Isoaspartate in ribosomal protein S11 of Escherichia coli.
Isoaspartyl sites, in which an aspartic acid residue is linked to its C-flanking neighbor via its beta-carboxyl side chain, are generally assumed to be an abnormal modification arising as proteins age. The enzyme protein L-isoaspartate methyltransferase (PIMT), present in many bacteria, plants, and animals, catalyzes the conversion of isoaspartate to normal alpha-linked aspartyl bonds and is thought to serve an important repair function in cells. Having introduced a plasmid into Escherichia coli that allows high-level expression of rat PIMT, we explored the possibility that the rat enzyme reduces isoaspartate levels in E. coli proteins, a result predicted by the repair hypothesis. The present study demonstrates that this is indeed the case; E. coli cells expressing rat PIMT had significantly lower isoaspartate levels than control cells, especially in stationary phase. Moreover, the distribution of isoaspartate-containing proteins in E. coli differed dramatically between logarithmic- and stationary-phase cultures. In stationary-phase cells, a number of proteins in the molecular mass range of 66 to 14 kDa contained isoaspartate, whereas in logarithmic-phase cells, nearly all of the detectable isoaspartate resided in a single 14-kDa protein which we identified as ribosomal protein S11. The near stoichiometric levels of isoaspartate in S11, estimated at 0.5 mol of isoaspartate per mol of S11, suggests that this unusual modification may be important for S11 function.
David
C L
CL
Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, California 92697-3900, USA.
Keener
J
J
Aswad
D W
DW
D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D007536:Isomerism; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase; D011496:Protein Methyltransferases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism*
D011994:Recombinant Proteins; D012269:Ribosomal Proteins; C045454:ribosomal protein S11; D001224:Aspartic Acid; D011496:Protein Methyltransferases; D026601:Protein D-Aspartate-L-Isoaspartate Methyltransferase