R1 Motif is the Major Actin Binding Domain of TRIOBP-4

Abstract

The objective of this study is to identify the actin-binding domains of TRIOBP, an actin-bundling protein associated with human deafness DFNB28. Previous study shows that in vitro, TRIOBP isoform 4 (TRIOBP 4) forms dense F-actin bundles resembling the inner ear hair cell rootlet structure. Deletion of TRIOBP isoforms 4 and 5 leads to hearing loss in mice due to their inability to form rootlets. Despite the importance of TRIOBP in hearing, the mechanism of actin bundle formation by TRIOBP is not fully understood. The amino acid sequences of TRIOBP isoforms 4 and 5 contain two repeated motifs, referred to as R1 and R2, respectively. To examine the potential role of R1 and R2 motifs in F-actin binding, we generated TRIOBP-4 mutant proteins deleted with R2, and/or R1, and assessed their F-actin binding activity and bundle formation in vitro by actin co-sedimentation assay, fluorescence and electron microscopy. Cellular distributions of the TRIOBP-4 mutants were examined by confocal microscopy. We showed that deletion of both R1 and R2 motifs completely disrupted actin binding activity of TRIOBP-4 and impaired its localization to cellular actin cytoskeleton structure including filopodia. By contrast, TRIOBP-4 lacking only R2 motif retained F-actin binding and bundling ability and localized to actin filaments in cells, similar to full length TRIOBP-4. Moreover, the R1 motif-deleted TRIOBP-4 mutant which mainly contains R2 motif, formed thin F-actin bundle in vitro, but failed to co-localize to actin filaments in cells. These results indicate that R1 motif is the major actin-binding domain of TRIOBP-4 and R2 motif may make secondary contacts with actin filaments within a bundle.