Abstract
Myosin V is a molecular motor that transports a variety of cellular cargo, including organelles, vesicles, and messenger RNA. The proper peripheral distribution of melanosomes, a dense pigment-containing organelle, is dependent on actin and the activity of myosin Va. The recruitment of myosin Va to the melanosome and proper transport of the melanosome requires melanophilin, which directly binds to myosin Va and is tethered to the melanosome membrane via Rab27a. Here we use highly purified proteins to demonstrate that the globular tail domain of myosin Va binds directly to an intrinsically unstructured domain of melanophilin. The myosin Va binding domain of melanophilin lacks stable secondary structure, and (1)H NMR measurements indicate that the protein is unfolded. This domain is extremely sensitive to mild proteolysis and has a hydrodynamic radius that is consistent with a random coil-like polypeptide. We show that myosin Va binding does not induce the global folding of melanophilin. Truncations of melanophilin were utilized to define a short peptide sequence (26 residues) within melanophilin that is critical for myosin Va binding. We demonstrate that a peptide corresponding to these residues binds directly to the globular tail domain with the same affinity as melanophilin. We discuss the possible implications of protein intrinsic disorder in recruitment and maintenance of myosin Va on melanosome membranes.
Highlights
Mutations in Rab27a and melanophilin cause a similar melanosome transport defect, suggesting that these two proteins function in the same pathway
Melanophilin does not activate myosin Va as well as the addition of Ca2ϩ (Vmax ϭ 6.6 versus 20 sϪ1 headϪ1, respectively). It is not clear whether melanophilin and Ca2ϩ activate the myosin Va motor by the same mechanism, but melanosome transport and activation of the myosin Va motor require the direct interaction of the globular tail domain and melanophilin
We found that purified melanophilin is very sensitive to proteolysis
Summary
Mutations in Rab27a and melanophilin (ashen and leaden, respectively) cause a similar melanosome transport defect, suggesting that these two proteins function in the same pathway. Hume et al [12] showed that the exon F binding site of melanophilin is required to rescue the melanosome transport defect in leaden melanocytes. In the same study the authors found that the globular tail domain binding site is not required for melanosome transport and membrane localization of myosin Va but may function to stabilize the complex in vivo. Melanophilin does not activate myosin Va as well as the addition of Ca2ϩ (Vmax ϭ 6.6 versus 20 sϪ1 headϪ1, respectively) It is not clear whether melanophilin and Ca2ϩ activate the myosin Va motor by the same mechanism, but melanosome transport and activation of the myosin Va motor require the direct interaction of the globular tail domain and melanophilin. Most cellular proteins must fold into a single well defined structure to fulfill their biological function, modern genomic and proteomic approaches have led to the VOLUME 282 NUMBER 29 JULY 20, 2007
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