MyRIP, a novel Rab effector, enables myosin VIIa recruitment to retinal melanosomes

Abstract

Defects of the myosin VIIa motor protein cause deafness and retinal anomalies in humans and mice. We report on the identification of a novel myosin-VIIa-interacting protein that we have named MyRIP (myosin-VIIa- and Rab-interacting protein), since it also binds to Rab27A in a GTP-dependent manner. In the retinal pigment epithelium cells, MyRIP, myosin VIIa and Rab27A are associated with melanosomes. In transfected PC12 cells, overexpression of MyRIP was shown to interfere with the myosin VIIa tail localization. We propose that a molecular complex composed of Rab27A, MyRIP and myosin VIIa bridges retinal melanosomes to the actin cytoskeleton and thereby mediates the local trafficking of these organelles. The defect of this molecular complex is likely to account for the perinuclear mislocalization of the melanosomes observed in the retinal pigment epithelium cells of myosinVIIa-defective mice.

Fig. 1. MyRIP, a novel Rab effector that binds to myosin VIIa. (A) Structure of the MyRIP predicted protein. The N-terminal FYVE domain (Rab-binding domain) and the region corresponding to the A7 prey (i.e. binding to myosin VIIa) are indicated. (B) Clustal W sequence alignment of the FYVE domains of MyRIP and other Rab effectors. The eight conserved cysteine residues responsible for Zn²⁺ binding are indicated. The SGAWF(F/Y) motif that is involved in the binding of Rabphilin3A and Noc2 to Rab3A is shown. The MyRIP FYVE domain shows 50 and 30% aa identity with the Rab-binding domains of melanophilin and Rabphilin3A, respectively. (C) Immunoprecipitation experiment. Lane 1 contains soluble protein extract from HEK293 cells producing the myosin VIIa tail alone. Extracts from cotransfected HEK293 cells producing both the myosin VIIa tail and myc-MyRIP (lane 2) were used for immunoprecipitations (lanes 3 and 4). The two proteins are coprecipitated by the anti-hA7 MyRIP antibody (lane 4) but not by the corresponding preimmune serum (lane 3). Extracts from untransfected cells (lane 5) and from cells expressing the myosin VIIa tail alone (lane 6) were used as negative controls for the immunoprecipitation experiment with the anti-hA7 antibody. The myosin VIIa tail coprecipitated also with MyRIP–myBD, using the anti-hA7 antibody (lane 7). The mouse anti-myosin-VIIa (anti-SSI) and anti-myc (9E10) mouse monoclonal antibodies were used to detect the myosin VIIa tail (top), and the myc-MyRIP and myc-MyRIP–myBD (bottom), respectively. (D) In vitro binding assay. The bait (lane 1) and chloramphenicol acetyltransferase (CAT, lane 2)-bound resins were incubated with bacterial extracts containing a MyRIP fragment corresponding to aa 138–403. Only the bait binds to the MyRIP fragment. Horseradish-peroxidase-conjugated streptavidin was used to detect biotinylated proteins.

Fig. 2. MyRIP binds to Rab27A–GTP. (A) A filter binding assay was used to measure the binding of various Rab proteins to the GST-tagged MyRIP–RabBD. Non-specific binding to GST was subtracted. Only Rab27A binds to MyRIP–RabBD. (B) Recombinant Rab27A (80 nM) was loaded either with [³H]GTP or [³H]GDP and incubated with 150 pmol immobilized GST–MyRIP–RabBD (grey bars) or GST alone (white bars). Only GTP-bound Rab27A binds to MyRIP–RabBD. (C) Rab27A–GTP binds equally well to the mutated form MyRIP–RabBDAAA, carrying 3 aa substitutions in the SLEWFY motif. Data are shown as the percentage of the total radioactivity (mean ± SE, n = 3). ** indicates significant difference (p < 0.01) with the negative control GST.

Fig. 3. Localization of MyRIP and Rab27A in the mouse retina. (A) MyRIP is detected at the synaptic region (S) of photoreceptor cells (PhR) and on the microvilli of retinal pigment epithelium (RPE) cells. (B) Rab27A is present on the microvilli of RPE cells. In RPE cells, MyRIP (C and D), myosin VIIa (E) and Rab27A (F) silver-enhanced immunogold labellings are associated with the membrane of melanosomes. IS, inner segment; OS, outer segment. Scale bars: (A and B), 12 µm; (C), 175 nm; (D), 100 nm; (E), 235 nm; (F and inset), 140 nm.

Fig. 4. In PC12 cells producing the GFP–myosin-VIIa tail alone (A), the labelling is observed essentially in the cell body, whereas the neurites are unstained (arrows). In contrast, in PC12 cells coproducing MyRIP and the GFP–myosin-VIIa tail (B), the two proteins are entirely colocalized not only in the cell body, but also at the tip of the neurites (arrows). Different MyRIP truncated forms (C) are produced in PC12 cells. In PC12 cells producing MyRIP–myBD (D), a cytosolic diffuse labelling is observed in the cell body; no labelling is detected at the neurite tips (arrows). In contrast, in cells producing MyRIP–RabBD (E), an enrichment of the immunolabelling is observed at the tip of the neurites (arrows). No such enrichment is observed in PC12 cells producing MyRIP–ΔRabBD (F); immunolabelling is associated with several spines extending from the neurites (arrows). The myc-tagged MyRIP fusion proteins are detected using the anti-myc (9E10) antibody. Scale bar = 10 µm.

Fig. 5. Model of the trafficking of melanosomes in the retinal pigment epithelium cells. (1) Melanosomes display fast, bidirectional, microtubule-dependent long-range movements in the cell body. (2) Rab27A targeted to the melanosome membrane interacts with its effector, MyRIP, which in turn binds to the actin-based motor protein myosin VIIa. (3) Myosin VIIa then enables the local movement of the melanosomes along the actin filaments of the microvilli.