Studies on the role of actin's aspartic acid 3 and aspartic acid 11 using oligodeoxynucleotide-directed site-specific mutagenesis.
One or more of the five acidic amino-terminal residues of skeletal muscle actin have been implicated as being important in a number of actin-related processes. We have constructed a series of actins containing mutations at Asp3 and Asp11 and tested these mutant proteins for their ability to bind to DNase I-agarose, polymerize with rabbit skeletal muscle actin, undergo amino-terminal processing, and bind to the myosin-S1 subfragment. The mutant actins were expressed in vitro using a coupled transcription/translation system which involves the synthesis of mutant RNAs with SP6 RNA polymerase followed by their translation in a rabbit reticulocyte lysate. When Asp3 was changed to Ala, His, or Asn there was no difference in the tested properties as compared to wild type actin. These results suggest that an acidic residue at position 3 is not critical for the actin functions measured. When Asp11 was changed to Glu, Asn, or His or if the conserved Asp-Asn sequence at positions 11 and 12 was reversed, the mutants were able to copolymerize with rabbit skeletal muscle actin and be cross-linked to myosin-S1 to nearly the same extent as wild type actin. However, the amount of in vitro-synthesized actin capable of binding to DNase I-agarose with high affinity or undergoing amino-terminal processing was reduced significantly relative to the wild type actin synthesized in vitro. The Asp11 mutants ran anomalously on native polyacrylamide gels suggestive of a conformational change induced in the actin. Together, these results suggest that Asp11 may be important in proper actin folding and function.
Solomon
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TL
Department of Biochemistry, University of Iowa College of Medicine, Iowa City 52242.
Solomon
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LR
Gay
L S
LS
Rubenstein
P A
PA
D000199:Actins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001224:Aspartic Acid; D009132:Muscles / Q000378:metabolism; D009154:Mutation; D015879:Myosin Subfragments; D009218:Myosins / Q000378:metabolism; D015345:Oligonucleotide Probes; D010446:Peptide Fragments / Q000378:metabolism; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D014158:Transcription, Genetic
D000199:Actins; D015879:Myosin Subfragments; D015345:Oligonucleotide Probes; D010446:Peptide Fragments; D012333:RNA, Messenger; D001224:Aspartic Acid; D009218:Myosins