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J. Biol. Chem., Vol. 281, Issue 14, 9589-9599, April 7, 2006

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

From the Institute for Genetic Medicine and Department of Biochemistry and Molecular Biology, Keck School of Medicine of the University of Southern California, Los Angeles, California 90033

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 20-40%) from the G136A Tmod mutant but more substantially (reduced 50-100%) 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.

Received for publication, November 9, 2005 , and in revised form, January 9, 2006.

^* This work was supported in part by Grants DE12941 and HL52771 from the National Institutes of Health (to L. K.). This investigation was conducted in a facility constructed with support from the National Center for Research Resources, National Institutes of Health, Research Facilities Improvement Program Grants C06 RR014514-01, C06 RR10600-01, and C06 CA62528-01. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Institute for Genetic Medicine, 2250 Alcazar St., Los Angeles, CA 90033. Tel.: 323-442-1144; Fax: 323-442-2764; E-mail: kedes{at}usc.edu.

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