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

Bagnato P, Barone V, Giacomello E, Rossi D, Sorrentino V - J. Cell Biol. (2003)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Medicine Section, Department of Neuroscience, University of Siena, 53100 Siena, Italy.

ABSTRACT
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 approximately 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.

Show MeSH
The site in ank1.5 able to interact with obscurin is located in a sequence highly conserved among ankyrins. (A) A schematic representation of the structure of the small muscle–specific ankyrin isoforms is presented: a color code identifies the different exons that, by undergoing alternative splicing, generate the different ank1 isoforms. The cytosolic sequences of ank1.5, ank1.6, and ank1.7 were used as baits in two-hybrid system experiments against the obscurin subclone A7. In parallel, in vitro–translated obscurin subclone A7 was allowed to interact with GST–ank1.5, GST–ank1.6, and GST–ank1.7. Both two-hybrid and in vitro binding assays demonstrated that only ank1.5 is capable of binding the obscurin subclone A7. +, detectable activity; −, no detectable activity. (B) The alignment of COOH-terminal sequences of a skeletal muscle–specific ank3 isoform (AnkG107), ank2.2, ank1.3, and ank1.5. Conserved aa residues are in bold. (C) In vitro binding of a GST fusion protein containing the COOH-terminal region of ank2.2 (aa 1758–1872) against in vitro–transcribed and –translated obscurin clone A7. (D) Site-directed mutagenesis of aa 97–123 of ank1.5 was performed and the in vitro–transcribed and –translated mutant proteins were used in binding experiments with the GST–Obs6215–6353 fusion protein. Mutagenesis of any of four aa residues (T99, K101, R104, and K105) of ank1.5 strongly reduced the ability to bind to obscurin.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172649&req=5

fig2: The site in ank1.5 able to interact with obscurin is located in a sequence highly conserved among ankyrins. (A) A schematic representation of the structure of the small muscle–specific ankyrin isoforms is presented: a color code identifies the different exons that, by undergoing alternative splicing, generate the different ank1 isoforms. The cytosolic sequences of ank1.5, ank1.6, and ank1.7 were used as baits in two-hybrid system experiments against the obscurin subclone A7. In parallel, in vitro–translated obscurin subclone A7 was allowed to interact with GST–ank1.5, GST–ank1.6, and GST–ank1.7. Both two-hybrid and in vitro binding assays demonstrated that only ank1.5 is capable of binding the obscurin subclone A7. +, detectable activity; −, no detectable activity. (B) The alignment of COOH-terminal sequences of a skeletal muscle–specific ank3 isoform (AnkG107), ank2.2, ank1.3, and ank1.5. Conserved aa residues are in bold. (C) In vitro binding of a GST fusion protein containing the COOH-terminal region of ank2.2 (aa 1758–1872) against in vitro–transcribed and –translated obscurin clone A7. (D) Site-directed mutagenesis of aa 97–123 of ank1.5 was performed and the in vitro–transcribed and –translated mutant proteins were used in binding experiments with the GST–Obs6215–6353 fusion protein. Mutagenesis of any of four aa residues (T99, K101, R104, and K105) of ank1.5 strongly reduced the ability to bind to obscurin.

Mentions: To identify the sequence in ank1.5 responsible for the interaction with the COOH terminus of obscurin, we performed experiments designed to test the ability of the three small ankyrin isoforms (ank1.5, ank1.6, and ank1.7) to bind to the COOH terminus of obscurin. Two-hybrid experiments were performed using the cytosolic sequences of the ank1.5, ank1.6, and ank1.7 isoforms (cloned in the pGBKT7 vector as a bait) against the obscurin subclone A7 (cloned in the pACT2 vector). Both the ability to grow in selective media lacking histidine and β-galactosidase activity indicated that only ank1.5, not ank1.6 or ank1.7, was capable of binding the COOH terminus of obscurin (Fig. 2 A). From a quantitative point of view, it is worth noting that whereas the interaction of obscurin subclone A7 with ank1.5 yielded a very strong blue reaction in the β-galactosidase assay, interaction with ank1.6 and ank1.7 did not result in any activity at all. In parallel, pull-down experiments were performed using in vitro–translated obscurin subclone A7 and GST fusion proteins of the three ankyrin isoforms. As shown in Fig. 2 A, in agreement with two-hybrid results, binding to the GST–obscurin clone A7 was observed with ank1.5 and not with ank1.6 or ank1.7. The alignment of aa sequences of muscle-specific small ank1 isoforms revealed that ank1.5 contains a stretch of 22 aa (aa 102–123) that is absent in ank1.6 and ank1.7, suggesting that this region is involved in mediating the binding of ank1.5 with the COOH terminus of obscurin.


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

Bagnato P, Barone V, Giacomello E, Rossi D, Sorrentino V - J. Cell Biol. (2003)

The site in ank1.5 able to interact with obscurin is located in a sequence highly conserved among ankyrins. (A) A schematic representation of the structure of the small muscle–specific ankyrin isoforms is presented: a color code identifies the different exons that, by undergoing alternative splicing, generate the different ank1 isoforms. The cytosolic sequences of ank1.5, ank1.6, and ank1.7 were used as baits in two-hybrid system experiments against the obscurin subclone A7. In parallel, in vitro–translated obscurin subclone A7 was allowed to interact with GST–ank1.5, GST–ank1.6, and GST–ank1.7. Both two-hybrid and in vitro binding assays demonstrated that only ank1.5 is capable of binding the obscurin subclone A7. +, detectable activity; −, no detectable activity. (B) The alignment of COOH-terminal sequences of a skeletal muscle–specific ank3 isoform (AnkG107), ank2.2, ank1.3, and ank1.5. Conserved aa residues are in bold. (C) In vitro binding of a GST fusion protein containing the COOH-terminal region of ank2.2 (aa 1758–1872) against in vitro–transcribed and –translated obscurin clone A7. (D) Site-directed mutagenesis of aa 97–123 of ank1.5 was performed and the in vitro–transcribed and –translated mutant proteins were used in binding experiments with the GST–Obs6215–6353 fusion protein. Mutagenesis of any of four aa residues (T99, K101, R104, and K105) of ank1.5 strongly reduced the ability to bind to obscurin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172649&req=5

fig2: The site in ank1.5 able to interact with obscurin is located in a sequence highly conserved among ankyrins. (A) A schematic representation of the structure of the small muscle–specific ankyrin isoforms is presented: a color code identifies the different exons that, by undergoing alternative splicing, generate the different ank1 isoforms. The cytosolic sequences of ank1.5, ank1.6, and ank1.7 were used as baits in two-hybrid system experiments against the obscurin subclone A7. In parallel, in vitro–translated obscurin subclone A7 was allowed to interact with GST–ank1.5, GST–ank1.6, and GST–ank1.7. Both two-hybrid and in vitro binding assays demonstrated that only ank1.5 is capable of binding the obscurin subclone A7. +, detectable activity; −, no detectable activity. (B) The alignment of COOH-terminal sequences of a skeletal muscle–specific ank3 isoform (AnkG107), ank2.2, ank1.3, and ank1.5. Conserved aa residues are in bold. (C) In vitro binding of a GST fusion protein containing the COOH-terminal region of ank2.2 (aa 1758–1872) against in vitro–transcribed and –translated obscurin clone A7. (D) Site-directed mutagenesis of aa 97–123 of ank1.5 was performed and the in vitro–transcribed and –translated mutant proteins were used in binding experiments with the GST–Obs6215–6353 fusion protein. Mutagenesis of any of four aa residues (T99, K101, R104, and K105) of ank1.5 strongly reduced the ability to bind to obscurin.
Mentions: To identify the sequence in ank1.5 responsible for the interaction with the COOH terminus of obscurin, we performed experiments designed to test the ability of the three small ankyrin isoforms (ank1.5, ank1.6, and ank1.7) to bind to the COOH terminus of obscurin. Two-hybrid experiments were performed using the cytosolic sequences of the ank1.5, ank1.6, and ank1.7 isoforms (cloned in the pGBKT7 vector as a bait) against the obscurin subclone A7 (cloned in the pACT2 vector). Both the ability to grow in selective media lacking histidine and β-galactosidase activity indicated that only ank1.5, not ank1.6 or ank1.7, was capable of binding the COOH terminus of obscurin (Fig. 2 A). From a quantitative point of view, it is worth noting that whereas the interaction of obscurin subclone A7 with ank1.5 yielded a very strong blue reaction in the β-galactosidase assay, interaction with ank1.6 and ank1.7 did not result in any activity at all. In parallel, pull-down experiments were performed using in vitro–translated obscurin subclone A7 and GST fusion proteins of the three ankyrin isoforms. As shown in Fig. 2 A, in agreement with two-hybrid results, binding to the GST–obscurin clone A7 was observed with ank1.5 and not with ank1.6 or ank1.7. The alignment of aa sequences of muscle-specific small ank1 isoforms revealed that ank1.5 contains a stretch of 22 aa (aa 102–123) that is absent in ank1.6 and ank1.7, suggesting that this region is involved in mediating the binding of ank1.5 with the COOH terminus of obscurin.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Medicine Section, Department of Neuroscience, University of Siena, 53100 Siena, Italy.

ABSTRACT
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 approximately 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.

Show MeSH