Key result
Smyd1b_tv1 is localized on the M-line of sarcomeres in skeletal muscle fibers, and this strong sarcomeric localization requires the highly conserved residues Phe223 and Ser225 within its specific 13-amino acid insertion.
Smyd1b_tv1 localizes to the M-line of skeletal muscle sarcomeres, a process dependent on the Phe223 and Ser225 residues within its specific 13-amino acid insertion.
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Hypothesis-generating for sarcomere protein targeting in skeletal muscle; leaves open cardiac relevance and human disease implications pending further studies.
BACKGROUND: Smyd1b is a member of the Smyd family that plays a key role in sarcomere assembly during myofibrillogenesis. Smyd1b encodes two alternatively spliced isoforms, smyd1b_tv1 and smyd1b_tv2, that are expressed in skeletal and cardiac muscles and play a vital role in myofibrillogenesis in skeletal muscles of zebrafish embryos. METHODOLOGY/PRINCIPAL FINDINGS: To better understand Smyd1b function in myofibrillogenesis, we analyzed the subcellular localization of Smyd1b_tv1 and Smyd1b_tv2 in transgenic zebrafish expressing a myc-tagged Smyd1b_tv1 or Smyd1b_tv2. The results showed a dynamic change of their subcellular localization during muscle cell differentiation. Smyd1b_tv1 and Smyd1b_tv2 were primarily localized in the cytosol of myoblasts and myotubes at early stage zebrafish embryos. However, in mature myofibers, Smyd1b_tv1, and to a small degree of Smyd1b_tv2, exhibited a sarcomeric localization. Double staining with sarcomeric markers revealed that Smyd1b_tv1was localized on the M-lines. The sarcomeric localization was confirmed in zebrafish embryos expressing the Smyd1b_tv1-GFP or Smyd1b_tv2-GFP fusion proteins. Compared with Smyd1b_tv1, Smyd1b_tv2, however, showed a weak sarcomeric localization. Smyd1b_tv1 differs from Smyd1b_tv2 by a 13 amino acid insertion encoded by exon 5, suggesting that some residues within the 13 aa insertion may be critical for the strong sarcomeric localization of Smyd1b_tv1. Sequence comparison with Smyd1b_tv1 orthologs from other vertebrates revealed several highly conserved residues (Phe223, His224 and Gln226) and two potential phosphorylation sites (Thr221 and Ser225) within the 13 aa insertion. To determine whether these residues are involved in the increased sarcomeric localization of Smyd1b_tv1, we mutated these residues into alanine. Substitution of Phe223 or Ser225 with alanine significantly reduced the sarcomeric localization of Smyd1b_tv1. In contrast, other substitutions had no effect. Moreover, replacing Ser225 with threonine (S225T) retained the strong sarcomeric localization of Smyd1b_tv1. CONCLUSION/SIGNIFICANCE: Together, these data indicate that Phe223 and Ser225 are required for the M-line localization of Smyd1b_tv1.
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Li et al. (2011) studied Sarcomere assembly in skeletal muscle. Smyd1b_tv1 and Smyd1b_tv2 expression and mutation vs. Wild-type or alternative isoforms/mutants was evaluated on Subcellular localization of Smyd1b isoforms. Smyd1b_tv1 is localized on the M-line of sarcomeres in skeletal muscle fibers, and this strong sarcomeric localization requires the highly conserved residues Phe223 and Ser225 within its specific 13-amino acid insertion.
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