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The NH2 terminus of titin spans the Z-disc: its interaction with a novel 19-kD ligand (T-cap) is required for sarcomeric integrity.

Gregorio CC, Trombitás K, Centner T, Kolmerer B, Stier G, Kunke K, Suzuki K, Obermayr F, Herrmann B, Granzier H, Sorimachi H, Labeit S - J. Cell Biol. (1998)

Bottom Line: In vitro binding studies reveal that mammalian titins have at least four potential binding sites for alpha-actinin within their Z-line spanning region.Furthermore, we demonstrate that the NH2-terminal titin Ig repeats Z1 and Z2 in the periphery of the Z-line bind to a novel 19-kD protein, referred to as titin-cap.Using dominant-negative approaches in cardiac myocytes, both the titin Z1-Z2 domains and titin-cap are shown to be required for the structural integrity of sarcomeres, suggesting that their interaction is critical in titin filament-regulated sarcomeric assembly.

View Article: PubMed Central - PubMed

Affiliation: Departments of Cell Biology and Anatomy, and Molecular and Cellular Biology, University of Arizona, Tucson, Arizona 85724, USA. gregorio@u.arizona.edu

ABSTRACT
Titin is a giant elastic protein in vertebrate striated muscles with an unprecedented molecular mass of 3-4 megadaltons. Single molecules of titin extend from the Z-line to the M-line. Here, we define the molecular layout of titin within the Z-line; the most NH2-terminal 30 kD of titin is located at the periphery of the Z-line at the border of the adjacent sarcomere, whereas the subsequent 60 kD of titin spans the entire width of the Z-line. In vitro binding studies reveal that mammalian titins have at least four potential binding sites for alpha-actinin within their Z-line spanning region. Titin filaments may specify Z-line width and internal structure by varying the length of their NH2-terminal overlap and number of alpha-actinin binding sites that serve to cross-link the titin and thin filaments. Furthermore, we demonstrate that the NH2-terminal titin Ig repeats Z1 and Z2 in the periphery of the Z-line bind to a novel 19-kD protein, referred to as titin-cap. Using dominant-negative approaches in cardiac myocytes, both the titin Z1-Z2 domains and titin-cap are shown to be required for the structural integrity of sarcomeres, suggesting that their interaction is critical in titin filament-regulated sarcomeric assembly.

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In vitro interaction of Z-repeats with  α-actinin. (A) Binding of four Z-repeat fragments, Zr1, 2, 3, and 7 to an α-actinin fusion protein. A Coomassie blue–stained 15% Laemmli  SDS gel of a whole-cell lysate of BL21 cells expressing the Z-repeat fragment Zr1, 2, 3, and 7,  which corresponds to the splice variant of psoas  muscle (lane Lys). These cells were mixed with  cells expressing a GST-His double-tagged α-actinin (GST-actn) fusion peptide and lysed together, and lysates were passed over Ni-NTA  columns. Interaction of α-actinin with the  Z-repeats is indicated by also retaining the nontagged Z-repeats on an Ni-NTA column (arrow Zr1,2,3,7). (B) As in A, but here the Z-repeats were expressed as GST-His6 double-tagged fusions, and the expressed α-actinin COOH-terminal peptide was tagless. Interaction of the 15-kD from the α-actinin COOH  terminus with the Z-repeats is indicated by retaining the α-actinin 15 kD band on the column (arrow actn). Here, Lys indicates a whole-cell lysate of BL21 cells overexpressing the α-actinin COOH terminus; control (C), Zr1-His6-GST fusion peptide alone loaded on the  column, demonstrating that a 15-kD protein from E. coli does not bind nonspecifically on the column. Lanes Zr1–Zr7, interaction of  each of the seven Z-repeats alone with the COOH terminus of α-actinin; lane Zr4-6, interaction of the three fused Z-repeats with the  COOH terminus of α-actinin. The Z-repeats Zr1, 2, 3, and 7 alone are sufficient for binding to the α-actinin COOH terminus under  the conditions used. The Zr4, 5, and 6 alone do not interact with the α-actinin COOH terminus, but as a fusion peptide, they also bind to  the COOH terminus of α-actinin. Lane M, molecular mass markers corresponding to 97, 60, 40, 30, 20, and 15 kD.
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Figure 2: In vitro interaction of Z-repeats with α-actinin. (A) Binding of four Z-repeat fragments, Zr1, 2, 3, and 7 to an α-actinin fusion protein. A Coomassie blue–stained 15% Laemmli SDS gel of a whole-cell lysate of BL21 cells expressing the Z-repeat fragment Zr1, 2, 3, and 7, which corresponds to the splice variant of psoas muscle (lane Lys). These cells were mixed with cells expressing a GST-His double-tagged α-actinin (GST-actn) fusion peptide and lysed together, and lysates were passed over Ni-NTA columns. Interaction of α-actinin with the Z-repeats is indicated by also retaining the nontagged Z-repeats on an Ni-NTA column (arrow Zr1,2,3,7). (B) As in A, but here the Z-repeats were expressed as GST-His6 double-tagged fusions, and the expressed α-actinin COOH-terminal peptide was tagless. Interaction of the 15-kD from the α-actinin COOH terminus with the Z-repeats is indicated by retaining the α-actinin 15 kD band on the column (arrow actn). Here, Lys indicates a whole-cell lysate of BL21 cells overexpressing the α-actinin COOH terminus; control (C), Zr1-His6-GST fusion peptide alone loaded on the column, demonstrating that a 15-kD protein from E. coli does not bind nonspecifically on the column. Lanes Zr1–Zr7, interaction of each of the seven Z-repeats alone with the COOH terminus of α-actinin; lane Zr4-6, interaction of the three fused Z-repeats with the COOH terminus of α-actinin. The Z-repeats Zr1, 2, 3, and 7 alone are sufficient for binding to the α-actinin COOH terminus under the conditions used. The Zr4, 5, and 6 alone do not interact with the α-actinin COOH terminus, but as a fusion peptide, they also bind to the COOH terminus of α-actinin. Lane M, molecular mass markers corresponding to 97, 60, 40, 30, 20, and 15 kD.

Mentions: For the titin and α-actinin interaction studies, Z-repeat and α-actinin (ACTN2; Beggs et al., 1992) cDNA fragments were isolated by PCR and inserted into modified pET9D vectors (Studier and Moffat, 1991). Unmodified pET9D was used to express tagless proteins, whereas modified pET9D vectors were used to express NH2-terminal His6-tagged and His6-GST–double tagged fusion proteins. The glutathione-S-transferase (GST) fusion component was included to improve solubility of the expressed peptides, whereas the His6-tag was used to monitor interactions. Induction, expression, and cell lysis were carried out as previously described (Studier et al., 1990). E. coli cells expressing the four repeat fragment Zr1, 2, 3, and 7 were mixed with cells expressing His6-GST–tagged α-actinin (Fig. 2 A). Additionally, cells expressing the COOH-terminal 15 kD of α-actinin (containing residues 760–864) were mixed with His6-GST– tagged Z-repeats (Fig. 2 B). After cell lysis, proteins were passed over Ni-NTA resin.


The NH2 terminus of titin spans the Z-disc: its interaction with a novel 19-kD ligand (T-cap) is required for sarcomeric integrity.

Gregorio CC, Trombitás K, Centner T, Kolmerer B, Stier G, Kunke K, Suzuki K, Obermayr F, Herrmann B, Granzier H, Sorimachi H, Labeit S - J. Cell Biol. (1998)

In vitro interaction of Z-repeats with  α-actinin. (A) Binding of four Z-repeat fragments, Zr1, 2, 3, and 7 to an α-actinin fusion protein. A Coomassie blue–stained 15% Laemmli  SDS gel of a whole-cell lysate of BL21 cells expressing the Z-repeat fragment Zr1, 2, 3, and 7,  which corresponds to the splice variant of psoas  muscle (lane Lys). These cells were mixed with  cells expressing a GST-His double-tagged α-actinin (GST-actn) fusion peptide and lysed together, and lysates were passed over Ni-NTA  columns. Interaction of α-actinin with the  Z-repeats is indicated by also retaining the nontagged Z-repeats on an Ni-NTA column (arrow Zr1,2,3,7). (B) As in A, but here the Z-repeats were expressed as GST-His6 double-tagged fusions, and the expressed α-actinin COOH-terminal peptide was tagless. Interaction of the 15-kD from the α-actinin COOH  terminus with the Z-repeats is indicated by retaining the α-actinin 15 kD band on the column (arrow actn). Here, Lys indicates a whole-cell lysate of BL21 cells overexpressing the α-actinin COOH terminus; control (C), Zr1-His6-GST fusion peptide alone loaded on the  column, demonstrating that a 15-kD protein from E. coli does not bind nonspecifically on the column. Lanes Zr1–Zr7, interaction of  each of the seven Z-repeats alone with the COOH terminus of α-actinin; lane Zr4-6, interaction of the three fused Z-repeats with the  COOH terminus of α-actinin. The Z-repeats Zr1, 2, 3, and 7 alone are sufficient for binding to the α-actinin COOH terminus under  the conditions used. The Zr4, 5, and 6 alone do not interact with the α-actinin COOH terminus, but as a fusion peptide, they also bind to  the COOH terminus of α-actinin. Lane M, molecular mass markers corresponding to 97, 60, 40, 30, 20, and 15 kD.
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Figure 2: In vitro interaction of Z-repeats with α-actinin. (A) Binding of four Z-repeat fragments, Zr1, 2, 3, and 7 to an α-actinin fusion protein. A Coomassie blue–stained 15% Laemmli SDS gel of a whole-cell lysate of BL21 cells expressing the Z-repeat fragment Zr1, 2, 3, and 7, which corresponds to the splice variant of psoas muscle (lane Lys). These cells were mixed with cells expressing a GST-His double-tagged α-actinin (GST-actn) fusion peptide and lysed together, and lysates were passed over Ni-NTA columns. Interaction of α-actinin with the Z-repeats is indicated by also retaining the nontagged Z-repeats on an Ni-NTA column (arrow Zr1,2,3,7). (B) As in A, but here the Z-repeats were expressed as GST-His6 double-tagged fusions, and the expressed α-actinin COOH-terminal peptide was tagless. Interaction of the 15-kD from the α-actinin COOH terminus with the Z-repeats is indicated by retaining the α-actinin 15 kD band on the column (arrow actn). Here, Lys indicates a whole-cell lysate of BL21 cells overexpressing the α-actinin COOH terminus; control (C), Zr1-His6-GST fusion peptide alone loaded on the column, demonstrating that a 15-kD protein from E. coli does not bind nonspecifically on the column. Lanes Zr1–Zr7, interaction of each of the seven Z-repeats alone with the COOH terminus of α-actinin; lane Zr4-6, interaction of the three fused Z-repeats with the COOH terminus of α-actinin. The Z-repeats Zr1, 2, 3, and 7 alone are sufficient for binding to the α-actinin COOH terminus under the conditions used. The Zr4, 5, and 6 alone do not interact with the α-actinin COOH terminus, but as a fusion peptide, they also bind to the COOH terminus of α-actinin. Lane M, molecular mass markers corresponding to 97, 60, 40, 30, 20, and 15 kD.
Mentions: For the titin and α-actinin interaction studies, Z-repeat and α-actinin (ACTN2; Beggs et al., 1992) cDNA fragments were isolated by PCR and inserted into modified pET9D vectors (Studier and Moffat, 1991). Unmodified pET9D was used to express tagless proteins, whereas modified pET9D vectors were used to express NH2-terminal His6-tagged and His6-GST–double tagged fusion proteins. The glutathione-S-transferase (GST) fusion component was included to improve solubility of the expressed peptides, whereas the His6-tag was used to monitor interactions. Induction, expression, and cell lysis were carried out as previously described (Studier et al., 1990). E. coli cells expressing the four repeat fragment Zr1, 2, 3, and 7 were mixed with cells expressing His6-GST–tagged α-actinin (Fig. 2 A). Additionally, cells expressing the COOH-terminal 15 kD of α-actinin (containing residues 760–864) were mixed with His6-GST– tagged Z-repeats (Fig. 2 B). After cell lysis, proteins were passed over Ni-NTA resin.

Bottom Line: In vitro binding studies reveal that mammalian titins have at least four potential binding sites for alpha-actinin within their Z-line spanning region.Furthermore, we demonstrate that the NH2-terminal titin Ig repeats Z1 and Z2 in the periphery of the Z-line bind to a novel 19-kD protein, referred to as titin-cap.Using dominant-negative approaches in cardiac myocytes, both the titin Z1-Z2 domains and titin-cap are shown to be required for the structural integrity of sarcomeres, suggesting that their interaction is critical in titin filament-regulated sarcomeric assembly.

View Article: PubMed Central - PubMed

Affiliation: Departments of Cell Biology and Anatomy, and Molecular and Cellular Biology, University of Arizona, Tucson, Arizona 85724, USA. gregorio@u.arizona.edu

ABSTRACT
Titin is a giant elastic protein in vertebrate striated muscles with an unprecedented molecular mass of 3-4 megadaltons. Single molecules of titin extend from the Z-line to the M-line. Here, we define the molecular layout of titin within the Z-line; the most NH2-terminal 30 kD of titin is located at the periphery of the Z-line at the border of the adjacent sarcomere, whereas the subsequent 60 kD of titin spans the entire width of the Z-line. In vitro binding studies reveal that mammalian titins have at least four potential binding sites for alpha-actinin within their Z-line spanning region. Titin filaments may specify Z-line width and internal structure by varying the length of their NH2-terminal overlap and number of alpha-actinin binding sites that serve to cross-link the titin and thin filaments. Furthermore, we demonstrate that the NH2-terminal titin Ig repeats Z1 and Z2 in the periphery of the Z-line bind to a novel 19-kD protein, referred to as titin-cap. Using dominant-negative approaches in cardiac myocytes, both the titin Z1-Z2 domains and titin-cap are shown to be required for the structural integrity of sarcomeres, suggesting that their interaction is critical in titin filament-regulated sarcomeric assembly.

Show MeSH