Dispensability of the actin-binding site and spectrin repeats for targeting sarcomeric alpha-actinin into maturing Z bands in vivo: implications for in vitro binding studies.
To explore the roles of specific domains of sarcomeric alpha-actinin (s-alpha-actinin) in the assembly and maintenance of striated myofibrils, myogenic cultures were transfected with four MYC-tagged s-alpha-actinin peptides. They were: (1) full-length sarcomeric alpha-actinin, (2) an N-terminal deletion that removed the actin-binding site only (MYC/A-), (3) a peptide that consisted of the actin-binding site only (MYC/A+), and (4) an N-terminal deletion that removed the EF-hands and titin-binding domains (MYC/EFT-). While cytotoxic in replicating myogenic cells, as they were in PtK2 cells, the four MYC peptides were not cytotoxic in postmitotic myotubes. In myotubes each of the four different MYC peptides were promptly and selectively incorporated into normal Z bands. The incorporation of MYC/A-, MYC/A+, and MYC/EFT- into Z bands suggests that (a) the actin-binding site, (b) the spectrin-repeats believed to be responsible for anti-parallel dimerization, and (c) the C-terminal EF-hands and titin-binding domains are each dispensable for targeting s-alpha-actinin/MYC peptides into Z bands. These findings could not have been predicted from the behavior of alpha-actinin (a) in binding assays in cell-free systems or (b) when expressed in transfected nonmuscle cells.
Department of Cell Biology, Beijing Institute for Cancer Research, Beijing Medical University, China.
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Lin
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Hijikata
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Zhang
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Choi
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Holtzer
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Sweeney
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Holtzer
D000185:Actinin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000199:Actins / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D002642:Chick Embryo; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D018482:Muscle, Skeletal / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D012518:Sarcomeres / Q000378:metabolism*; D013049:Spectrin / Q000737:chemistry; D014162:Transfection
D000199:Actins; D010446:Peptide Fragments; D016271:Proto-Oncogene Proteins c-myc; D000185:Actinin; D013049:Spectrin