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

Abstract

TRIOBP is an actin-bundling protein. Mutations of TRIOBP are associated with human deafness DFNB28. 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 the absence of stereocilia rootlets. 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 here as R1 and R2. To examine the potential role of R1 and R2 motifs in F-actin binding, we generated TRIOBP-4 mutant proteins deleted for R1 and/or R2, and then assessed their actin-binding activity and bundle formation in vitro using actin cosedimentation assays, and 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 the actin binding/bundling activities of TRIOBP-4 and impaired its localization to cellular actin cytoskeleton structures. By contrast, TRIOBP-4, lacking only R2 motif, retained its F-actin bundling ability and remained localized to actin filaments in cells, similar to full length TRIOBP-4. On the contrary, the R1 motif-deleted TRIOBP-4 mutant, which mainly consists of the R2 motif, formed thin F-actin bundles in vitro but failed to colocalize to actin filaments in cells. These results indicate that R1 motif is the major actin-binding domain of TRIOBP-4, and the binding of R2 motif with actin filaments is nonspecific.

TRIOBP-5 sculpts stereocilia rootlets and stiffens supporting cells enabling hearing

“…Using live cell NanoSPD assays, we show that coiled-coil domains are essential for oligomerization of TRIOBP-5, which appears to be a mechanism for holding actin bundles together. We propose that oligomerized TRIOBP-5 embraces filaments, allowing them to slide past one another within the bundle, thus providing flexible durability of rootlets during life-long deflection of stereocilia (8,18). We also speculate that oligomerized TRIOBP-5 interacts with and integrates rootlets into the actin meshwork of the cuticular plate ( Figure 9B), a function not fulfilled by TRIOBP-4.…”

“…A mature rootlet has the appearance of a double-pointed needle centrally positioned at the pivot point of a WT stereocilium, extending into its F-actin core about a third to half the length of a stereocilium (referred here as the upper half of a rootlet) and approximately the same distance into the actin meshwork of the cuticular plate (the lower half of a rootlet) (26). Actin filaments within the rootlets are tightly packed with no noticeable space between them, an observation that we recapitulated in vitro using F-actin bundled by purified TRIOBP-4 (8,18).…”

“…TRIOBP-1 has 5 predicted coiled-coil domains and a pleckstrin homology domain (PH) ( Figure 1A), binds and stabilizes actin filaments, and is required for embryonic viability (8,(14)(15)(16)(17). TRIOBP-4 is predicted to be a disordered protein that binds F-actin by its R1-repeat motifs (18) and has no amino acid sequence in common with TRIOBP-1, while TRIOBP-5 includes the entire amino acid sequence of TRIOBP-4, most of the sequence of TRIOBP-1, and more ( Figure 1A). Thus, TRIOBP-5 contains all of the actin-binding repeat motifs of TRIOBP-4 (18) and additional ones (14).…”

“…TRIOBP-4 is predicted to be a disordered protein that binds F-actin by its R1-repeat motifs (18) and has no amino acid sequence in common with TRIOBP-1, while TRIOBP-5 includes the entire amino acid sequence of TRIOBP-4, most of the sequence of TRIOBP-1, and more ( Figure 1A). Thus, TRIOBP-5 contains all of the actin-binding repeat motifs of TRIOBP-4 (18) and additional ones (14). TRIOBP-4 and TRIOBP-5 are expressed in human and mouse retina, brain, and inner ear (5,6,8).…”

Aggregation of the Protein TRIOBP-1 and Its Potential Relevance to Schizophrenia

“…Additionally long isoforms of more than 200 kDa exist such as TRIOBP-5 which incorporates both the TRIOBP-1 and TRIOBP-4 exons [10] . Both the protein encoded for by the major 3′ isoform TRIOBP-1, also known as Tara, and the major 5′ isoform of mouse, TRIOBP-4, have been shown to be vital for actin polymerisation [9] , [11] , with knockdown of TRIOBP by siRNA being useable as a tool to block F-actin formation in the cell [12] – [14] . Of these variants, the 3′ variant TRIOBP-1 is the most ubiquitously expressed, while the 5′ variants are mainly found in the retina and inner ear [10] .…”