Purification of human β- and γ-actin from budding yeast

被引:5
作者
Haarer, Brian K. [1 ]
Pimm, Morgan L. [1 ]
de Jong, Ebbing P. [2 ]
Amberg, David C. [1 ]
Henty-Ridilla, Jessica L. [1 ,3 ]
机构
[1] SUNY Upstate Med Univ, Dept Biochem & Mol Biol, Syracuse, NY 13210 USA
[2] SUNY Upstate Med Univ, Mass Spectrometry Core Facil, Syracuse, NY 13210 USA
[3] SUNY Upstate Med Univ, Dept Neurosci & Physiol, Syracuse, NY 13210 USA
基金
美国国家卫生研究院;
关键词
Cytoplasmic actin; Non-muscle actin; beta-actin; gamma-actin; PROFILIN; BINDING; EXPRESSION; COMPLEX; ALPHA; SUBSTITUTION; FILAMENTS; PROTEINS; RESIDUES; MUTATION;
D O I
10.1242/jcs.260540
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
Biochemical studies of human actin and its binding partners rely heavily on abundant and easily purified a-actin from skeletal muscle. Therefore, muscle actin has been used to evaluate and determine the activities of most actin regulatory proteins but there is an underlying concern that these proteins perform differently from actin present in non-muscle cells. To provide easily accessible and relatively abundant sources of human beta- or.-actin (i.e. cytoplasmic actins), we developed Saccharomyces cerevisiae strains that express each as their sole source of actin. Both beta- or.-actin purified in this system polymerize and interact with various binding partners, including profilin, mDia1 ( formin), fascin and thymosin-beta 4 (T beta 4). Notably, T beta 4 and profilin bind to beta- or.-actin with higher affinity than to a-actin, emphasizing the value of testing actin ligands with specific actin isoforms. These reagents will make specific isoforms of actin more accessible for future studies on actin regulation.
引用
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页数:9
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