A comprehensive understanding of the secretory pathway of filamentous fungi and its interplay with polar growth and cell wall integrity is still lacking in industrial fungi such as Aspergillus niger. GTP-binding proteins such as members of the Arf family are major switches controlling coated vesicle formation during intracellular trafficking in eukaryotes. The function of all Arf family proteins have been studied in S. cerevisiae, whereas only SarA of the seven predicted ones have been examined in A. niger so far. In the current study, we have investigated the function of ArfA, the orthologue of the S. cerevisiae ARF1 and ARF2 genes in A. niger, as the encoding gene is specifically up-regulated in A. niger under conditions that lead to high secretion of glucoamylase (Jørgensen et al 2009). ARF1 and ARF2 have multiple roles within the secretory pathway of S. cerevisiae and the closet human orthologue was recently shown to be a key factor ensuring the hypersecretion phenotype of neuroendocrine tumor cells (Münzberg et al 2015). The GTP-binding protein ArfA seems to be an essential protein for A. niger as deletion of the arfA gene (An08g03690 ) turned out to be lethal. We thus generated a conditional arfA mutant with the help of the Tet-On expression system (Meyer et al 2007) in a fluorescently labelled v-SNARE background strain (GFP-SncA; Kwon et al. 2013). This approach allowed us to follow the effect of arfA downregulation and over-expression on post-Golgi cargo distribution (v-SNARE) and glucoamylase secretion in a single isolate. We could also show that ArfA is able to complement the lethal phenotype of a S. cerevisiae arf1 arf2 double null mutant.
Full conference title:
- Asperfest 13 (2016)