Obscurin is a ligand for small ankyrin 1 in skeletal muscle

Aikaterini Kontrogianni-Konstantopoulos, Ellene M. Jones, Damian B. Van Rossum, Robert J. Bloch

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144 Scopus citations

Abstract

The factors that organize the internal membranes of cells are still poorly understood. We have been addressing this question using striated muscle cells, which have regular arrays of membranes that associate with the contractile apparatus in stereotypic patterns. Here we examine links between contractile structures and the sarcoplasmic reticulum (SR) established by small ankyrin 1 (sAnk1), a ∼17.5-kDa integral protein of network SR. We used yeast two-hybrid to identify obscurin, a giant Rho-GEF protein, as the major cytoplasmic ligand for sAnk1. The binding of obscurin to the cytoplasmic sequence of sAnk1 is mediated by a sequence of obscurin that is C-terminal to its last Ig-like domain. Binding was confirmed in two in vitro assays. In one, GST-obscurin, bound to glutathione-matrix, specifically adsorbed native sAnk1 from muscle homogenates. In the second, MBP-obscurin bound recombinant GST-sAnk1 in nitrocellulose blots. Kinetic studies using surface plasmon resonance yielded a KD = 130 nM. On subcellular fractionation, obscurin was concentrated in the myofibrillar fraction, consistent with its identification as sarcomeric protein. Nevertheless, obscurin, like sAnk1, concentrated around Z-disks and M-lines of striated muscle. Our findings suggest that obscurin binds sAnk1, and are the first to document a specific and direct interaction between proteins of the sarcomere and the SR.

Original languageEnglish (US)
Pages (from-to)1138-1148
Number of pages11
JournalMolecular biology of the cell
Volume14
Issue number3
DOIs
StatePublished - Mar 1 2003

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Cell Biology

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