Brain Actin Synthesized In Vitro Undergoes Two Different and Sequential Posttranslational Modifications

Abstract

The have studied the posttranslational processing of actin molecules synthesized in a cell-free system. The results of these experiments indicate that during the in vitro synthesis of the actins from rat brain the primary translational products undergo two different and sequential posttranslational modifications. These modifications are accompanied by slight changes in the isoelectric points of the proteins and can be detected by isoelectric focusing analysis. The same posttranslational modifications can be detected during the in vitro synthesis of chick embryo skeletal muscle actin. The evidence presented suggest that the first posttranslational modification may correspond to the methylation of a histidine residue, and the second modification most likely corresponds to the acetylation of the NH(2)-terminal amino acid residues of actin molecules.

Molecular genetics of actin function

“…Although it may not be fully processed [32,121], such actin behaves the same as the product of the same gene expressed in vivo [32]. Mutant actins have been expressed from the genes for human a-skeletal muscle actin [28][29][30], rat brain cytoplasmic actin [27] and Drosophila Act88F [31][32][33]35,36,148] and used in co-polymerization and protein binding studies. Although the costs are prohibitive for making large amounts of actin this way, expression is quickly and easily achieved and binding assays can be used to screen rapidly large numbers of mutations and pinpoint those of interest for more detailed study.…”

“…Actin mutations have been described in the yeast Saccharomyces cerevisiae [15,16], the fruitfly Drosophila melanogaster [17][18][19][20][21][22], the nematode Caenorhabditis elegans [23,24] and cultured human cells [25,26]. Mutations made in cloned actin genes have been studied after expression either by transcription and translation in vitro [27][28][29][30][31][32][33][34][35][36] or in vivo following transformation of suitable organisms such as S. cerevisiae [37][38][39][40][41][42][43], Dictyostelium discoideum [44], D. melanogaster [20,33,[45][46][47][48][49], or cultured cell lines [25,26,28,50].…”

Post‐translational processing of the amino terminus affects actin function

“…In addition to the processing of the N terminus, His73 is methylated (Vanderkerckhove and Weber, 1979). It has been suggested that actin translated in vitro is completely processed (Saborio and Palmer, 1981) and we have confirmed that it is possible for the N terminus of actin to be completely processed in vitro.…”

“…We have not determined whether or not methylation of actin also occurs in rabbit reticulocyte lysate. Redman and have questioned the results obtained by Saborio and Palmer (1981), and a lack of methylation would certainly account for the more acidic charge of the actin translation in vitro.…”

Actin structure‐function relationships in vitro using oligodeoxynucleotide‐directed site‐specific mutagenesis

“…We first used this system to try to determine the functional importance of the 3-methylhistidine residue at position 73 referred to earlier [Solomon and Rubenstein, MET-CYS-ASP-GLU , , , , I 1987al. This modified amino acid had been implicated as being important in three processes: the binding of DNAse I to actin [Sussman, 1984;Sutoh, 19841, in actin polymerization [Collins and Elzinga, 19751, and in actin amino-terminal processing involving the acetylation-dependent removal of amino acids from the precursor form of the protein [Saborio and Palmer, 1981;Redman, 19851. We substituted the histidine at this position with arginine, which maintained the positive charge at this position, and with tyrosine, which eliminated the positive charge but still maintained a hydrophilic environment at this site.…”