, these results demonstrated that mutation in the CgERG6 gene may lead to a reduced susceptibility to polyenes and to a pseudohyphal growth due to the subsequent changes in sterol content of the plasma membrane.

Azole resistance has been insufficiently investigated in the yeast Candida tropicalis. Here we determined the molecular mechanisms responsible for azole resistance in a clinical isolate of this pathogenic yeast. Antifungal susceptibility testing performed by a disk diffusion method showed resistance or markedly decreased susceptibility to azoles, which was confirmed by determination of MICs. Considering the relationship between azole susceptibility and the respiration reported for other yeast species, the respiratory activity of this isolate was investigated. Flow cytometry using rhodamine 123 and oxygraphy demonstrated an increased respiratory activity, which was not linked to an overexpression or increased number of copies of the mitochondrial genome. Among previously described resistance mechanisms, an increased activity of efflux pumps was investigated by flow cytometry using rhodamine 6G. However, the efflux of rhodamine 6G was lower in the resistant isolate than in susceptible ones. Likewise, real-time reverse transcription-PCR quantification of the expression of C. tropicalis MDR1 (CtMDR1), which encodes an efflux protein belonging to the major facilitator superfamily, did not show overexpression of this gene. In contrast, the resistant isolate overexpressed the CtERG11 gene coding for lanosterol 14alpha-demethylase. This was in agreement with the larger amount of ergosterol found in this isolate. Moreover, sequencing of CtERG11 showed a point mutation leading to a tyrosine substitution in the protein sequence, which might lead to decreased binding affinity for azoles. In conclusion, overexpression of CtERG11 associated with a missense mutation in this gene seemed to be responsible for the acquired azole resistance of this clinical isolate.

Efficient sterol influx in the yeast Saccharomyces cerevisiae is restricted to anaerobiosis or to haem deficiency resulting from mutations. Constitutive expression of SUT1, an hypoxic gene encoding a transcriptional regulator, induces sterol uptake in aerobiosis. A genome-wide approach using DNA microarray was used to identify the mediators of SUT1 effects on aerobic sterol uptake. A total of 121 ORFs (open reading frames) were significantly and differentially expressed after SUT1 overexpression, 61 down-regulated and 60 up-regulated. Among these genes, the role of the putative ABC transporter (ATP-binding-cassette transporter) Aus1, and of the cell-wall mannoprotein Dan1, was characterized better. These two genes play an essential role in aerobic sterol uptake, since their deletion compromised the SUT1 effects, but individual overexpression of either of these genes in a wild-type background was not sufficient for this process. However, constitutive co-expression of AUS1 and DAN1 in a wild-type background resulted in sterol influx in aerobiosis. These results suggest that the corresponding proteins may act synergistically in vivo to promote sterol uptake.

We previously showed that resistant colonies of Candida glabrata inside the azole inhibition zones had respiratory deficiency due to mutations in mitochondrial DNA. Here, we analyzed the mechanisms of azole resistance in petite mutants of C. glabrata obtained by exposure to fluconazole or induced by ethidium bromide. The respiratory deficiency of these mutants was confirmed by oxygraphy and flow cytometric analysis with rhodamine 123, and its mitochondrial origin was demonstrated by transmission electron microscopy and restriction endonuclease analysis of the mitochondrial DNA. Flow cytometry with rhodamine 6G suggested an increased drug efflux in mutant cells, which was further supported by Northern blot analysis of the expression of the C. glabrata CDR1 (CgCDR1) and CgCDR2 genes, encoding efflux pumps. Conversely, the expression of CgERG11, which encodes the azole target, was not affected by petite mutations, and no differences were seen in the sequence of this gene between parent isolates and mutants. Moreover, sterol analysis showed similar overall amount of sterols in parent and mutant cells, but quantitative modifications were observed in the mutants, with almost undetectable biosynthesis intermediates. Further analysis performed after separation of free sterols from steryl esters revealed a defect in sterol esterification in mutant cells, with free ergosterol representing 92% of the overall sterol content. Thus, resistance or decreased susceptibility to azoles in petite mutants of C. glabrata is associated with increased expression of CgCDR1 and, to a lesser extent, of CgCDR2. In addition, the marked increase in free ergosterol content would explain their increased susceptibility to polyenes.

In the yeast Saccharomyces cerevisiae, UFA (unsaturated fatty acids) and ergosterol syntheses are aerobic processes that require haem. We took advantage of a strain affected in haem synthesis ( hem1 Delta) to starve specifically for one or the other of these essential lipids in order to examine the consequences on the overall lipid composition. Our results demonstrate that reserve lipids (i.e. triacylglycerols and steryl esters) are depleted independently of haem availability and that their UFA and sterol content is not crucial to sustain residual growth under lipid depletion. In parallel to UFA starvation, a net accumulation of SFA (saturated fatty acids) is observed as a consequence of haem biosynthesis preclusion. Interestingly, the excess SFA are not mainly stored within triacylglycerols and steryl esters but rather within specific phospholipid species, with a marked preference for PtdIns. This results in an increase in the cellular PtdIns content. However, neutral lipid homoeostasis is perturbed under haem starvation. The contribution of two lipid particle-associated proteins (namely Tgl1p and Dga1p) to this process is described.

Over the past two decades, the incidence of infections due to Candida glabrata, a yeast with intrinsic low susceptibility to azole antifungals, has increased markedly. Respiratory deficiency due to mutations in mitochondrial DNA (mtDNA) associated with resistance to azoles frequently occurs in vitro in this species. In order to specify the relationships between respiration and azole susceptibility, the effects of respiratory chain inhibitors on a wild-type isolate of C. glabrata were evaluated. Respiration of blastoconidia was immediately blocked after extemporaneous addition of potassium cyanide, whereas a 4-h preincubation was required for sodium azide. Antifungal susceptibility determined by a disk diffusion method on Casitone agar containing sodium azide showed a significant decrease in the susceptibility to azoles. Biweekly subculturing on Casitone agar supplemented with sodium azide was therefore performed. This resulted after 40 passages in the isolation of a respiration-deficient mutant, as suggested by its lack of growth on glycerol-containing agar. This respiratory deficiency was confirmed by flow cytometric analysis of blastoconidia stained with rhodamine 123 and by oxygraphy. Moreover, transmission electron microscopy and restriction endonuclease analysis of the mtDNA of mutant cells demonstrated the mitochondrial origin of the respiratory deficiency. Finally, this mutant exhibited cross-resistance to all the azoles tested. In conclusion, blockage of respiration in C. glabrata induces decreased susceptibility to azoles, culminating in azole resistance due to the deletion of mtDNA. This mechanism could explain the induction of petite mutations by azole antifungals which have been demonstrated to act directly on the mitochondrial respiratory chain.

SUT1 constitutive expression in aerobiosis suppressed the ts phenotype of the sec14-1 mutation, restored growth of the sec14-null mutant and corrected the translocation defect of the vacuolar carboxypeptidase Y. Therefore SUT1 was shown to be a novel potent sec14-1 suppressor. Further, the hypoxic gene CSR1 (YLR380W), a Sec14 homolog, was upregulated upon SUT1 constitutive expression. In addition, SUT1 effects on both sec14-1 suppression and on free sterol composition were abolished in a csr1-null background, showing that this gene acts downstream of SUT1.

SUT1 is a hypoxic gene encoding a nuclear protein that belongs to the Zn[II]2Cys-6 family. It has been shown that constitutive expression of SUT1 induces exogenous sterol uptake in aerobically growing Saccharomyces cerevisiae cells. A differential display approach was used to identify genes whose transcription is modified upon SUT1 induction. Within the promoter sequence of one of these genes, DAN1, we identified the region responsive to SUT1 and showed that it has a strong repressive activity when cloned in the vicinity of distinct promoters. Upon SUT1 constitutive expression in aerobiosis, the repression is released, allowing enhanced transcription of the reporter gene. We provide evidence that the repression is promoted by the Cyc8p(Ssn6p)-Tup1p co-repressor and that release of repression is the result of a physical interaction between Sut1p and Cyc8p. Moreover, genetic data suggest that complete derepression of the reporter gene requires a functional Cyc8p. In addition, we show that Sut1p is involved in the induction of hypoxic gene transcription when the cells are shifted from aerobiosis to anaerobiosis.

Budding yeast Saccharomyces cerevisiae is a facultative anaerobe whose growth upon oxygen starvation depends on its capacity to import exogenously supplied sterols, whereas the cells are not permeable to these molecules when grown aerobically. Few genes have been identified as being involved in sterol uptake. A higher SUT1 gene dosage leads to a modest, but significant, increase in sterol uptake under aerobic conditions. Based on sequence and physiological data, SUT1 is a hypoxic gene negatively regulated when the cells are grown in the presence of oxygen. We replaced the SUT1 promoter with the constitutive PMA1 gene promoter in order to enhance its transcription. We observed that sterol uptake was then comparable with that obtained with a sterol importing hem1 mutant, although the heme status of the strain was not modified in a process which still occurs when the cells are not growing. Unexpectedly, SUT1 constitutive expression led to a parallel significant increase in endogenous sterol biosynthesis. Moreover, here we present new data showing that the structurally related YPR009 gene (SUT2) is a functional homologue of SUT1, and that both gene products may represent two novel yeast regulatory proteins involved in sterol uptake.

The wild-type ERG19 gene of the yeast Saccharomyces cerevisiae encoding mevalonate diphosphate decarboxylase (MVD) and the mutated recessive erg19-34 allele leading to a decrease of sterol production and to a thermosensitive phenotype have been characterized [2]. The mutated erg19-34 allele bears a single amino acid leucine 79-to-proline (L79P) substitution. It was shown that this mutation does not affect the level of production of the enzyme. We performed a two-hybrid assay to show that the yeast Saccharomyces cerevisiae MVD forms homodimers in vivo and that the single point mutation drastically impairs the oligomerization of the protein, thereby explaining the deficiency of MVD activity observed in the temperature-sensitive strain.

We report the cloning of a cDNA encoding human syntaxin 8 (STX8), using the regulator (R) domain of the cystic fibrosis transmembrane conductance regulator (CFTR) as a bait to screen a human fetal lung cDNA library by the yeast two-hybrid system. This gene was found broadly transcribed and its mRNA size is about 1.3 kb. The STX8 gene maps to chromosomal band 17p12 and it encodes a 236-amino-acid protein. Syntaxin 8 contains in its C-terminal half a coiled-coil domain found highly conserved in the t-SNARE (SNAP receptor on target membrane) superfamily of proteins, which are involved in vesicular trafficking and docking. In syntaxin 8, a C-terminal hydrophobic domain may constitute a transmembrane anchor. It was recently shown that CFTR-mediated chloride currents can be regulated by syntaxin 1A, a t-SNARE family member, through direct protein-protein interaction. This raises the possibility that syntaxin 8 may also be involved in such regulations., (Copyright 1999 Academic Press.)

Sequence comparison with the mevalonate diphosphate decarboxylase (MVD) amino acid sequence of Saccharomyces cerevisiae identified an EST clone corresponding to a cDNA that may encode Arabidopsis thaliana MVD (AtMVD1). This enzyme catalyses the synthesis of isopentenyl diphosphate, the building block of sterol and isoprenoid biosynthesis, and uses mevalonate diphosphate as a substrate. Sequencing of the full-length cDNA was performed. The predicted amino acid sequence presents about 55% identity with the yeast, human and rat MVDs. The sequence of the genomic region of A. thaliana MVD was also obtained and Southern blot analysis on genomic DNA showed that A. thaliana could have at least one homologous MVD gene. In order to allow heterologous expression in S. cerevisiae, the MVD open reading frame (ORF) was then cloned under the control of the yeast PMA1 strong promoter. When expressed in yeast, the A. thaliana cDNA complemented both the thermosensitive MN19-34 strain deficient in MVD, and the lethal phenotype of an ERG19 deleted strain. However, the wild-type sterol content was not fully restored suggesting that the A. thaliana MVD activity may not be optimal in yeast. A two-hybrid assay was also performed to evaluate homodimer formation of the A. thaliana MVD and heterodimer formation between the plant and yeast heterologous enzymes.

The filamentous fungus Aspergillus niger produces two glycosylated forms of the sucrose-hydrolysing enzyme, invertase. In contrast, some Trichoderma species lack invertase and are unable to utilise sucrose as a sole carbon source. Using an A. niger genomic library constructed in a cosmid vector containing the ura5 gene of Podospora anserina as a selectable marker, and the T. reesei ura5- strain as a sucrose-minus recipient strain, an A. niger invertase gene (suc1) has been cloned by a sib selection procedure. PAGE and enzyme analysis confirmed that transformants had acquired invertase activity. The cloned gene contained DNA sequences which were complementary to the amino-acid sequences of tryptic peptides found in invertase purified from A. niger. The suc1 invertase gene can be used as a dominant selectable marker for the transformation of Trichoderma strains.

Uridine auxotrophs of the filamentous fungus Trichoderma reesei have been selected using a positive screening procedure with 5-fluoro orotate. Mutants deficient for the orotidine-5'-phosphate decarboxylase gene (ura3 mutants) and for the orotate phosphoribosyl transferase gene (ura5 mutants) have been characterized. The homologous ura3 and ura5 genes have been isolated and used to transform the auxotrophic mutants. Transformation efficiency with these homologous systems is very high (greater than 10(4) transformants per micrograms DNA). Transformation occurred by integration of vector DNA at homologous and ectopic loci. Mitotic instability was observed among some of the transformants. Sequence analysis at the protein level, of the T. reesei ura3 and ura5 genes showed extensive blocks of homology, with the corresponding genes from other organisms. The ura3 gene from T. reesei contains an insertion of 103 aa. A similar sequence is also found inserted in OMPdecase from the pyrenomycetes Neurospora crassa and Cephalosporium acremonium.

We have developed an improved transformation procedure for the filamentous fungus Podospora anserina. This procedure is based on the observation that a heat shock at an elevated temperature (48 degrees C) improves the competence of P. anserina protoplasts for transformation 5- to 10-fold. This is observable only if the heat shock is applied before the addition of transforming DNA. An increase in competence is observed immediately after the heat shock, and heat-shocked cells are still competent after 20-30 min. The mechanism by which heat shock improves competence remains unclear. The modified transformation procedure gives as many as 200-500 stable transformants per microgram of plasmid DNA containing the P. anserina ura5 gene. This should allow direct cloning of P. anserina genes from a cosmid library.