We have started research to analyse the molecular mechanism underlying protein secretion in A. niger. In the first instance we have isolated 9 distinct GTPase encoding genes from A. niger (sarA, sagA-H) corresponding to GTPases involved in most stages of the secretory pathway. Interestingly, several of these genes homologues are present in higher eukaryotes, but not in S. cerevisiae. These genes are used to generate a set of (conditional/deletion) mutants imposing defined blocks in the secretory pathway. To analyse transport and secretion of proteins we have developed a GFP-based secretion reporter system by fusing GFP to a carrier protein, glucoamylase (GLA). Expression of a g1aA::gfp fusion construct resulted in fluorescence of the cell wall, probably representing secreted GLA::GFP fusion protein that is retained within the extracellular matrix. Periplasmic fluorescence was only observed in young mycelium. No periplasmic fluorescence is observed in older mycelia probably due to acidification of the medium, and/or increased protease activity. Targeting of the GLA::GFP fusion protein to the ER by fusing the ER retention signal (HDEL) to the fusion protein, resulted in intracellular, punctuated fluorescence, indicating retention of the fusion protein. The GFP-fusion proteins will be introduced into the various secretion mutants to validate and complement the results obtained with the analysis of secretion defects in our secretion mutants.
Fungal Genet. Newsl. 46 (Supl):
Full conference title:
Fungal Genetics Conference 20th
- Fungal Genetics Conference 20th (1999)