Overexpression of EAR1 and SSH4 that encode PPxY proteins in the multivesicular body provides stability to tryptophan permease Tat2, allowing yeast cells to grow under high hydrostatic pressure

Abstract

Tryptophan uptake in yeast Saccharomyces cerevisiae is susceptible to high hydrostatic pressure and it limits the growth of tryptophan auxotrophic (Trp⁻) strains under pressures of 15–25 MPa. The susceptibility of tryptophan uptake is accounted for by the pressure-induced degradation of tryptophan permease Tat2 occurring in a Rsp5 ubiquitin ligase-dependent manner. Ear1 and Ssh4 are multivesicular body proteins that physically interact with Rsp5. We found that overexpression of either of the EAR1 or SSH4 genes enabled the Trp⁻ cells to grow at 15–25 MPa. EAR1 and SSH4 appeared to provide stability to the Tat2 protein when overexpressed. The result suggests that Ear1 and Ssh4 negatively regulate Rsp5 on ubiquitination of Tat2. Currently, high hydrostatic pressure is widely used in bioscience and biotechnology for structurally perturbing macromolecules such as proteins and lipids or in food processing and sterilizing microbes. We suggest that hydrostatic pressure is an operative experimental parameter to screen yeast genes specifically for regulation of Tat2 through the function of Rsp5 ubiquitin ligase.

Keywords:

• high hydrostatic pressure
• tryptophan permease Tat2
• EAR1
• SSH4
• Rsp5 ubiquitin ligase
• PPxY motif
• ubiquitination

Acknowledgements

We thank Per O. Ljungdahl for providing plasmids and valuable comments, and Sébastien Léon, Yoshiko Kikuchi, Daniéle Urban-Grimal and Pierre Morsomme for helpful discussions. This work was supported by the Japan Society for the Promotion of Science (No. 18658039 to F. Abe).