The Aspergillus nidulans rcoA gene is required for veA-dependent sexual development

The Aspergillus nidulans rcoADelta mutant exhibits growth and developmental defects. We show that the rcoADelta mutant lacks cleistothecia and is self-sterile. In crosses with wild-type strains, rcoADelta nuclei do not contribute to the cleistothecial walls. Furthermore, sexual development resulting from veA overexpression is rcoA dependent, indicating that rcoA lies downstream of veA in the sexual development pathway.     

The rpl16a gene for ribosomal protein L16A identified from expressed sequence tags is differentially expressed during sexual development of Aspergillus nidulans

We obtained 305 expressed sequence tags (ESTs), which are from the poly(A) site to the most proximal MboI site, from mycelia at the early sexual developmental (ESD) stage of Aspergillus nidulans. By comparison of these ESTs with those obtained previously from the vegetative stage and from the late sexual developmental stage followed by Northern blot analyses, genes of 17 ESTs were identified as being expressed more abundantly at the ESD stage than at the vegetative stage. Five of 17 genes were expressed more abundantly in the presence of the veA gene or the nsdD gene, suggesting that these 5 genes may be involved in sexual development. In a gene of one EST, appearing three times among 305 ESTs and identified by GenBank, polyadenylation seemed to occur at two sites. Nucleotide sequences of the gene having the EST and its cDNA revealed that the gene can code for a 202-amino-acid polypeptide with an estimated molecular mass of 23 kDa. The deduced amino acid showed 73% identity to Saccharomyces cerevisiae ribosomal protein L16A (RPL16A), and therefore the gene was named rpl16a. A. nidulans RPL16A had a putative leucine zipper motif and a basic leucine zipper motif like those of other organisms. The expression level of the rpl16a gene, present as a single copy in this organism, reached a maximum after 2 h, decreased thereafter, and increased again 30 to 50 h after the end of induction of sexual development. These results clearly indicated that the rpl16a gene is expressed differentially during sexual development.     

The VeA regulatory system and its role in morphological and chemical development in fungi

In fungi, the velvet gene, or veA, is involved in the regulation of diverse cellular processes, including control of asexual and sexual development as well as secondary metabolism. This global regulator is conserved in numerous fungal species. Interestingly, in Aspergilli, where most of the studies on veA have been carried out, this gene has been described to mediate development in response to light. In recent years the knowledge of this important regulatory system has expanded through the use of Aspergillus nidulans as a model organism, and through the study of veA orthologs across fungal genera. This review includes information on the current understanding of veA function and its mechanism of action. The fact that veA has only been found in fungi, together with advances in the elucidation of the veA mechanism, might be useful in designing future control strategies to decrease the detrimental effects of fungi while enhancing those qualities that are beneficial.     

冠突散囊菌野生型与△veA突变菌株的差异蛋白研究

为了分离和鉴定冠突散囊菌野生型与veA基因缺失菌株的差异表达蛋白,寻找并比较与veA基因相关的产孢蛋白,为进一步研究丝状真菌产孢机理打下基础。经veA基因缺失,利用双向电泳技术分离差异表达蛋白,经凝胶银染显色后,Bio-Rad凝胶扫描仪扫描,Imagemaster图像软件分析,差异蛋白点进行质谱鉴定。所获肽序列与生物信息数据库匹配,在NCBI及Uniprot数据库中查找蛋白质信息,并归纳分析。结果显示,野生型菌株中出现表达上调的蛋白点77个,veA缺失型菌株中出现表达上调的蛋白点有116个,得到鉴定的30个功能各异的蛋白点,其中大多数蛋白与代谢相关。     

The NPEY sequence is not necessary for endocytosis and processing of insulin-receptor complexes.

The tetrameric amino acid sequence AsnProXTyr (NPXY), where X represents any amino acid, is conserved in the intracytoplasmic domains of several membrane proteins and has been postulated to play a role in receptor-mediated endocytosis. The human insulin receptor (hIR) contains a single copy of the sequence AsnProGluTyr (NPEY) in its intracytoplasmic domain. To determine if this putative consensus sequence is necessary for endocytic functions of hIR, we constructed a mutant receptor, hIR delta NPEY, that lacks NPEY sequence, stably expressed this mutant receptor in Chinese hamster ovary cells, and then studied its endocytic functions. When compared to wild type hIR similarly expressed in Chinese hamster ovary cells, the hIR delta NPEY mutant exhibited: 1) normal subunit organization and insulin binding affinity; 2) essentially normal internalization of covalent photoaffinity labeled insulin-receptor complexes; and 3) normal internalization of receptor-bound [125I]insulin as well as normal degradation and release of the internalized insulin. Therefore, we conclude that the NPEY sequence in the juxtamembrane domain of hIR is not necessary for its endocytic function.     

Aspergillus nidulans VeA subcellular localization is dependent on the importin alpha carrier and on light

The veA gene is a light-dependent regulator governing development and secondary metabolism in Aspergillus nidulans. We have identified a putative bipartite nuclear localization signal (NLS) motif in the A. nidulans VeA amino acid sequence and demonstrated its functionality when expressed in yeast. Furthermore, migration of VeA to the nucleus was dependent on the importin alpha. This bipartite NLS is also functional when VeA is expressed in A. nidulans. Interestingly, we found that VeA migration to the nucleus is light-dependent. While in the dark VeA is located mainly in the nuclei, under light VeA is found abundantly in the cytoplasm. The VeA1 mutant protein (lacking the first 36 amino acids at the N-terminus) was found predominantly in the cytoplasm independent of illumination. This indicates that the truncated bipartite NLS in VeA1 is not functional and fails to respond to light. These results might explain the lack of the morphological light-dependent response in strains carrying the veA1 allele. We also evaluated the effect of light on production of the mycotoxin sterigmatocystin in a veA wild-type and the veA1 mutant strains and found that the highest amount of toxin was produced by the veA+ strain growing in the dark, condition favouring accumulation of VeA in the nucleus.     

SakA MAP kinase is involved in stress signal transduction,sexual development and spore viability in Aspergillus nidulans

In eukaryotic cells, environmental stress signals are transmitted by evolutionarily conserved MAPKs, such as Hog1 in the budding yeast Saccharomyces cerevisiae, Spc1 in the fission yeast Schizosaccharomyces pombe and p38/JNK in mammalian cells. Here, we report the identification of the Aspergillus nidulans sakA gene, which encodes a member of the stress MAPK family. The sakA gene is able to complement the S. pombe spc1- defects in both osmo-regulation and cell cycle progression. Moreover, SakA MAPK is activated in response to osmotic and oxidative stress in both S. pombe and A. nidulans. However, in contrast to hog1 and spc1 mutants, the sakA null mutant is not sensitive to high osmolarity stress, indicating a different regulation of the osmostress response in this fungus. On the other hand, the DeltasakA mutant shows development and cell-specific phenotypes. First, it displays premature steA-dependent sexual development. Second, DeltasakA mutant produces asexual spores that are highly sensitive to oxidative and heat shock stress and lose viability upon storage. Indeed, SakA is transiently activated early after induction of conidiation. Our results indicate that SakA MAPK is involved in stress signal transduction and repression of sexual development, and is required for spore stress resistance and survival.