Localization of wild type and mutant class I myosin proteins in Aspergillus nidulans using GFP-fusion proteins.
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.
Yamashita
R A
RA
Division of Pathology and Laboratory Medicine, University of Texas, M.D. Anderson Cancer Center, Houston, TX 77030, USA.
Osherov
N
N
May
G S
GS
D001233:Aspergillus nidulans / Q000166:cytology / Q000378:metabolism*; D015139:Blotting, Southern; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D024461:Myosin Type I; D009218:Myosins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013172:Spores, Fungal / Q000166:cytology / Q000502:physiology
D005656:Fungal Proteins; D008164:Luminescent Proteins; C092129:MyoA protein, Aspergillus nidulans; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D024461:Myosin Type I; D009218:Myosins