Selective carboxyl methylation of structurally altered calmodulins in Xenopus oocytes.

R R Desrosiers,E A Romanik,C M O'Connor

doi:10.1016/s0021-9258(17)45370-1

Abstract

The eucaryotic protein carboxyl methyltransferase specifically modifies atypical D-aspartyl and L-isoaspartyl residues which are generated spontaneously as proteins age. The selectivity of the enzyme for altered proteins in intact cells was explored by co-injecting Xenopus laevis oocytes with S-adenosyl-L-[methyl-3H]methionine and structurally altered calmodulins generated during a 14-day preincubation in vitro. Control experiments indicated that the oocyte protein carboxyl methyltransferase was not saturated with endogenous substrates, since protein carboxyl methylation rates could be stimulated up to 8-fold by increasing concentrations of injected calmodulin. The oocyte protein carboxyl methyltransferase showed strong selectivities for bovine brain and bacterially synthesized calmodulins which had been preincubated in the presence of 1 mM EDTA relative to calmodulins which had been preincubated with 1 mM CaCl2. Radioactive methyl groups were incorporated into base-stable linkages with recombinant calmodulin as well as into carboxyl methyl esters following its microinjection into oocytes. This base-stable radioactivity most likely represents the trimethylation of lysine 115, a highly conserved post-translational modification which is present in bovine and Xenopus but not in bacterially synthesized calmodulin. Endogenous oocyte calmodulin incorporates radioactivity into both carboxyl methyl esters and into base-stable linkages following microinjection of oocytes with S-adenosyl-[methyl-3H]methionine alone. The rate of oocyte calmodulin carboxyl methylation in injected oocytes is calculated to be similar to that of lysine 115 trimethylation, suggesting that the rate of calmodulin carboxyl methylation is similar to that of calmodulin synthesis. At steady state, oocyte calmodulin contains approximately 0.0002 esters/mol of protein, which turn over rapidly. The results suggest the quantitative significance of carboxyl methylation in the metabolism of oocyte calmodulin.

Highlights

The results suggest the quantitative significance of carboxyl methylation in the metabolism of oocyte calmodulin
We have examined the selectivity of the oocyte protein carboxyl methyltransferase for native and altered calmodulins from both bovine brain and recombinant bacteria following their microinjection into oocytes
Data are compared for both calf brain calmodulin and recombinant calmodulin isolated from bacteria overexpressing the chicken calmodulin gene

Summary

PROCEDURES

Materials-S-Adenosyl-L-[methyl-“Hlmethionine (11.2 Ci/mmol) was purchased from Du Pont-New England Nuclear. Enzyme Purification and Assay-Protein carboxyl methyltransferase was purified from calf brain as described by Aswad and Deight [11]. Calmodulin was purified from calf brain cytosol by isoelectric precipitation and phenyl-Sepharose chromatography [27]. Calmodulin was purified from the cytosol fraction obtained by centrifugation at 13,000 X g for 15 min using phenyl-Sepharose chromatography. The phenyl-Sepharose procedure was modified from that described above for calf brain calmodulin by substituting 100 mM. ’ The abbreviations used are: AdoMet, S-adenosylmethionine; QAE-Sephadex, quarternary aminoethyl-Sephadex; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; MES, 2-(Nmorpholino)ethanesulfonic acid; PIPES, piperazine-N,N’-bis(2-ethanesulfonic acid); EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid; CaM, calmodulin. Oocytes were homogenized as described above, and the cytosol was stored at -20 “C prior to analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at pH 2.4. Fluorographic analysis was performed as described previously [35]

Characterization of Calmodulin

Slice number

TABLE I

Recombinant calmodulin