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 a-actinin (s-al pha-aetinin) in the assembly and maintenance of striated myofibrils, m yogenic 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 tit in-binding domains (MYC/EFT-). While cytotoxic in replicating myogenic cells, as they were in PtK2 cells, the four MYC peptides were not cyt otoxic 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 b ands suggests that (a) the actin-binding site, (b) the spectrin-repeat s believed to be responsible for anti-parallel dimerization, and (c) t he C-terminal EF-hands and titin-binding domains are each dispensable for targeting s-alpha-actinin/MYC peptides into Z bands. These finding s could not have been predicted from the behavior of alpha-actinin (a) in binding assays in cell-free systems or (b) when expressed in trans fected nonmuscle cells. (C) 1998 Academic Press.