CARMIL, also known as Acan 125, is a multidomain protein that was originally identified on the basis of its interaction with the Src homology 3 (SH3) domain of type I myosins from Acanthamoeba. In a subsequent study of CARMIL from Dictyostelium, pull-down assays indicated that the protein also bound capping protein and the Arp2/3 complex. Here we present biochemical evidence that Acanthamoeba CARMIL interacts tightly with capping protein. In biochemical preparations, CARMIL copurified extensively with two polypeptides that were shown by microsequencing to be the ␣-and -subunits of Acanthamoeba capping protein. The complex between CARMIL and capping protein, which is readily demonstratable by chemical cross-linking, can be completely dissociated by size exclusion chromatography at pH 5.4. Analytical ultracentrifugation, surface plasmon resonance and SH3 domain pull-down assays indicate that the dissociation constant of capping protein for CARMIL is ϳ0.4 M or lower. Using CARMIL fusion proteins, the binding site for capping protein was shown to reside within the carboxyl-terminal, ϳ200 residue, proline-rich domain of CARMIL. Finally, chemical cross-linking, analytical ultracentrifugation, and rotary shadowed electron microscopy revealed that CARMIL is asymmetric and that it exists in a monomer 7 dimer equilibrium with an association constant of 1.0 ؋ 10 6 M ؊1 . Together, these results indicate that CARMIL selfassociates and interacts with capping protein with affinities that, given the cellular concentrations of the proteins (ϳ1 and 2 M for capping protein and CARMIL, respectively), indicate that both activities should be physiologically relevant.In 1995, Zot (1) and colleagues identified a ϳ125-kDa protein from Acanthamoeba on the basis of its ability to bind to the isolated Src homology 3 (SH3) 1 domain of Acanthamoeba myosin IC (1). This protein, which they called Acan 125, coimmunoprecipitated with myosin IC and appeared to colocalize with the myosin in cellular surface projections involved in pinocytosis. The subsequent cloning of the Acanthamoeba gene for Acan 125 (2) revealed a multidomain protein dominated by a central, ϳ460-residue leucine-rich repeat (LRR) domain. LRRs are ϳ29-residue sequences that contain a loose consensus dominated by leucines, form amphipathic ␣--structural units and mediate protein-protein interactions, either by serving as the ligand binding sites themselves or by increasing the affinity and/or specificity of binding at a separate site (3). The second most striking structural feature of Acan 125 is its ϳ200-residue, proline-rich COOH-terminal domain. Consistent with the fact that SH3 domains mediate protein-protein interactions by binding to proline-rich target sequences containing the core element PXXP (4), Xu et al. (2) showed that a fusion protein containing the COOH-terminal 344 residues of Acan 125 bound to the isolated SH3 domain of myosin IC and that this interaction was abrogated by an 18-residue deletion spanning two PXXP motifs fitting the consensus for SH3 domain tar...
The peripheral accumulation of melanosomes characteristic of wild-type mouse melanocytes is driven by a cooperative process involving long-range, bidirectional, microtubule-dependent movements coupled to capture and local movement in the actin-rich periphery by myosin Va, the product of the dilute locus. Genetic evidence suggests that Rab27a, the product of the ashen locus, functions with myosin Va in this process. Here we show that ashen melanocytes, like dilute melanocytes, exhibit normal dendritic morphology and melanosome biogenesis, an abnormal accumulation of end-stage melanosomes in the cell center, and rapid, bidirectional, microtubule-dependent melanosome movements between the cell center and the periphery. This phenotype suggests that ashen melanocytes, like dilute melanocytes, are defective in peripheral melanosome capture. Consistent with this, introduction into ashen melanocytes of cDNAs encoding wild-type and GTP-bound versions of Rab27a restores the peripheral accumulation of melanosomes in a microtubule-dependent manner. Conversely, introduction into wild-type melanocytes of the GDP-bound version of Rab27a generates an ashen/dilute phenotype. Rab27a colocalizes with end-stage melanosomes in wild-type cells, and is most concentrated in melanosome-rich dendritic tips, where it also colocalizes with myosin Va. Finally, neither endogenous myosin Va nor an expressed, GFP-tagged, myosin Va tail domain fusion protein colocalize with melanosomes in ashen melanocytes, in contrast to that seen previously in wild-type cells. These results argue that Rab27a serves to enable the myosinVa-dependent capture of melanosomes delivered to the periphery by bidirectional, microtubule-dependent transport, and that it does so by recruiting the myosin to the melanosome surface. We suggest that Rab27a, in its GTP-bound and melanosome-associated form, predominates in the periphery, and that it is this form that recruits the myosin, enabling capture. These results argue that Rab27a serves as a myosin Va ‘receptor’, and add to the growing evidence that Rab GTPases regulate vesicle motors as well as SNARE pairing.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.