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

Abstract

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.

Tropomodulin Binds Two Tropomyosins: A Novel Model for Actin Filament Capping

“…Original evidence of a second TM binding site was shown in the ability of 125 I-labeled erythrocyte TM (a heterodimer of α and γ short TM) to bind to Tmod1 95-359 immobilized on nitrocellulose (28). Since we began our work, Vera et al (29) mapped a binding site for TM5 (a short erythrocyte TM encoded by the γ-TM gene) to residues 105-127 and Kong et al (42) found that residues L134 and L135 are crucial for TM5 binding. However, all three studies used qualitative assays to measure interaction, methods that were unable to quantify the affinity or stoichiometry of the complex.…”

Mammalian Tropomodulins Nucleate Actin Polymerization via Their Actin Monomer Binding and Filament Pointed End-capping Activities*

“…We also introduced four mutations (L29G, L73D, L134D, and L138V) that were reported previously to disrupt each of the three functional ␣-helices in N-terminal half of Tmod3 (supplemental Fig. S1) (29,30,44). We tested the ability of Tmod3 mutant proteins to bind G-actin by performing chemical cross-linking assays with the zero-length cross-linker, EDC/sulfo-NHS, followed by SDS-PAGE and Coomassie staining.…”

“…Indeed, disruption of each tropomyosin-binding site by introducing a point mutation in ␣-helix1 or ␣-helix3 of Tmod3 has no effect on its actin-nucleating activity (Fig. 7 and data not shown) while completely abolishing its tropomyosin binding and tropomyosin-dependent actin capping activities (29,30,44). However, it is possible that tropomyosin could bind to the N-terminal domain of a Tmod whose C-terminal domain caps the pointed end of an actin nucleus, which then may further bind and stabilize actin filament growth from that nucleus.…”

“…Both Tmod3-L134D and Tmod3-L138V cross-link to G-actin in a cross-linking assay (Fig. 2), and the L134D and L138V mutations disrupt ␣-helix3 in Tmod1, thereby abolishing tropomyosin-binding activity (29,44). We also introduced one or two point mutations in four C-terminal Tmod3 residues (K317A, K344A, and R345A/R346A) that are highly conserved among Tmod isoforms (Figs.…”

Leiomodin 3 and Tropomodulin 4 have overlapping functions during skeletal myofibrillogenesis

“…In striated muscle, in cooperation with tropomyosin, Tmod1 binds to and limits the exchange of actin monomers at the pointed end of the actin thin filaments (Weber et al, 1994). The localization and capping functions of Tmod1 appear to be dependent on two actin-binding and two tropomyosin-binding domains, given that mutations in any of these regions affect these functions Kostyukova et al, 2005;Kostyukova et al, 2006;Greenfield et al, 2005;Kong and Kedes, 2006;Tsukada et al, 2011). Overexpression of Tmod1 in primary myocyte cultures results in a shortening of actin filaments (Sussman et al, 1999;Littlefield et al, 2001), whereas depletion of Tmod1 leads to abnormally long thin filaments (Gregorio et al, 1995;Sussman et al, 1998;Mudry et al, 2003).…”