RESUMO
Autophagy is a tightly controlled degradation process of all eukaryotes. It includes the sequestration of cytoplasmic contents and organelles within a double-membraned autophagosome. Autophagy involves core autophagy related (atg) genes as well as genes regulating vesicle trafficking. Previously, we analyzed the impact of proteins of the core autophagic machinery SmATG7, SmATG8 and SmATG4 on the sexual and vegetative development of the filamentous ascomycete Sordaria macrospora. While deletion of Smatg8 and Smatg4 abolished fruiting-body formation and impaired vegetative growth, Smatg7 is required for viability. In yeast, the phosphatidylinositol 3-kinase vacuolar protein sorting 34 (Vps34) and its myristoylated membrane targeting unit, the protein kinase Vps15 have been shown to be important regulators of autophagy and vacuolar protein sorting. However, their exact role in filamentous ascomycetes remains elusive. To determine the function of Smvps34 and Smvps15 we isolated genes with high sequence similarity to Saccharomyces cerevisiae VPS34 and VPS15. For both genes we were not able to generate a homokaryotic knockout mutant in S. macrospora, suggesting that Smvps34 and Smvps15 are required for viability. Furthermore, we analyzed the repertoire of vps genes encoded by S. macrospora and could identify putative homologs of nearly all of the 61 VPS genes of S. cerevisiae.
Assuntos
Autofagia , Classe III de Fosfatidilinositol 3-Quinases/metabolismo , Viabilidade Microbiana , Sordariales/citologia , Sordariales/enzimologia , Proteína VPS15 de Distribuição Vacuolar/metabolismo , Sequência de Aminoácidos , Classe III de Fosfatidilinositol 3-Quinases/química , Classe III de Fosfatidilinositol 3-Quinases/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência , Sordariales/química , Sordariales/genética , Proteína VPS15 de Distribuição Vacuolar/química , Proteína VPS15 de Distribuição Vacuolar/genéticaRESUMO
Autophagy is a precisely controlled degradation process in eukaryotic cells, during which the bulk of the cytoplasm is engulfed by a double membrane vesicle, the autophagosome. Fusion of the autophagosome with the vacuole leads to breakdown of its contents, such as proteins and organelles, and the recycling of nutrients. Earlier studies of autophagic genes of the core autophagic machinery in the filamentous ascomycete Sordaria macrospora elucidated the impact of autophagy on fungal viability, vegetative growth and fruiting-body development. To gain further knowledge about the regulation of autophagy in S. macrospora, we analyzed the function of the bZIP transcription factor SmJLB1, a homolog of the Podospora anserina basic zipper-type transcription factor induced during incompatibility 4 (IDI-4) and the Aspergillus nidulans transcription factor jun-like bZIP A (JlbA). Generation of the homokaryotic deletion mutant demonstrated S. macrospora Smjlb1 is associated with autophagy-dependent processes. Deletion of Smjlb1 abolished fruiting-body formation and impaired vegetative growth. SmJLB1 is localized to the cytoplasm and to nuclei. Quantitative real-time PCR experiments revealed an upregulated expression of autophagy-related genes Smatg8 and Smatg4 in the Smjlb1 deletion mutant, suggesting a transcriptional repression function of SmJLB1.
Assuntos
Autofagia , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Carpóforos/crescimento & desenvolvimento , Regulação Fúngica da Expressão Gênica , Sordariales/crescimento & desenvolvimento , Sordariales/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Deleção de Genes , Perfilação da Expressão Gênica , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Hac1 is the activator of the cellular response to the accumulation of unfolded proteins in the endoplasmic reticulum. Hac1 function requires the activity of Gcn4, which mainly acts as a regulator of the general amino acid control network providing Saccharomyces cerevisiae cells with amino acids. Here, we demonstrate novel functions of Hac1 and describe a mutual connection between Hac1 and Gcn4. Hac1 is required for induction of Gcn4-responsive promoter elements in haploid as well as diploid cells and therefore participates in the cellular amino acid supply. Furthermore, Hac1 and Gcn4 mutually influence their mRNA expression levels. Hac1 is also involved in FLO11 expression and adhesion upon amino acid starvation. Hac1 and Gcn4 act through the same promoter regions of the FLO11 flocculin. The results indicate an indirect effect of both transcription factors on FLO11 expression. Our data suggest a complex mutual cross talk between the Hac1- and Gcn4-controlled networks.
