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Published online 13 January 2003. doi:10.1083/jcb.200208109
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© The Rockefeller University Press, 0021-9525/2003/1/245 $5.00
The Journal of Cell Biology, Volume 160, Number 2, 245-253

Article

 Binding of an ankyrin-1 isoform to obscurin suggests a molecular link between the sarcoplasmic reticulum and myofibrils in striated muscles

Paola Bagnato1, Virigina Barone2, Emiliana Giacomello1, Daniela Rossi1 and Vincenzo Sorrentino1,2

1 Molecular Medicine Section, Department of Neuroscience, University of Siena, 53100 Siena, Italy
2 San Raffaele Scientific Institute, Milano, Italy

Address correspondence to Vincenzo Sorrentino, Molecular Medicine Section, Department of Neuroscience, University of Siena, via Aldo Moro 5, 53100 Siena, Italy. Tel.: 39-0577-234-079. Fax: 39-0577-234-191. E-mail: v.sorrentino{at}unisi.it

Assembly of specialized membrane domains, both of the plasma membrane and of the ER, is necessary for the physiological activity of striated muscle cells. The mechanisms that mediate the structural organization of the sarcoplasmic reticulum with respect to the myofibrils are, however, not known. We report here that ank1.5, a small splice variant of the ank1 gene localized on the sarcoplasmic reticulum membrane, is capable of interacting with a sequence of 25 aa located at the COOH terminus of obscurin. Obscurin is a giant sarcomeric protein of ~800 kD that binds to titin and has been proposed to mediate interactions between myofibrils and other cellular structures. The binding sites and the critical aa required in the interaction between ank1.5 and obscurin were characterized using the yeast two-hybrid system, in in vitro pull-down assays and in experiments in heterologous cells. In differentiated skeletal muscle cells, a transfected myc-tagged ank1.5 was found to be selectively restricted near the M line region where it colocalized with endogenous obscurin. The M line localization of ank1.5 required a functional obscurin-binding site, because mutations of this domain resulted in a diffused distribution of the mutant ank1.5 protein in skeletal muscle cells. The interaction between ank1.5 and obscurin represents the first direct evidence of two proteins that may provide a direct link between the sarcoplasmic reticulum and myofibrils.

In keeping with the proposed role of obscurin in mediating an interaction with ankyrins and sarcoplasmic reticulum, we have also found that a sequence with homology to the obscurin-binding site of ank1.5 is present in the ank2.2 isoform, which in striated muscles has been also shown to associate with the sarcoplasmic reticulum. Accordingly, a peptide containing the COOH terminus of ank2.2 fused with GST was found to bind to obscurin. Based on reported evidence showing that the COOH terminus of ank2.2 is necessary for the localization of ryanodine receptors and InsP3 receptors in the sarcoplasmic reticulum, we propose that obscurin, through multiple interactions with ank1.5 and ank2.2 isoforms, may assemble a large protein complex that, in addition to a structural function, may play a role in the organization of specific subdomains in the sarcoplasmic reticulum.

Key Words: sarcoplasmic reticulum; calcium release; ryanodine receptors; InsP3 receptors; endoplasmic reticulum


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