Studies on the role of actin's N tau-methylhistidine using oligodeoxynucleotide-directed site-specific mutagenesis.
The primary structure of all actins except that isolated from Naegleria gruberi contains a unique N tau-methylhistidine (MeHis) at position 73. This modified residue has been implicated as possibly being important for the post-translational processing of actin's amino terminus, the binding of actin to DNase I, and in the polymerization of G-actin. We have investigated the potential role of MeHis in each of these processes by utilizing site-directed mutagenesis to change His-73 of skeletal muscle actin to Arg and Tyr. Wild type and mutant actins were synthesized in vivo, using non-muscle cells transfected with mutant cDNAs, and in vitro by translating mutant RNAs synthesized using SP6 RNA polymerase in a rabbit reticulocyte lysate. We have found that actins containing Arg or Tyr at position 73 undergo amino-terminal processing, bind to DNase I-agarose, and become incorporated into the cytoskeleton of a nonmuscle cell as efficiently as wild type actin. Furthermore, using an in vitro copolymerization assay we have found that although there is no difference between the Arg mutant and the wild type actins, the Tyr mutant has a slightly greater critical concentration for polymerization. These results show that MeHis is not absolutely required for any of these processes.
Solomon
L R
LR
Rubenstein
P A
PA
D000199:Actins / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001435:Bacteriophages / Q000235:genetics; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism; D004274:DNA, Recombinant; D003850:Deoxyribonuclease I / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000031:analogs & derivatives; D006801:Humans; D008762:Methylhistidines; D009132:Muscles / Q000032:analysis; D009154:Mutation; D010957:Plasmids; D011108:Polymers; D011499:Protein Processing, Post-Translational; D013329:Structure-Activity Relationship; D014162:Transfection
D000199:Actins; D004274:DNA, Recombinant; D008762:Methylhistidines; D011108:Polymers; D006639:Histidine; D003850:Deoxyribonuclease I; C028118:3-methylhistidine