Proteasome and Autophagy Degradative Pathways Do Not Significantly Limit Isoaspartyl Protein Damage in Saccharomyces cerevisiae

The most common type of non‐oxidative protein damage under physiological conditions is the conversion of asparaginyl or aspartyl amino acids to abnormal isoaspartyls. Although the protein L‐isoaspartyl O ‐methyltransferase (PIMT) repairs this modification and is essential to most organisms, radiochemistry approaches show isoaspartyl damage in Saccharomyces cerevisiae , which lacks PIMT, is strikingly low (60 pmol/mg of total protein) and decreases over fifty‐percent in stationary phase. Although MG‐132 and 3‐methyladenine, proteasome and autophagy inhibitors, respectively, ruled out these pathways as principal factors limiting damage, incubating cells in pH 3 and 9 buffered media resulted in significant isoaspartyl accumulation. Interestingly, PIMT transformed S. cerevisiae have decreased isoaspartyl damage without apparent changes in S ‐adenosylmethionine/ S ‐adenosylhomocysteine ratios. Finally, investigating the possibility of a novel degradation pathway, enzymological approaches identified a protease activity in yeast extracts directed at isoaspartyl‐containing synthetic peptides. Further investigations are planned to identify and characterize the L‐isoaspartyl‐directed protease activity and to elucidate the role of intracellular and vacuolar pH on isoaspartyl levels. This research was supported by the UCLA Cellular and Molecular Training Grant.