Amino-terminal processing of actins mutagenized at the Cys-1 residue.
Most actins examined to date undergo a unique posttranslational modification termed processing, catalyzed by the actin N-acetylaminopeptidase. Processing is the removal of acetylmethionine from the amino terminus in class I actins with Met-Asp(Glu) amino termini. For class II actins with Met-X-Asp(Glu) amino termini, processing is the removal of the second residue as an N-acetylamino acid. Other cytosolic proteins with these amino termini are not processed suggesting that the reaction may be specific for actins. In actin, X is usually cysteine. However, there are some class II actins in which this residue is other than cysteine, suggesting a broader substrate specificity for actin N-acetylaminopeptidase than acetylmethionine or acetylcysteine. We constructed mutant actins in which this cysteine was replaced with serine, asparagine, glycine, aspartic acid, histidine, phenylalanine, and tyrosine and used these to determine the substrate specificity of rat liver actin N-acetylaminopeptidase in vitro. Amino-terminal acetylmethinonine was cleaved from adjacent aspartic acid, asparagine, or histidine, but not serine, glycine, phenylalanine, or tyrosine. Of the acetylated actin amino termini tested, only acetylmethionine and acetylcysteine were cleaved. Histidine was never N-acetylated and was not cleaved. When phenylalanine and tyrosine were adjacent to the initiator methionine, no initiator methionine was cleaved even though it was acetylated. These results suggest a narrow substrate specificity for the rat liver actin N-acetylaminopeptidase. They also demonstrate that the adjacent residue can effect actin N-acetylaminopeptidase specificity.
Sheff
D R
DR
Department of Biochemistry, University of Iowa College of Medicine, Iowa City 52242.
Rubenstein
P A
PA
D000199:Actins / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000378:metabolism; D000818:Animals; D003545:Cysteine / Q000378:metabolism*; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D051381:Rats; D013379:Substrate Specificity
D000199:Actins; D004247:DNA; D000626:Aminopeptidases; C072563:actin N-acetylaminopeptidase; D003545:Cysteine