Analysis of acid-phosphatase localization in Aspergillus fumigatus bya secreted chimeric green fluorescent protein as reporter

Abstract:

[Objective] In yeast glycosylphosphatidylinositol (GPI) anchoring is a signal directing localization of GPI proteins to the plasma membrane or cell wall. Some of the cis-requirements for the localization of GPI proteins are now understood, however, little is known the signals directing distribution of the GPI proteins in filamentous fungi. Previously, AfPhoA, a GPI-anchored acid phosphatase in filamentous fungus Aspergillus fumigatus, was first isolated from the cell membrane and latter found to be associated with the cell wall. The actual distribution of the AfPhoA remains unclear. Meanwhile, the signature amino acid motif that determines the distribution of GPI protein in yeast is not found in the C-terminal sequence of the AfPhoA. We aimed to elucidate the cell distribution of the AfPhoA. [Methods] The green fluorescent protein (GFP) was used as reporter to track the localization of the AfPhoA. The C-terminal sequence of the AfPhoA was fused to the C-terminus of the GFP. [Results] We first constructed the expression plasmid pchiGFP, in which the N-terminal signal sequence of the A. fumigatus AfChiB1 was fused to the N-terminus of the GFP. After transformation, a secreted expression of the GFP was achieved in A. fumigatus. Based on this construct, The C-terminal sequence of the AfPhoA was fused to the C-terminus of the GFP to construct a chimeric GFP. After the co-transformation of the fusion construct with plasmid pCDA14, a transformant was confirmed to harbor the chimeric GFP in its genome and could express the chimeric GFP. The transformant cultivated with or without chitin induction could express the chimeric GFP mainly attached to the cell membrane, a prolonged cultivation led to a minor distribution of the chimeric GFP in the cell wall. Although a 30KD of GFP fragment, instead of an intact 43.5KDa chimeric GFP, was also detected in the culture supernatant, which might be released by the cleavage between the fusion protein and its GPI anchor. [Conclusion] Our results suggest that GPI anchoring determines the distribution of the AfPhoA in the cell membrane. In addition to our investigation of the GPI anchoring, an expression vector was also constructed, which would be useful for analyses of the function and regulation of the genes and proteins in A. fumigatus.