Glucoamylase :: green fluorescent protein fusions to monitor protein secretion in Aspergillus niger

A glucoamylase::green fluorescent protein fusion (GLA::sGFP) was constructe d which allows the green fluorescent protein to be used as an in vivo repor ter of protein secretion in Aspergillus niger. Two secretory fusions were d esigned for secretion of GLA::sGFP which employed slightly different length s of the glucoamylase protein (GLA499 and CLA514). Expression of GLA::sGFP revealed that fluorescence was localized in the hyphal cell walls and septa , and that fluorescence was most intense at hyphal apices, Extracellular GL A::sGFP was detectable by Western blotting only in the supernatant of young cultures grown in soya milk medium. In older cultures, acidification of th e medium and induction of proteases were probably responsible for the loss of extracellular and cell wall fluorescence and the inability to detect CLA ::sGFP by Western analysis, A strain containing the CLA::sGFP construct was subjected to UV mutagenesis and survivors screened for mutations in the ge neral secretory pathway. Three mutants were isolated that were unable to fo rm a halo on either starch or gelatin medium, All three mutants grew poorly compared to the parental strain. Fluorescence microscopy revealed that for two of the mutants, CLA::sGFP accumulated intracellularly with no evidence of wall fluorescence, whereas for the third mutant, wall fluorescence was observed with no evidence of intracellular accumulation, These results indi cate that the CLA::sGFP fusion constructs can be used as convenient fluores cent markers to study the dynamics of protein secretion in vivo and as a to ol in the isolation of mutants in the general secretory pathway,