Nebulette and nebulin are homologous proteins associated with the Z-lines of cardiac and skeletal muscle sarcomeres. Although these proteins share ~70% sequence homology and an identical domain layout, nebulette is one-tenth the size of nebulin. To define structurally important features of these proteins in terms of the Z-line architecture, we have analyzed the primary structure of nebulette and nebulin from a variety of species and developmental stages. Alignment of the 35 residue nebulin-like modules from both proteins demonstrates that the individual modules share 30-90% homology across the six proteins analyzed. In addition, this analysis demonstrates a number of areas in which the identity across the six proteins is as high as 75%. These areas may be important signals for Z-line assembly and function in the striated muscles. Significantly, most of the areas of high identity also coincide with consensus phosphorylation sites. To evaluate if nebulette, like nebulin, exhibits tissue-specific and developmental specific polymorphism, a series of immunoblot assays were performed. These data demonstrate that nebulettes from different portions of the heart are the same size. Comparison of nebulette from embryonic and adult cardiac muscle also demonstrates that this protein does not appear to vary in size with developmental stage. Consistent with the large number of consensus phosphorylation sites identified in the nebulette primary structure, we find that nebulette is phosphorylated in the cardiac muscle at serine and threonine residues. These data and sequence analyses are discussed in terms of current models for Z-line architecture.
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