TOLERANCE OF LOW PH IN SCHIZOSACCHAROMYCES-POMBE REQUIRES A FUNCTIONING PUB1 UBIQUITIN LIGASE

A strain of Schizosaccharomyces pombe carrying a disrupted Na+/H+ anti porter gene (sod2::sup3-5), in addition to the common auxotrophic muta tions, ade6-216, ura4-D18 and leu1-32, is highly sensitive to media ad justed to pH 6.9. Reversion analysis of this strain yielded a group of revertants capable of growth at pH 6.9. Two of the revertants elongat ed and failed to form colonies at pH 3.5. Genetic characterization of one of the pH-sensitive elongated strains, J227, showed the presence o f two independently segregating mutations. One, pub1 (protein ubiquiti n ligase 1), has recently been reported as an E3 protein ubiquitin lig ase involved in cdc25 turnover. The second has been named elp3-1 (elon gated at low pH). Genetic dissection of the original strain revealed t hat poor growth at high pH was due to the presence of the auxotrophic markers, suggesting a possible inhibitory effect of high pH on the fun ction of permeases responsible for uptake of the necessary nutrients. Suppression of the high pH sensitivity required the presence of both t he pub1-1 and elp3-1 mutations. While the pub1-1 mutation reduced the capacity of cells to tolerate relatively moderate concentrations of Li Cl (3 mM) in liquid culture, it was capable of partially suppressing t he extreme Li+ sensitivity caused by the sod2 disruption. Under these conditions, the growth of pub1-1 sod2::ura4 double mutant cells was im proved over that of either pub1-1 or sod2::ura4 cells. The elp3-1 muta tion had no effect on the Li+ tolerance in either wild-type or sod2::u ra4 backgrounds. pub1-1 cells are elongated and incapable of colony fo rmation at pH 3.5. In contrast, elp3-1 cells are elongated at pH 3.5 a nd pH 5.5 (the normal pH of minimal medium) but can form colonies unde r both conditions. J227 cells are significantly longer than either sin gle mutant at pH 3.5 and do not form colonies but are visually similar to elp3-1 cells at pH 5.5. Complementation cloning in the J227 backgr ound yielded a genomic clone of pub1, allowing us to define the intron -exon structure of the gene. Sequences with high homology to the predi cted amino acid sequence of pub1 have been identified in Saccharomyces cerevisiae (RSP5/NPI1), human (hRPF1), mouse (mNedd4), and rat (rNedd 4). Based on the nature of our mutant selection, the pH-sensitive phen otype of the strains selected, and the known involvement of RSP5/NPI1 in membrane permease turnover in S. cerevisiae, we hypothesize a role for pub1, either directly or indirectly, in regulating membrane transp ort processes. This is further supported by the broad range of effects that the pub1-1 mutation exerts on overall performance of cells at hi gh and low external pH, and in the presence of toxic levels of Li+.