In vitro and in vivo characterization of four fibroblast tropomyosins produced in bacteria: TM-2, TM-3, TM-5a, and TM-5b are co-localized in interphase fibroblasts.

Pittenger, Mark F.; Helfman, David M.

doi:10.1083/jcb.118.4.841

Cited by 71 publications

(84 citation statements)

References 55 publications

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“…TM6b-9d (smooth muscle ␣-TM) bound to actin with high affinity, as previously reported (17,18). Replacing 21 residues of exon 6b with 6a (TM6a-9d) resulted in a 2-fold increase in actin affinity, consistent with previous results (41). Replacement of exon 6b with a GCN4 leucine zipper sequence (TM6zip-9d) or deletion of half an actin binding period (TM⌬6-9d) reduced the actin affinity so it could not be accurately measured.…”

Section: Rationale-supporting

confidence: 91%

“…The two native TM sequences, encoded by exon 6a and 6b, both function normally with a striated or smooth/non-muscle COOH terminus, although there is about a 2-fold difference in actin affinity, as previously reported (41). However, the effect of deleting exon 6 or replacing it with a leucine zipper-encoding sequence depends on the COOH-terminal sequence; neither can bind to actin with an exon 9d-encoded COOH terminus in any condition tested.…”

Section: Discussionsupporting

confidence: 73%

“…Interestingly, whereas replacement of exon 6a for 6b in ␣-TM alters actin affinity (these results) (41), in ␤-TM it does not affect actin affinity (49), even though exon 6a and 6b are about 70 and 85% identical, respectively, in rat ␣-versus ␤-TM. Substitution of all 25 residues of exon 6 with the GCN4 leucine zipper sequence changed all but 5 residues, 2 of which are in interface positions.…”

Section: Discussionmentioning

confidence: 83%

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The Sequence of the Alternatively Spliced Sixth Exon of α-Tropomyosin Is Critical for Cooperative Actin Binding but Not for Interaction with Troponin

Hammell

Hitchcock‐DeGregori

1997

Journal of Biological Chemistry

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Tropomyosins, a family of highly conserved coiled-coil actin binding proteins, can differ as a consequence of alternative expression of several exons (Lees-Miller, J., and Helfman, D. (1991) BioEssays 13, 429 -437). Exon 6, which encodes residues 189 -213 in long, 284-residue tropomyosins, has two alternative forms, exon 6a or 6b, both highly conserved throughout evolution. In ␣-tropomyosin, exon 6a or 6b is not specific to any one of the nine isoforms. Exon 6b encodes part of a putative Ca 2؉ -sensitive troponin binding site in striated muscle tropomyosins, suggesting that the exon 6-encoded region may be specialized for certain tropomyosin functions.A series of recombinant, unacetylated tropomyosin exon 6 deletion and substitution mutants and chimeras was expressed in Escherichia coli to determine the requirements of exon 6 for tropomyosin function. Functional properties of the tropomyosins were defined by actin affinity measured by cosedimentation, troponin T affinity using a newly developed biosensor assay, and regulation of the actomyosin MgATPase. The region of tropomyosin encoded by exon 6 affects actin affinity but not thin filament assembly, troponin T binding, or regulation with troponin. The tropomyosins with exon 6a or 6b function normally whether a striated muscle exon 9a or smooth/non-muscle exon 9d is present. However, the effect of deleting 21 amino acids encoded by exon 6 or replacing it with a GCN4 leucine zipper sequence depends on the COOH-terminal sequence. Tropomyosins (TM)1 are a family of highly conserved coiledcoil actin binding proteins present in most eukaryotic cells. At least 15 different isoforms arise through the use of alternative promoters and alternative RNA splicing of the transcripts of a small number of genes (three to four in vertebrates; reviewed in Ref. 1). These isoforms are expressed in developmentally and tissue-specific patterns and differ in actin affinity.A function common to all TMs is cooperative binding to F-actin (2). Tropomyosin molecules are aligned head-to-tail in the grooves of the helical actin filament (3, 4). The role of TM is best understood in striated muscle where it regulates Ca 2ϩ -dependent muscle contraction with Tn (reviewed in Refs. 5-7). Structural studies have shown that Tn, found only in striated muscles, extends along at least the COOH-terminal third of the TM molecule (8 -13). Troponin I, TnC, and the COOH terminus of TnT are positioned near Cys-190 of TM. The elongated NH 2 terminus of TnT extends beyond the COOH terminus of one TM to the NH 2 -terminal 10 -30 residues of the next TM along the actin filament (13,14).Troponin greatly increases the affinity of TM for actin in the presence of Ca 2ϩ , with a further increase upon removal of Ca 2ϩ (15). Based on binding studies with TM and Tn peptides, Mak and Smillie (16) proposed that the ␣-TM-Tn interaction in the region of Cys-190 is weakened in the presence of Ca 2ϩ (Ca 2ϩ -sensitive), whereas that at the COOH terminus and the overlap region is strong in the presence and absence of Ca 2ϩ (...

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Section: Rationale-supporting

confidence: 91%

Section: Discussionsupporting

confidence: 73%

Section: Discussionmentioning

confidence: 83%

See 1 more Smart Citation

The Sequence of the Alternatively Spliced Sixth Exon of α-Tropomyosin Is Critical for Cooperative Actin Binding but Not for Interaction with Troponin

Hammell

Hitchcock‐DeGregori

1997

Journal of Biological Chemistry

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“…The sequences of these tropomyosins differ from those of their lower-actin-affinity counterparts only at positions 189-213 (see refs. [53][54][55]. In these tropomyosins, in addition to the four conserved apolar residues in period 5 described above (Fig.…”

Section: Azimuthal Positions and Polarity Of Tropomyosin On Actinmentioning

confidence: 96%

Structure of the mid-region of tropomyosin: Bending and binding sites for actin

Brown

Zhou

Reshetnikova

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

142

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Tropomyosin is a two-chain ␣-helical coiled coil whose periodic interactions with the F-actin helix are critical for thin filament stabilization and the regulation of muscle contraction. Here we deduce the mechanical and chemical basis of these interactions from the 2.3-Å-resolution crystal structure of the middle three of tropomyosin's seven periods. Geometrically specific bends of the coiled coil, produced by clusters of core alanines, and variable bends about gaps in the core, produced by isolated alanines, occur along the molecule. The crystal packing is notable in signifying that the functionally important fifth period includes an especially favorable protein-binding site, comprising an unusual apolar patch on the surface together with surrounding charged residues. Based on these and other results, we have constructed a specific model of the thin filament, with the N-terminal halves of each period (i.e., the so-called ''␣ zones'') of tropomyosin axially aligned with subdomain 3 of each monomer in F-actin.alanine ͉ ␣-helix ͉ cardiomyopathy ͉ coiled coil ͉ packing

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“…The original TM5a and TM5b cDNA templates were also gifts from Dr. David Helfman (22). The ␣-and ␤-TM PCR templates were from LK clones 504 and 417, respectively.…”

Section: Plasmid Constructionmentioning

confidence: 99%

Leucine 135 of Tropomodulin-1 Regulates Its Association with Tropomyosin, Its Cellular Localization, and the Integrity of Sarcomeres

Kong

Kedes

2006

Journal of Biological Chemistry

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Tropomodulin-1 (Tmod-1) is a well defined actin-capping protein that interacts with tropomyosin (TM) at the pointed end of actin filaments. Previous studies by others have mapped its TM-binding domain to the amino terminus from amino acid 39 to 138. In this study, we have identified several amino acid residues on Tmod-1 that are important for its interaction with TM5 (a nonmuscle TM isoform). Glutathione S-transferase affinity chromatography and immunoprecipitation assays reveal that Tmod sense mutations of either amino acid 134, 135, or 136 causes various degrees of loss of function of Tmod TM-binding ability. The reduction of TM-binding ability was relatively mild (reduced ϳ20 -40%) from the G136A Tmod mutant but more substantially (reduced ϳ50 -100%) from the I134D, L135E, and L135V Tmod mutants. In addition, mutation at any of these three sites dramatically alters the subcellular location of Tmod-1 when introduced into mammalian cells. Further analysis of these three mutants uncovered a previously unknown nuclear trafficking function of Tmod-1, and residues 134, 135, and 136 are located within a nuclear export signal motif. As a result, mutation on either residue 134 or residue 135 not only will cause a significant reduction of the Tmod-1 ability to bind to TM5 but also lead to predominant nuclear localization of Tmod-1 by crippling its nuclear export mechanism. The failure of the Tmod mutations to fully associate with TM5 when introduced into neonatal rat cardiomyocytes was also associated with an accelerated and severe fragmentation of sarcomeric structures compared with overexpression of wild type Tmod-1. The multiple losses of function of Tmod engendered by these missense mutations are most severe with the single substitution of residue 135.

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In vitro and in vivo characterization of four fibroblast tropomyosins produced in bacteria: TM-2, TM-3, TM-5a, and TM-5b are co-localized in interphase fibroblasts.

Cited by 71 publications

References 55 publications

The Sequence of the Alternatively Spliced Sixth Exon of α-Tropomyosin Is Critical for Cooperative Actin Binding but Not for Interaction with Troponin

The Sequence of the Alternatively Spliced Sixth Exon of α-Tropomyosin Is Critical for Cooperative Actin Binding but Not for Interaction with Troponin

Structure of the mid-region of tropomyosin: Bending and binding sites for actin

Leucine 135 of Tropomodulin-1 Regulates Its Association with Tropomyosin, Its Cellular Localization, and the Integrity of Sarcomeres

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