Abstract
A protein carboxyl methyltransferase (PCMT), which specifically modifies atypical protein Lโisoaspartyl and Dโaspartyl residues, is widely distributed in eucaryotic cells, but the factors that regulate its activity in vivo have not been identified. It has been proposed that the PCMT initiates the repair of structurally damaged proteins. To test the possibility that the concentration of structurally abnormal cellular proteins affects PCMT activity, protein carboxyl methylation reactions were studied in HeLa cells exposed to various stresses that increase the extent of protein unfolding in cells. Protein carboxyl methylation rates increased 70โ80% during incubations at 42ยฐC and remained elevated for periods of up to 8 hr. This sustained increase was greater than that predicted from thermal effects on the enzyme alone and may relect the exposure of atypical aspartyl sites as proteins unfold as well as increased rates of protein deamidation and isomerization at elevated temperatures. Methylation rates showed no increases following 12 hr incubations with the amino acid analogs Lโazetidineโ2โcarboxylic acid or Lโcanavanine. Northern blot analysis of RNA preparations from control and stressed cells revealed three major transcripts for the PCMT in HeLa cells, which are 1.6, 2.6, and 4.5 kb in length. The concentrations of all three transcripts decreased by โผ 20% from control levels during heat shock. No changes in PCMT transcript concentrations were observed during incubation with the amino acid analogs. By contrast, large increases in the concentrations of hsp70 and ubiquitin trascripts were observed following either heat or chemical stresses. The results demonstrate that the PCMT is a constitutive component of cells whose function is required under normal conditions as well as during stress conditions, which accelerate structural damage to cellular proteins. ยฉ 1992 WileyโLiss, Inc.