Localization of wild type and mutant class I myosin proteins in Aspergillus nidulans using GFP-fusion proteins

Abstract

We have examined the distribution of MYOA, the class I myosin protein of the filamentous fungus Aspergillus nidulans, as a GFP fusion protein. Wild type GFP-MYOA expressed from the myoA promoter is able to rescue a conditional myoA null mutant. Growth of a strain expressing GFP-MYOA as the only class I myosin was approximately 50% that of a control strain, demonstrating that the fusion protein retains substantial myosin function. The distribution of the wild type GFP-MYOA fusion is enriched in growing hyphal tips and at sites of septum formation. In addition, we find that GFP-MYOA is also found in patches at the cell cortex. We have also investigated the effects of deletion or truncation mutations in the tail domain on MYOA localization. Mutant GFP-MYOA fusions that lacked either the C-terminal SH3 or a portion of the C-terminal proline-rich domain had subcellular distributions like wild type MYOA, consistent with their ability to complement a myoA null mutant. In contrast, mutants lacking all of the C-terminal proline-rich domain or the TH-1-like domain were mainly localized diffusely throughout the cytoplasm, but could less frequently be found in patches, and were unable to complement a myoA null mutant. The GFP-MYOA DeltaIQ mutant was localized into large bright fluorescent patches in the cytoplasm. This mutant protein was subsequently found to be insoluble.