Drosophila ACT88F indirect flight muscle-specific actin is not N-terminally acetylated: A mutation in N-terminal processing affects actin function

Many eukaryotic proteins are co and post-translationally modified at their N termini by removal of one or two amino acid residues and N-infinity-acety lation. Actins show two different forms of N-terminal processing dependent on their N-terminal sequence. Ln class II actins, which include muscle acti ns, the common primary sequence of Met-Cys-Asp-actin is processed to acetyl -Asp-actin. The functional significance of this in vivo is unknown. We have studied the indirect flight muscle-specific actin, ACT88F, of Drosophila m elanogaster. Our results show that ACT88F is N-terminally processed in vivo as a class II actin by removal of the first two amino acid residues (Met a nd Cys), but that uniquely the N terminus is not acetylated. Ln addition we show that ACT88F is methylated, probably at His73. Flies carrying the mod(-) mutation fail to complete post-translational proc essing of ACT88F. We propose that the mon gene product is normally responsi ble for removing N-acetyl-cysteine from actin. The biological significance of this process is demonstrated by observations that retention of the N-ace tyl-cysteine in ACT88F affects the flight muscle function of mod(-) flies. This suggests that the extreme N terminus affects actomyosin interactions i n vivo, a proposal we have examined by in vitro motility assays of ACT88F F -actin from mod(-) flies. The mod(-) actin only moves in the presence of me thylcellulose, a viscosity-enhancing agent, where it moves at velocities sl ightly, but significantly, reduced compared to wild-type. These data confir m that N-acetyl-cysteine at the N terminus affects actomyosin interactions, probably by reducing formation of the initial actomyosin collision complex , a process known to involve the actin N terminus. (C) 2000 Academic Press.