We investigated the role of all arrestin-like proteins of Aspergillus nidulans in respect to growth, morphology, sensitivity to drugs and specifically for the endocytosis and turnover of the uric acid-xanthine transporter UapA. All arrestin null mutants are viable showing wild-type growth and morphology, except one which is affected in conidiospore production, but several have modified profiles in respect to N or C source utilization and drug sensitivity. A single arrestin, ArtA, is essential for HulARsp5-dependent ubiquitination and endocytosis of UapA in response to ammonium or substrates. Genetic analysis further showed that residues 545-561 of the UapA C-tail, which includes a critical di-acidic motif, is required for efficient UapA endocytosis. Mutational analysis of ArtA shows that the N-terminal region (2-123) and both PY elements are essential for its function. ArtA undergoes HulA-dependent ubiquitination at residue Lys343 and this modification is critical for the efficiency of UapA ubiquitination and endocytosis, especially in response to ammonium. Lastly, we show that ArtA is essential for vacuolar turnover of transporters specific for purines (AzgA) or L-proline (PrnB), but not for an aspartate/glutamate transporter (AgtA). Our results are discussed within the frame of recently proposed mechanisms on how arrestins are activated and recruited for ubiquitination of transporters in response to broad range signals, but also put the basis for understanding how arrestins, such as ArtA, regulate the turnover of a specific transporter in the presence of its substrates.
Full conference title:
27th Fungal Genetics Conference
- Fungal Genetics Conference 27th (2013)