Recovery from long-term stationary phase and stress survival in Escherichia coli require the l-isoaspartyl protein carboxyl methyltransferase at alkaline pH

The l-isoaspartyl protein carboxyl methyltransferase (pcm) can stimulate repair of isoaspartyl residues arising spontaneously in proteins to normal l-aspartyl residues. PCM is needed in Escherichia coli for maximal long-term survival when exposed to oxidative stress, osmotic stress, repeated heat stress or methanol. The effect of pH on a pcm mutant during long-term stationary phase was examined. PCM was not required for long-term survival of E. coli subjected to pH stress alone; however, PCM-deficient cells showed impaired resistance to paraquat and methanol only at elevated pH. The mutant also showed stress-survival phenotypes in minimal medium buffered to pH 9·0. Accumulation of isoaspartyl residues was accelerated at pH 8·0 or 9·0 in vivo, though PCM-deficient cells did not show higher levels of damage. However, the pcm mutant displayed an extended lag phase in recovering from stationary phase at pH 9·0. Protein repair by PCM thus plays a key role in long-term stress survival only at alkaline pH in E. coli, and it may function primarily to repair damage in cells that are recovering from nutrient limitation and in those cells that are able to divide during long-term stationary phase.

Trypanosoma brucei prenylated-protein carboxyl methyltransferase prefers farnesylated substrates

Carboxyl methylation of the C-terminal prenylated cysteine, which occurs in most farnesylated and geranylgeranylated proteins, is a reversible step and is implicated in the regulation of membrane binding and cellular functions of prenylated proteins such as GTPases. The gene coding for prenylated-protein carboxyl methyltransferase (PPMT) of the protozoan parasite Trypanosoma brucei has been cloned and expressed in the baculovirus/Sf9 cell system. The protein of 245 amino acids has 24—28% sequence identity to the orthologues from other species including human and Saccharomyces cerevisiae. Methyltransferase activity was detected in the membrane fraction from Sf9 cells infected with the recombinant baculovirus using N-acetyl-S-farnesylcysteine (AFC) and S-adenosyl[methyl-3H]methionine ([3H]AdoMet) as substrates. Recombinant T. brucei PPMT prefers AFC to N-acetyl-S-geranylgeranylcysteine (AGGC) by 10—50-fold based on the Vmax/Km values. Native PPMT activity detected in the membrane fraction from T. brucei procyclics displays similar substrate specificity (40-fold preference for AFC over AGGC). In contrast, mouse liver PPMT utilizes both AFC and AGGC as substrates with similar catalytic efficiencies. Several cellular proteins of the T. brucei bloodstream form were shown to be carboxyl methylated in a cell-free system. Incorporation of [3H]methyl group from [3H]AdoMet into most of the proteins was significantly inhibited by AFC but not AGGC at 20μM, suggesting that T. brucei PPMT acts on farnesylated proteins in the cell. Cells of the T. brucei bloodstream form show higher sensitivity to AFC and AGGC (EC50 = 70—80μM) compared with mouse 3T3 cells (EC50>150μM).

Immunochemical characterization of l-isoaspartyl-protein carboxyl methyltransferase from mammalian tissues

Polyclonal antibodies were raised against a synthetic peptide corresponding to a sequence of 14 amino acid residues found near the C-terminus of L-isoaspartyl (D-aspartyl)-protein carboxyl methyltransferase (PCMT). The affinity-purified antibodies were used to detect the methyltransferase by Western-blot analysis in cytosolic and membrane fractions from several mammalian tissues. A protein of 27 kDa was detected in the cytosol of most tissues; co-incubation with the peptide used for immunization abolished the detection. The identity of the 27 kDa protein as a PCMT was demonstrated by renaturation of PCMT activity from SDS/polyacrylamide gels. The methyltransferase from brain cytosol was immunoprecipitated by the anti-PCMT antibodies and Protein A-agarose, indicating that the native protein was recognized by the antibodies. PCMT was also immunodetected in crude membranes from brain, testes and heart, and in purified membranes from kidney cortex. The expression of the methyltransferase was higher in bovine and human brain than in rat tissues. The bovine enzyme had a greater electrophoretic mobility, suggesting small structural differences. The membrane-bound methyltransferase could be extracted with detergents above their critical micellar concentration, but not with salt, alkaline or urea solutions suggesting that the binding of the enzyme to membranes is hydrophobic by nature. Anti-PCMT antibodies provide an interesting tool for studies regarding the expression of these enzymes in both soluble and membrane fractions of various cell types.