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Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly.

Young P, Ehler E, Gautel M - J. Cell Biol. (2001)

Bottom Line: It was believed that these two proteins represented unique results of protein evolution in vertebrate muscle.Both proteins coassemble during myofibrillogenesis.The presence of a calmodulin-binding IQ motif, and a Rho guanine nucleotide exchange factor domain in the COOH-terminal region suggest that obscurin is involved in Ca(2+)/calmodulin, as well as G protein-coupled signal transduction in the sarcomere.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Structural Biology Division, 69117 Heidelberg, Germany.

ABSTRACT
Vertebrate-striated muscle is assumed to owe its remarkable order to the molecular ruler functions of the giant modular signaling proteins, titin and nebulin. It was believed that these two proteins represented unique results of protein evolution in vertebrate muscle. In this paper we report the identification of a third giant protein from vertebrate muscle, obscurin, encoded on chromosome 1q42. Obscurin is approximately 800 kD and is expressed specifically in skeletal and cardiac muscle. The complete cDNA sequence of obscurin reveals a modular architecture, consisting of >67 intracellular immunoglobulin (Ig)- or fibronectin-3-like domains with multiple splice variants. A large region of obscurin shows a modular architecture of tandem Ig domains reminiscent of the elastic region of titin. The COOH-terminal region of obscurin interacts via two specific Ig-like domains with the NH(2)-terminal Z-disk region of titin. Both proteins coassemble during myofibrillogenesis. During the progression of myofibrillogenesis, all obscurin epitopes become detectable at the M band. The presence of a calmodulin-binding IQ motif, and a Rho guanine nucleotide exchange factor domain in the COOH-terminal region suggest that obscurin is involved in Ca(2+)/calmodulin, as well as G protein-coupled signal transduction in the sarcomere.

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Binding of titin and obscurin in yeast two-hybrid system and in vitro. (A) Yeast two-hybrid analysis of the interaction between obscurin and titin. Titin domains Z7–Z10 (depicted as ovals) which were used as the primary two-hybrid bait is shown. One of the interacting clones obtained (clone no. 27) was used to further map the binding site on titin. The region Z9–Z10 interacted as strongly as the original bait whereas Z7–Z8 did not interact in this assay. Interactions were assayed by cotransformation of bait and prey plasmids into L40 yeast cells and monitoring reporter gene activation. His3 gene activation is marked as +/− and β-galactosidase activity is given as arbitrary units from liquid assays. Further separation of either titin Ig domains Z9 and Z10 or obscurin Ig 48 and 49 abolishes the interaction, demonstrating that the tandem domains of both proteins are required to form a functional binding site. (B) In vitro binding of titin to obscurin. The histidine-tagged titin fragment Z9–Z10 was assayed for binding to an untagged obscurin fragment Ig48–49 on mini Ni-NTA agarose columns. Lane 1 shows a mixture of both proteins as used in the assay. Either such a mixture of both proteins (lanes 5–7), or the obscurin fragment alone (lanes 2–4), were loaded on the column. The obscurin fragment is retained on the column when mixed with the titin Z9–Z10 (lane 7, asterisk), whereas there is no unspecific binding (lane 4). Lanes 2 and 5, flow through fractions; lanes 3 and 6, wash fraction; lanes 4 and 7, eluate fraction; M, marker lane (sizes given in kD). (C) Ob48–51 is targeted to the Z-disk in neonatal rat cardiomyocytes. The T7-tagged fragment was transfected into neonatal rat cardiomyocytes and detected by a tag-specific monoclonal antibody at the sarcomeric Z-disk (green), as demonstrated by the counterstain with myomesin at the M-band (red) in the overlay. Occasional weak M-band localization can also be observed (arrowhead). Overexpressed protein is also accumulating in the nuclei. Note that expression of Ob48–51 does not disrupt myofibrils. Bar, 8 μm.
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fig4: Binding of titin and obscurin in yeast two-hybrid system and in vitro. (A) Yeast two-hybrid analysis of the interaction between obscurin and titin. Titin domains Z7–Z10 (depicted as ovals) which were used as the primary two-hybrid bait is shown. One of the interacting clones obtained (clone no. 27) was used to further map the binding site on titin. The region Z9–Z10 interacted as strongly as the original bait whereas Z7–Z8 did not interact in this assay. Interactions were assayed by cotransformation of bait and prey plasmids into L40 yeast cells and monitoring reporter gene activation. His3 gene activation is marked as +/− and β-galactosidase activity is given as arbitrary units from liquid assays. Further separation of either titin Ig domains Z9 and Z10 or obscurin Ig 48 and 49 abolishes the interaction, demonstrating that the tandem domains of both proteins are required to form a functional binding site. (B) In vitro binding of titin to obscurin. The histidine-tagged titin fragment Z9–Z10 was assayed for binding to an untagged obscurin fragment Ig48–49 on mini Ni-NTA agarose columns. Lane 1 shows a mixture of both proteins as used in the assay. Either such a mixture of both proteins (lanes 5–7), or the obscurin fragment alone (lanes 2–4), were loaded on the column. The obscurin fragment is retained on the column when mixed with the titin Z9–Z10 (lane 7, asterisk), whereas there is no unspecific binding (lane 4). Lanes 2 and 5, flow through fractions; lanes 3 and 6, wash fraction; lanes 4 and 7, eluate fraction; M, marker lane (sizes given in kD). (C) Ob48–51 is targeted to the Z-disk in neonatal rat cardiomyocytes. The T7-tagged fragment was transfected into neonatal rat cardiomyocytes and detected by a tag-specific monoclonal antibody at the sarcomeric Z-disk (green), as demonstrated by the counterstain with myomesin at the M-band (red) in the overlay. Occasional weak M-band localization can also be observed (arrowhead). Overexpressed protein is also accumulating in the nuclei. Note that expression of Ob48–51 does not disrupt myofibrils. Bar, 8 μm.

Mentions: Obscurin was identified in a systematic search for proteins interacting with the peripheral Z-disk region of titin, using the bait Z7-Z10 to screen a skeletal muscle cDNA library in the two-hybrid system. The bait is ultrastructurally located at the comb-like transition region of the peripheral Z-disk (Gautel et al., 1996a; Yajima et al., 1996). The yeast two-hybrid screen yielded over 200 HIS3 and β-galactosidase positive clones from several complexities of the library. Nine of these were sequenced and all were found to encode Ig domains 48 and 49 of obscurin followed by either a truncated (e.g., clone no. 27) or complete (e.g., clone no. 25) Fn3 domain (Ob50; Fig. 4 A).


Obscurin, a giant sarcomeric Rho guanine nucleotide exchange factor protein involved in sarcomere assembly.

Young P, Ehler E, Gautel M - J. Cell Biol. (2001)

Binding of titin and obscurin in yeast two-hybrid system and in vitro. (A) Yeast two-hybrid analysis of the interaction between obscurin and titin. Titin domains Z7–Z10 (depicted as ovals) which were used as the primary two-hybrid bait is shown. One of the interacting clones obtained (clone no. 27) was used to further map the binding site on titin. The region Z9–Z10 interacted as strongly as the original bait whereas Z7–Z8 did not interact in this assay. Interactions were assayed by cotransformation of bait and prey plasmids into L40 yeast cells and monitoring reporter gene activation. His3 gene activation is marked as +/− and β-galactosidase activity is given as arbitrary units from liquid assays. Further separation of either titin Ig domains Z9 and Z10 or obscurin Ig 48 and 49 abolishes the interaction, demonstrating that the tandem domains of both proteins are required to form a functional binding site. (B) In vitro binding of titin to obscurin. The histidine-tagged titin fragment Z9–Z10 was assayed for binding to an untagged obscurin fragment Ig48–49 on mini Ni-NTA agarose columns. Lane 1 shows a mixture of both proteins as used in the assay. Either such a mixture of both proteins (lanes 5–7), or the obscurin fragment alone (lanes 2–4), were loaded on the column. The obscurin fragment is retained on the column when mixed with the titin Z9–Z10 (lane 7, asterisk), whereas there is no unspecific binding (lane 4). Lanes 2 and 5, flow through fractions; lanes 3 and 6, wash fraction; lanes 4 and 7, eluate fraction; M, marker lane (sizes given in kD). (C) Ob48–51 is targeted to the Z-disk in neonatal rat cardiomyocytes. The T7-tagged fragment was transfected into neonatal rat cardiomyocytes and detected by a tag-specific monoclonal antibody at the sarcomeric Z-disk (green), as demonstrated by the counterstain with myomesin at the M-band (red) in the overlay. Occasional weak M-band localization can also be observed (arrowhead). Overexpressed protein is also accumulating in the nuclei. Note that expression of Ob48–51 does not disrupt myofibrils. Bar, 8 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196875&req=5

fig4: Binding of titin and obscurin in yeast two-hybrid system and in vitro. (A) Yeast two-hybrid analysis of the interaction between obscurin and titin. Titin domains Z7–Z10 (depicted as ovals) which were used as the primary two-hybrid bait is shown. One of the interacting clones obtained (clone no. 27) was used to further map the binding site on titin. The region Z9–Z10 interacted as strongly as the original bait whereas Z7–Z8 did not interact in this assay. Interactions were assayed by cotransformation of bait and prey plasmids into L40 yeast cells and monitoring reporter gene activation. His3 gene activation is marked as +/− and β-galactosidase activity is given as arbitrary units from liquid assays. Further separation of either titin Ig domains Z9 and Z10 or obscurin Ig 48 and 49 abolishes the interaction, demonstrating that the tandem domains of both proteins are required to form a functional binding site. (B) In vitro binding of titin to obscurin. The histidine-tagged titin fragment Z9–Z10 was assayed for binding to an untagged obscurin fragment Ig48–49 on mini Ni-NTA agarose columns. Lane 1 shows a mixture of both proteins as used in the assay. Either such a mixture of both proteins (lanes 5–7), or the obscurin fragment alone (lanes 2–4), were loaded on the column. The obscurin fragment is retained on the column when mixed with the titin Z9–Z10 (lane 7, asterisk), whereas there is no unspecific binding (lane 4). Lanes 2 and 5, flow through fractions; lanes 3 and 6, wash fraction; lanes 4 and 7, eluate fraction; M, marker lane (sizes given in kD). (C) Ob48–51 is targeted to the Z-disk in neonatal rat cardiomyocytes. The T7-tagged fragment was transfected into neonatal rat cardiomyocytes and detected by a tag-specific monoclonal antibody at the sarcomeric Z-disk (green), as demonstrated by the counterstain with myomesin at the M-band (red) in the overlay. Occasional weak M-band localization can also be observed (arrowhead). Overexpressed protein is also accumulating in the nuclei. Note that expression of Ob48–51 does not disrupt myofibrils. Bar, 8 μm.
Mentions: Obscurin was identified in a systematic search for proteins interacting with the peripheral Z-disk region of titin, using the bait Z7-Z10 to screen a skeletal muscle cDNA library in the two-hybrid system. The bait is ultrastructurally located at the comb-like transition region of the peripheral Z-disk (Gautel et al., 1996a; Yajima et al., 1996). The yeast two-hybrid screen yielded over 200 HIS3 and β-galactosidase positive clones from several complexities of the library. Nine of these were sequenced and all were found to encode Ig domains 48 and 49 of obscurin followed by either a truncated (e.g., clone no. 27) or complete (e.g., clone no. 25) Fn3 domain (Ob50; Fig. 4 A).

Bottom Line: It was believed that these two proteins represented unique results of protein evolution in vertebrate muscle.Both proteins coassemble during myofibrillogenesis.The presence of a calmodulin-binding IQ motif, and a Rho guanine nucleotide exchange factor domain in the COOH-terminal region suggest that obscurin is involved in Ca(2+)/calmodulin, as well as G protein-coupled signal transduction in the sarcomere.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Structural Biology Division, 69117 Heidelberg, Germany.

ABSTRACT
Vertebrate-striated muscle is assumed to owe its remarkable order to the molecular ruler functions of the giant modular signaling proteins, titin and nebulin. It was believed that these two proteins represented unique results of protein evolution in vertebrate muscle. In this paper we report the identification of a third giant protein from vertebrate muscle, obscurin, encoded on chromosome 1q42. Obscurin is approximately 800 kD and is expressed specifically in skeletal and cardiac muscle. The complete cDNA sequence of obscurin reveals a modular architecture, consisting of >67 intracellular immunoglobulin (Ig)- or fibronectin-3-like domains with multiple splice variants. A large region of obscurin shows a modular architecture of tandem Ig domains reminiscent of the elastic region of titin. The COOH-terminal region of obscurin interacts via two specific Ig-like domains with the NH(2)-terminal Z-disk region of titin. Both proteins coassemble during myofibrillogenesis. During the progression of myofibrillogenesis, all obscurin epitopes become detectable at the M band. The presence of a calmodulin-binding IQ motif, and a Rho guanine nucleotide exchange factor domain in the COOH-terminal region suggest that obscurin is involved in Ca(2+)/calmodulin, as well as G protein-coupled signal transduction in the sarcomere.

Show MeSH
Related in: MedlinePlus