UBI4, the polyubiquitin gene of Saccharomyces cerevisiae, is a heat shock gene that is also subject to catabolite derepression control

Carbon and nitrogen regulation of UBI4, the stress-inducible polyubiquitin gene of Saccharomyces cerevisiae, was investigated using a UBI4 promoter-LacZ fusion gene (UBI4-LacZ). Expression of this gene in cells grown on different media indicated that the UBI4 promoter is more active during growth on respiratory than on fermentable carbon sources but is not subject to appreciable control by nitrogen catabolite repression. UBI4-LacZ expression was virtually identical in cells having constitutively high (ras2, sra1-13) or constitutively low (ras2) levels of cyclic AMP-dependent protein kinase activity, indicating that this kinase does not exert a major influence on UBI4 expression. Catabolite derepression control of the UBI4 promoter was confirmed by measurements of UBI4-LacZ expression in hap mutant and wild-type strains before and after transfer from glucose to lactate. Mutagenesis of the perfect consensus for HAP2/3/4 complex binding at position ?542 resulted in considerable reduction of UBI4 promoter derepression with respiratory adaptation in HAP wild-type cells and abolished the reduced UBI4-LacZ derepression normally seen when aerobic cultures of the hap1 mutant are transferred from glucose to lactate. This HAP2/3/4 binding site is therefore a major element contributing to catabolite derepression of the UBI4 promoter, although data obtained with hap1 mutant cells indicated that HAP1 also contributes to this derepression. The HAP2/3/4 and HAP1 systems are normally found to activate genes for mitochondrial (respiratory) functions. Their involvement in mediating higher activity of the UBI4 promoter during respiratory growth may reflect the contribution of UBI4 expression to tolerance of oxidative stress.     

The CCAAT box-binding factor stimulates ammonium assimilation in Saccharomyces cerevisiae, defining a new cross-pathway regulation between nitrogen and carbon metabolisms.

In Saccharomyces cerevisiae, carbon and nitrogen metabolisms are connected via the incorporation of ammonia into glutamate; this reaction is catalyzed by the NADP-dependent glutamate dehydrogenase (NADP-GDH) encoded by the GDH1 gene. In this report, we show that the GDH1 gene requires the CCAAT box-binding activator (HAP complex) for optimal expression. This conclusion is based on several lines of evidence: (1) overexpression of GDH1 can correct the growth defect of hap2 and hap3 mutants on ammonium sulfate as a nitrogen source, (ii) Northern (RNA) blot analysis shows that the steady-state level of GDH1 mRNA is strongly lowered in a hap2 mutant, (iii) expression of a GDH1-lacZ fusion is drastically reduced in hap mutants, (iv) NADP-GDH activity is several times lower in the hap mutants compared with that in the isogenic wild-type strain, and finally, (v) site-directed mutagenesis of two consensual HAP binding sites in the GDH1 promoter strongly reduces expression of GDH1 and makes it HAP independent. Expression of GDH1 is also regulated by the carbon source, i.e., expression is higher on lactate than on ethanol, glycerol, or galactose, with the lowest expression being found on glucose. Finally, we show that a hap2 mutation does not affect expression of other genes involved in nitrogen metabolism (GDH2, GLN1, and GLN3 encoding, respectively, the NAD-GDH, glutamine synthetase, and a general activator of several nitrogen catabolic genes). The HAP complex is known to regulate expression of several genes involved in carbon metabolism; its role in the control of GDH1 gene expression, therefore, provides evidence for a cross-pathway regulation between carbon and nitrogen metabolisms.     

Transcription of yeast COX6, the gene for cytochrome c oxidase subunit VI, is dependent on heme and on the HAP2 gene

The COX6 gene encodes subunit VI of cytochrome c oxidase. Previously, this gene and its mRNAs were characterized, and its expression has been shown to be subject to glucose repression/derepression. In this study we have examined the effects of heme and the HAP1 (CYP1) and HAP2 genes on the expression of COX6. By quantitating COX6 RNA levels and assaying beta-galactosidase activity in yeast cells carrying COX6-lacZ fusion genes, we have found that COX6 is regulated positively by heme and HAP2, but is unaffected by HAP1. Through 5' deletion analysis we have also found that the effects of heme and HAP2 on COX6 are mediated by sequences between 135 and 590 base pairs upstream of its initiation codon. These findings identify COX6 as the fourth respiratory protein gene that is known to be regulated positively by heme and HAP2. The other three, CYC1, COX4, and COX5a, encode iso-1-cytochrome c, cytochrome c oxidase subunit IV, and an isolog, Va, of cytochrome c oxidase subunit V, respectively. Thus, it appears that the biogenesis of two interacting proteins, cytochrome c and cytochrome c oxidase, in the mitochondrial respiratory chain, are under the control of common factors.     

Molecular characterization of a cellulase-negative mutant of Hypocrea jecorina

The "cbh2 activating element," CAE, consisting of two separate boxes (ATTGG = CCAAT and GTAATA, respectively) is essential for cellobiohydrolase II gene expression in the filamentous fungus Hypcrea jecorina. Here we report that cell-free extracts from a cellulase-negative mutant form CAE-protein complexes with higher mobility and lower binding-strength compared to the wild type. EMSA analysis demonstrated an increased mobility of the GTAATA-binding protein complex and, supported by in vivo footprinting, a lowered binding strength of the HAP2/3/5 proteins. However, the hap2/hap3/hap5 genes of the mutant are unaltered and transcribed normally. A nucleotide fragment of the cbh1 promoter containing a (GG)CTAATA motif without an adjacent CCAAT box is also bound by cell-free extracts of H. jecorina, and the protein-DNA complex of the mutant shows the characteristic increase in mobility. We conclude that this mutant is defective in the functional formation of the CAE-protein complexes but not in their binding to the target sequences itself.     

Arabidopsis HAP2 (GCS1) is a sperm-specific gene required for pollen tube guidance and fertilization

In flowering plants, sperm cells develop in the pollen cytoplasm and are transported through floral tissues to an ovule by a pollen tube, a highly polarized cellular extension. After targeting an ovule, the pollen tube bursts, releasing two sperm that fertilize an egg and a central cell. Here, we identified the gene encoding Arabidopsis HAP2, demonstrating that it is allelic to GCS1. HAP2 is expressed only in the haploid sperm and is required for efficient pollen tube guidance to ovules. We identified an insertion (hap2-1) that disrupts the C-terminal portion of the protein and tags mutant pollen grains with the beta-glucuronidase reporter. By monitoring reporter expression, we showed that hap2-1 does not diminish pollen tube length in vitro or in the pistil, but it reduces ovule targeting by twofold. In addition, we show that the hap2 sperm that are delivered to ovules fail to initiate fertilization. HAP2 is predicted to encode a protein with an N-terminal secretion signal, a single transmembrane domain and a C-terminal histidine-rich domain. These results point to a dual role for HAP2, functioning in both pollen tube guidance and in fertilization. Moreover, our findings suggest that sperm, long considered to be passive cargo, are involved in directing the pollen tube to its target.     

Expression of the ech42 (endochitinase) gene of Trichoderma atroviride under carbon starvation is antagonized via a BrlA-like cis-acting element

Expression of the endochitinase encoding ech42 gene of the mycoparasite Trichoderma atroviride is subject to control by several environmental signals, including derepression by carbon starvation. In order to identify promoter areas involved in control by this condition, we prepared fusions of several mutant forms of the ech42 promoter to the Aspergillus niger goxA gene as a reporter. Removal of a 130-bp fragment comprising a binding site for the carbon catabolite repressor Cre1, an AGGGG element and three separate binding sites identical and highly similar, respectively, to those for the Aspergillus nidulans regulator of conidiation BrlA resulted in a three-fold increase in derepression under carbon starvation. A truncation of the promoter to 196 bp, which removed all of the observed DNA binding motifs, resulted in five-fold derepression. In vitro protein-DNA binding analyses showed that only the BrlA-like sites, but neither the AGGGG element nor the Cre1 binding site, bound proteins from cell-free extracts from carbon-starved mycelia of T. atroviride. Thus this study identifies a new regulator of chitinase gene expression in Trichoderma, a BrlA-like binding motif.     

The Candida albicans UBI3 gene encoding a hybrid ubiquitin fusion protein involved in ribosome biogenesis is essential for growth

We have constructed a conditional null mutant Candida albicans strain for the UBI3 gene which encodes a ubiquitin fusion protein involved in ribosome biogenesis. A one-step gene disruption procedure, using the plasmid pCaDis, was designed to place the second copy of the UBI3 gene under the control of the tightly regulated MET3 promoter in a C. albicans heterozygous strain (UBI3/Deltaubi3::hisG), previously isolated in the first step of the ura-blaster protocol. Analysis of the conditional null mutant in repressing and inducing conditions indicates that UBI3 is an essential gene whose expression is required for growth of C. albicans.     

Depletion of polyubiquitin encoded by the UBI4 gene confers pleiotropic phenotype to Candida albicans cells

We have studied the roles of polyubiquitin in Candida albicans physiology. Heterologous expression of the C. albicans polyubiquitin (UBI4) gene in a ubi4 Saccharomyces cerevisiae strain suppressed the mutant phenotype (hypersensitivity to heat shock). A heterozygous strain UBI4/Deltaubi4::hisG, obtained following the ura-blaster procedure, was used to construct a conditional mutant using a pCaDis derivative plasmid. By serendipity we isolated the UBI4 conditional mutant as well as a UBI4 mutant containing a non-functional MET3 promoter. Depletion of polyubiquitin conferred pleiotropic effects to mutant cells: (i) a limited increased sensitivity to mild heat shock; (ii) increased formation of colony morphology variants; and (iii) induction of hyphal and pseudohypal development. These results indicate that polyubiquitin in C. albicans is involved in the negative control of switching, as well as in maintaining the yeast cell morphology, probably by silencing mechanisms triggering the hyphal and pseudohyphal development in the absence of environmental inducers.