Role of ATP-dependent Mechanisms and pH on Isoaspartyl Accumulation in S. cerevisiae

As organisms age, the most common form of non-oxidative protein damage is the conversion of asparaginyl or aspartyl amino acids to abnormal isoaspartyls. These residues change the primary structure of proteins, introducing kinks into the polypeptide backbones, which may lead to protein aggregation and eventual cell death.  The protein L-isoaspartyl O-methyltransferase (PIMT) repairs this conversion, and is essential to almost all Kingdoms of life. However, Saccharomyces cerevisiae lacks PIMT, but is able to maintain low isoaspartyl damage levels (less than 60 pmol per mg of protein). It is hypothesized that this maintenance is a result of ATP-dependent pumps, which regulate cytosolic and vacuoler pH. Incubating S. cerevisiae in media buffered to pH 3 or 9 has been shown by base labile volatility assays to increase isoaspartyl levels in cytosolic proteins compared to pH 6. This increase suggests a role for ATP-dependent pumps in regulating isoaspartyl group formation. To further confirm the correlation between these pumps and isoaspartyl formation, yeast cells incubated with varying concentrations of iodoacetic acid (IAA) exhibited 100-fold decreases in ATP in both exponential and stationary phases. These results correlated to the increase in isoaspartyl levels compared to an untreated control. Treatment with Concanamycin A, a potent inhibitor of vacuoler H⁺ ATPases, showed a 50-fold increase in isoaspartyl levels, which suggests that pH is a factor in isoaspartyl maintenance. These results may lead to therapeutics developed to counteract isoaspartyl accumulation and aging. Further studies are planned to elucidate the role of cytosolic and vacuoler pH on isoaspartyl levels.