Expression of actin mutants to study their roles in cardiomyopathy.
Mutations in the human cardiac actin gene (ACTC) have been implicated in the development of hypertrophic or dilated cardiomyopathy in humans. To determine the molecular mechanism for the disease development, a system for the expression of mutant cardiac actin proteins that may be lethal to eukaryotic cells must be developed. Here, we explore some of the advantages and disadvantages of human ACTC expression in yeast and insect cells. We show that human ACTC is incapable of rescuing a yeast endogenous actin (ACT1)-knockout in yeast cells and that coexpression of human ACTC in yeast results in slower growth, making yeast an unsuitable expression system. However, we show that it is possible for yeast cells to express a polymerization-deficient ACTI mutant, thereby allowing us to examine the cell biology of this mutation in the future. Finally, mutant forms of human cardiac actin can be expressed in and purified from insect cells in a properly folded and functional form, permitting important characterization of the biochemical mechanisms responsible for cardiomyopathy development in humans. These studies allow for further research into the biochemical characteristics of previously untenable actin mutant proteins.
Rutkevich
Lori A
LA
Department of Molecular and Cellular Biology, University of Guelph, ON, Canada.
Teal
David J
DJ
Dawson
John F
JF
D000199:Actins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D009202:Cardiomyopathies / Q000209:etiology* / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D007313:Insecta; D050505:Mutant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D017510:Protein Folding; D015003:Yeasts / Q000235:genetics
D000199:Actins; D050505:Mutant Proteins