Prion-forming ability of Ure2 of yeasts is not evolutionarily conserved

[URE3] is a prion (infectious protein) of the Saccharomyces cerevisiae Ure2p, a regulator of nitrogen catabolism. We show that wild S. paradoxus can be infected with a [URE3] prion, supporting the use of S. cerevisiae as a prion test bed. We find that the Ure2p of Candida albicans and C. glabrata also regulate nitrogen catabolism. Conservation of amino acid sequence within the prion domain of Ure2p has been proposed as evidence that the [URE3] prion helps its host. We show that the C. albicans Ure2p, which does not conserve this sequence, can nonetheless form a [URE3] prion in S. cerevisiae, but the C. glabrata Ure2p, which does have the conserved sequence, cannot form [URE3] as judged by its performance in S. cerevisiae. These results suggest that the sequence is not conserved to preserve prion forming ability.     

The glucose repressor CRE1 from Sclerotinia sclerotiorum is functionally related to CREA from Aspergillus nidulans but not to the Mig proteins from Saccharomyces cerevisiae.

We isolated the putative glucose repressor gene cre1 from the phytopathogenic fungus Sclerotinia sclerotiorum. cre1 encodes a 429 amino acid protein 59% similar to the carbon catabolite repressor CREA from Aspergillus nidulans. In addition to the overall amino acid sequence relatedness between CRE1 and CREA proteins, cre1 can functionally complement the A. nidulans creAd30 mutation as assessed by repression of the alcohol dehydrogenase I gene expression. The CREI region carrying the two zinc fingers is also very similar to the DNA binding domains of the Saccharomyces cerevisiae glucose repressors Mig1p and Mig2p. Despite the presence in the CRE1 protein of several motifs involved in the regulation of Miglp activity, cre1 cannot complement mig deficiencies in S. cerevisiae. These data suggest that glucose repression pathways may have evolved differently in yeasts and filamentous fungi.     

Conservation of the prion properties of Ure2p through evolution

The yeast inheritable [URE3] element corresponds to a prion form of the nitrogen catabolism regulator Ure2p. We have isolated several orthologous URE2 genes in different yeast species: Saccharomyces paradoxus, S. uvarum, Kluyveromyces lactis, Candida albicans, and Schizosaccharomyces pombe. We show here by in silico analysis that the GST-like functional domain and the prion domain of the Ure2 proteins have diverged separately, the functional domain being more conserved through the evolution. The more extreme situation is found in the two S. pombe genes, in which the prion domain is absent. The functional analysis demonstrates that all the homologous genes except for the two S. pombe genes are able to complement the URE2 gene deletion in a S. cerevisiae strain. We show that in the two most closely related yeast species to S. cerevisiae, i.e., S. paradoxus and S. uvarum, the prion domains of the proteins have retained the capability to induce [URE3] in a S. cerevisiae strain. However, only the S. uvarum full-length Ure2p is able to behave as a prion. We also show that the prion inactivation mechanisms can be cross-transmitted between the S. cerevisiae and S. uvarum prions.     

The URE2 protein regulates nitrogen catabolic gene expression through the GATAA-containing UASNTR element in Saccharomyces cerevisiae.

Many of the gene products that participate in nitrogen metabolism are sensitive to nitrogen catabolite repression (NCR), i.e., their expression is decreased to low levels when readily used nitrogen sources such as asparagine are provided. Previous work has shown this NCR sensitivity requires the cis-acting UASNTR element and trans-acting GLN3. Here, we extend the analysis to include the response of their expression to deletion of the URE2 locus. The expression of these nitrogen catabolic genes becomes, to various degrees, NCR insensitive in the ure2 deletion. This response is shown to be mediated through the GATAA-containing UASNTR element and supports the current idea that the NCR regulatory circuit involves the following steps: environmental signal-->URE2-->GLN3-->UASNTR operation-->NCR-sensitive gene expression. The various responses of the nitrogen catabolic genes' expression to deletion of the URE2 locus also indicate that not all NCR is mediated through URE2.     

The tamA gene of Aspergillus nidulans contains a putative zinc cluster motif which is not required for gene function.

Expression of many nitrogen catabolic enzymes is controlled by nitrogen metabolite repression in Aspergillus nidulans. Although the phenotypes of tamA mutants have implicated this gene in nitrogen regulation, its function is unknown. We have cloned the tamA gene by complementation of a new tamA allele. The tamA sequence shares significant homology with the UGA35/DAL81/DURL gene of Saccharomyces cerevisiae. In vitro mutagenesis of sequences encoding a putative zinc cluster DNA binding domain indicated that this motif is not required for in vivo TamA function.     

A role for creD, a carbon catabolite repression gene from Aspergillus nidulans, in ubiquitination

In Aspergillus nidulans, it is known that creB encodes a deubiquitinating enzyme that forms a complex with the WD40 motif containing protein encoded by creC, that mutations in these genes lead to altered carbon source utilization and that the creD34 mutation suppresses the phenotypic effects of mutations in creC and creB. Therefore, creD was characterized in order to dissect the regulatory network that involves the CreB-CreC deubiquitination complex. CreD contains arrestin domains and PY motifs and is highly similar to the Rod1p and Rog3p proteins from Saccharomyces cerevisiae. An additional gene was identified in the A. nidulans genome that also encodes an arrestin and PY motif-containing protein, which we have designated apyA, and thus two similar proteins also exist in A. nidulans. In S. cerevisiae, Rod1p and Rog3p interact with the ubiquitin ligase Rsp5p, and so the A. nidulans homologue of Rsp5p was identified, and the gene encoding this HECT ubiquitin ligase was designated hulA. CreD and ApyA were tested for protein-protein interactions with HulA via the bacterial two-hybrid system, and ApyA showed strong interaction, and CreD showed weak interaction, with HulA in this system.     

敲除MIG1和SNF1基因对酿酒酵母共利用葡萄糖和木糖的影响

Mig1和Snf1是酿酒酵母葡萄糖阻遏效应的两个关键调控因子。为了提高酿酒酵母工程菌同时利用葡萄糖和木糖的能力,分别对MIG1和SNF1基因进行了单敲除和双敲除,并通过摇瓶发酵实验和RNA-Seq转录组分析,初步揭示了Mig1和Snf1可能影响葡萄糖和木糖共利用表达差异基因的层级调控机制。研究结果表明,MIG1单敲除对混合糖的共利用影响不大;SNF1单敲除会加快混合糖中木糖的利用而且葡萄糖和木糖可以被同时利用,这可能归因于SNF1单敲除会解除对一些氮分解代谢阻遏基因表达的抑制,从而促进了细胞对氮源营养的利用;进一步敲除MIG1,会解除更多氮分解代谢阻遏基因表达的抑制,以及一些碳中心代谢途径基因表达上调。虽然MIG1和SNF1双敲除菌株利用葡萄糖加快而利用木糖变慢,但是葡萄糖和木糖可以被同时利用,进而加快乙醇的积累。综上所述,MIG1和SNF1的敲除导致氮分解阻遏基因表达上调,有助于促进葡萄糖和木糖的共利用;解析Mig1和Snf1对氮分解阻遏基因的层级调控作用,为进一步提高葡萄糖和木糖的共利用提供新的靶点。     

The CCAAT-binding complex of eukaryotes: evolution of a second NLS in the HapB subunit of the filamentous fungus Aspergillus nidulans despite functional conservation at the molecular level between yeast, A.nidulans and human

The heterotrimeric CCAAT-binding complex is evolutionarily conserved in eukaryotic organisms, including fungi, plants and mammals. In the filamentous fungus Aspergillus nidulans, the corresponding complex was designated AnCF (A.nidulans CCAAT-binding factor). AnCF consists of the subunits HapB, HapC and HapE. All three subunits are necessary for DNA binding. HapB contains two putative nuclear localisation signal sequences (NLSs) designated NLS1 and NLS2. Previously, it was shown that only NLS2 was required for nuclear localisation of HapB. Furthermore, HapC and HapE are transported to the nucleus only in complex with HapB via a piggy back mechanism. Here, by using various GFP constructs and by establishing a novel marker gene for transformation of A.nidulans, i.e. the pabaA gene encoding p-aminobenzoic acid synthase, it was shown that the HapB homologous proteins of both Saccharomyces cerevisiae (Hap2p) and human (NF-YA) use an NLS homologous to HapB NLS1 for nuclear localisation in S.cerevisiae. Interestingly, for A.nidulans HapB, NLS1 was sufficient for nuclear localisation in S.cerevisiae. In A.nidulans, HapB NLS1 was also functional when present in a different protein context. However, in A.nidulans, both S.cerevisiae Hap2p and human NF-YA entered the nucleus only when HapB NLS2 was present in the respective proteins. In that case, both proteins Hap2p and NF-YA complemented, at least in part, the hap phenotype of A.nidulans with respect to lack of growth on acetamide. Similarly, A.nidulans HapB and human NF-YA complemented a hap2 mutant of S.cerevisiae. In summary, HapB, Hap2p and NF-YA are interchangeable. Because the A.nidulans hapB mutant was complemented, at least in part, by both the human NF-YA and S.cerevisiae Hap2p this finding suggests that the piggy-back mechanism of nuclear transport found for A.nidulans is conserved in yeast and human.     

Characterization of the Amdr-Controlled Lama and Lamb Genes of Aspergillus Nidulans

Four Aspergillus nidulans genes are known to be under the control of the trans-acting regulatory gene amdR. We describe the isolation and initial characterization of one of these amdR-regulated genes, lamA. The lam locus, however, was found to consist of two divergently transcribed genes, the lamA gene, and a new gene, also under amdR control, which we have designated lamB. Using recombinant DNA techniques we have constructed a strain of A. nidulans lacking a functional lamB gene. Experiments conducted with this strain demonstrate that lamB, like lamA, is involved in utilization of 2-pyrrolidinone in A. nidulans. Metabolism of a related compound, gamma-amino butyric acid (GABA) is not affected. We also provide evidence that the conversion of exogenous 2-pyrrolidinone to endogenous GABA requires a functional lamB gene. The expression of both lamA and lamB is subject to carbon and nitrogen metabolite repression in addition to amdR-mediated induction by omega-amino acids.