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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.

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Exogenous and endogenous ank1.5 interacts with the COOH-terminal region of obscurin. (A) Microsomes from HEK293 cells transfected with myc-tagged full-length cDNAs of ank1.5 and ank1.7 were used in interaction studies with the GST–Obs6215–6353 fusion protein. (B) ank1.5 is present in the sarcoplasmic reticulum of skeletal muscle. ank1.5 reactivity was detected in the total microsomal fraction (lane 1), in sarcoplasmic reticulum fractions enriched in longitudinal tubules (lane 2), and in terminal cisternae (lane 3). No signal was detected by a preimmune sera when tested against the same preparations of the total microsomal fraction (lane 4), longitudinal tubules (lane 5), and terminal cisternae (lane 5). (C) Microsomes from mouse skeletal muscles were used as a source of endogenous ank1.5 to perform pull-down experiments against the MBP–Obs6215–6353 fusion protein.
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fig3: Exogenous and endogenous ank1.5 interacts with the COOH-terminal region of obscurin. (A) Microsomes from HEK293 cells transfected with myc-tagged full-length cDNAs of ank1.5 and ank1.7 were used in interaction studies with the GST–Obs6215–6353 fusion protein. (B) ank1.5 is present in the sarcoplasmic reticulum of skeletal muscle. ank1.5 reactivity was detected in the total microsomal fraction (lane 1), in sarcoplasmic reticulum fractions enriched in longitudinal tubules (lane 2), and in terminal cisternae (lane 3). No signal was detected by a preimmune sera when tested against the same preparations of the total microsomal fraction (lane 4), longitudinal tubules (lane 5), and terminal cisternae (lane 5). (C) Microsomes from mouse skeletal muscles were used as a source of endogenous ank1.5 to perform pull-down experiments against the MBP–Obs6215–6353 fusion protein.

Mentions: To further expand on the interaction between ank1.5 and the COOH-terminal part of obscurin, human embryonic kidney (HEK)* 293 cells were transfected with myc-tagged plasmids encoding full-length ank1.5 or ank1.7 cDNAs. Microsomes prepared from transfected cells were solubilized and the resulting supernatant was allowed to interact with a fusion protein containing aa 6215–6353 of obscurin in frame with GST. Western blot with anti-myc monoclonal antibodies revealed that ank1.5, but not ank1.7, expressed in HEK293 cells was retained by the GST–obscurin fusion protein (Fig. 3 A). Previous work had shown that ank1.5 is a membrane protein of the sarcoplasmic reticulum (Zhou et al., 1997). To further examine the localization of ank1.5 in the sarcoplasmic reticulum, skeletal muscle microsomes were further fractionated according to established procedures (Saito et al., 1984). Selected fractions, enriched in terminal cisternae and longitudinal tubules, were analyzed by Western blot with a rabbit antibody against ank1.5. A major band of an apparent molecular mass of 25 kD, which is compatible with the expected size of ank1.5, was found in fractions of the sarcoplasmic reticulum containing longitudinal tubules and terminal cisternae (Fig. 3 B). Using the ank1.5 antiserum, endogenous ank1.5 solubilized from skeletal muscle sarcoplasmic reticulum was found to bind to the maltose binding protein (MBP)–obscurin (aa 6215–6351) fusion protein, but not to MBP alone (Fig. 3 C).


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)

Exogenous and endogenous ank1.5 interacts with the COOH-terminal region of obscurin. (A) Microsomes from HEK293 cells transfected with myc-tagged full-length cDNAs of ank1.5 and ank1.7 were used in interaction studies with the GST–Obs6215–6353 fusion protein. (B) ank1.5 is present in the sarcoplasmic reticulum of skeletal muscle. ank1.5 reactivity was detected in the total microsomal fraction (lane 1), in sarcoplasmic reticulum fractions enriched in longitudinal tubules (lane 2), and in terminal cisternae (lane 3). No signal was detected by a preimmune sera when tested against the same preparations of the total microsomal fraction (lane 4), longitudinal tubules (lane 5), and terminal cisternae (lane 5). (C) Microsomes from mouse skeletal muscles were used as a source of endogenous ank1.5 to perform pull-down experiments against the MBP–Obs6215–6353 fusion protein.
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Related In: Results  -  Collection

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fig3: Exogenous and endogenous ank1.5 interacts with the COOH-terminal region of obscurin. (A) Microsomes from HEK293 cells transfected with myc-tagged full-length cDNAs of ank1.5 and ank1.7 were used in interaction studies with the GST–Obs6215–6353 fusion protein. (B) ank1.5 is present in the sarcoplasmic reticulum of skeletal muscle. ank1.5 reactivity was detected in the total microsomal fraction (lane 1), in sarcoplasmic reticulum fractions enriched in longitudinal tubules (lane 2), and in terminal cisternae (lane 3). No signal was detected by a preimmune sera when tested against the same preparations of the total microsomal fraction (lane 4), longitudinal tubules (lane 5), and terminal cisternae (lane 5). (C) Microsomes from mouse skeletal muscles were used as a source of endogenous ank1.5 to perform pull-down experiments against the MBP–Obs6215–6353 fusion protein.
Mentions: To further expand on the interaction between ank1.5 and the COOH-terminal part of obscurin, human embryonic kidney (HEK)* 293 cells were transfected with myc-tagged plasmids encoding full-length ank1.5 or ank1.7 cDNAs. Microsomes prepared from transfected cells were solubilized and the resulting supernatant was allowed to interact with a fusion protein containing aa 6215–6353 of obscurin in frame with GST. Western blot with anti-myc monoclonal antibodies revealed that ank1.5, but not ank1.7, expressed in HEK293 cells was retained by the GST–obscurin fusion protein (Fig. 3 A). Previous work had shown that ank1.5 is a membrane protein of the sarcoplasmic reticulum (Zhou et al., 1997). To further examine the localization of ank1.5 in the sarcoplasmic reticulum, skeletal muscle microsomes were further fractionated according to established procedures (Saito et al., 1984). Selected fractions, enriched in terminal cisternae and longitudinal tubules, were analyzed by Western blot with a rabbit antibody against ank1.5. A major band of an apparent molecular mass of 25 kD, which is compatible with the expected size of ank1.5, was found in fractions of the sarcoplasmic reticulum containing longitudinal tubules and terminal cisternae (Fig. 3 B). Using the ank1.5 antiserum, endogenous ank1.5 solubilized from skeletal muscle sarcoplasmic reticulum was found to bind to the maltose binding protein (MBP)–obscurin (aa 6215–6351) fusion protein, but not to MBP alone (Fig. 3 C).

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