The pub1 E3 ubiquitin ligase negatively regulates leucine uptake in response to NH4+ in fission yeast

Abstract

Fission yeast strains auxotrophic for leucine are unable to proliferate in normally supplemented minimal media adjusted to pH 6.4 or above. High-pH sensitivity can be suppressed by the loss of Pub1, an E3 ubiquitin ligase, or by the replacement of NH₄ ⁺ with a non-repressing source of nitrogen such as L-proline. In this report we show pub1 to be required for the rapid down-regulation of leucine uptake observed in response to the addition of NH₄ ⁺ to the growth media. Furthermore, we corroborate earlier results demonstrating the transport of leucine to be negatively influenced by high extracellular pH. pub1 is homologous to the budding yeast nitrogen permease inactivator, NPI1/RSP5, which mediates the ubiquitination and subsequent destruction of NH₄ ⁺-sensitive permeases. The high-pH sensitivity of cells auxotrophic for leucine thus seems to reflect an inability of NH₄ ⁺-insensitive permeases to transport sufficient leucine under conditions where the proton gradient driving nutrient transport is low, and NH₄ ⁺-sensitive permeases have been destroyed. Intriguingly, the partial suppression of both high pH sensitivity, and the inactivating effect of NH₄ ⁺ on leucine transport, seen in pub1-1 point mutants, becomes as complete as seen in pub1Δ backgrounds when cells have concomitantly lost the function of the spc1 stress-activated MAPK.