NH2-terminal processing of Drosophila melanogaster actin. Sequential removal of two amino acids.
Class II actins, such as Drosophila and mammalian skeletal muscle actins, have genes that code for a Met-X-Asp NH2 terminus where X is usually cysteine. These actins have an Ac-Asp NH2 terminus so two amino acids must be removed. To determine the nature of this processing, we labeled Drosophila Schneider L-2 cells with [35S]methionine or cysteine, isolated the actin, and analyzed the NH2-terminal actin tryptic peptides and their thermolysin digestion products. After a 4-h labeling period, we detected completed actin polypeptide chains with either an unblocked Asp or an Ac-Asp NH2 terminus. No intermediate with an NH2-terminal Cys or Met could be demonstrated. If, however, Drosophila mRNA was translated in a mRNA-dependent rabbit reticulocyte lysate system, an additional 43-kDa actin intermediate was observed. On the basis of thermolysin digestion studies and experiments using mild acid hydrolysis of a labeled actin NH2-terminal tryptic peptide fragment, we identified this intermediate as having an Ac-Cys-Asp NH2 terminus. In a time-dependent fashion, Ac-Cys was removed generating actin with an exposed NH2-terminal Asp which was subsequently acetylated to produce the mature form of actin. The removal of Met and the acetylation of Cys may occur early in translation while the nascent polypeptide chain is still attached to the ribosome. Subsequent processing occurs following completion of the synthesis of the actin polypeptide. The removal of Ac-Cys from Drosophila actin is thus similar to removal of Ac-Met from the NH2 terminus of class I actins although in the case of the class II actins, it is the second amino acid that is removed as an acetylated species.
D000199:Actins / Q000032:analysis* / Q000096:biosynthesis; D000595:Amino Acid Sequence; D000818:Animals; D003545:Cysteine / Q000032:analysis; D004331:Drosophila melanogaster; D008715:Methionine / Q000032:analysis; D013820:Thermolysin / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D000199:Actins; D008715:Methionine; D014357:Trypsin; D013820:Thermolysin; D003545:Cysteine