Myosin5a Tail Associates Directly with Rab3A-containing Compartments in Neurons

Torsten Wöllert,Anamika Patel,Ying-Lung Lee,D William Provance,Valarie E Vought,Michael S Cosgrove,John A Mercer,George M Langford

doi:10.1074/jbc.m110.187286

Abstract

Myosin-Va (Myo5a) is a motor protein associated with synaptic vesicles (SVs) but the mechanism by which it interacts has not yet been identified. A potential class of binding partners are Rab GTPases and Rab3A is known to associate with SVs and is involved in SV trafficking. We performed experiments to determine whether Rab3A interacts with Myo5a and whether it is required for transport of neuronal vesicles. In vitro motility assays performed with axoplasm from the squid giant axon showed a requirement for a Rab GTPase in Myo5a-dependent vesicle transport. Furthermore, mouse recombinant Myo5a tail revealed that it associated with Rab3A in rat brain synaptosomal preparations in vitro and the association was confirmed by immunofluorescence imaging of primary neurons isolated from the frontal cortex of mouse brains. Synaptosomal Rab3A was retained on recombinant GST-tagged Myo5a tail affinity columns in a GTP-dependent manner. Finally, the direct interaction of Myo5a and Rab3A was determined by sedimentation velocity analytical ultracentrifugation using recombinant mouse Myo5a tail and human Rab3A. When both proteins were incubated in the presence of 1 mm GTPγS, Myo5a tail and Rab3A formed a complex and a direct interaction was observed. Further analysis revealed that GTP-bound Rab3A interacts with both the monomeric and dimeric species of the Myo5a tail. However, the interaction between Myo5a tail and nucleotide-free Rab3A did not occur. Thus, our results show that Myo5a and Rab3A are direct binding partners and interact on SVs and that the Myo5a/Rab3A complex is involved in transport of neuronal vesicles.

Highlights

Previous studies have established that Rab3A, a highly abundant small GTPase on brain SVs in the presynaptic nerve terminal, controls the Ca2ϩ-mediated release of neurotransmitter [47,48]
It is not well understood whether and how SVs are actively transported between different compartments in the presynaptic nerve terminal
In this study we confirmed that Myo5a tail associates with SVs in the rat brain synaptosomal fraction and we showed for the first time that the globular tail domain of recombinant Myo5a forms a complex with Rab3A in a GTP-dependent manner

Summary

EXPERIMENTAL PROCEDURES

Antibodies—The following commercial primary antibodies were used in this study: rabbit polyclonal anti-myosin5a (DIL2, 36), anti-Rab3A and anti-Rabphilin3A (both from StressGen), anti-Rab4A and anti-Rab (both from Santa Cruz Biotechnology), anti-GST (GE Healthcare), anti-synapsin 1 (Invitrogen), and mouse monoclonal antibodies directed against Rab3A (Synaptic Systems), PSD95 (ABR), actin (Sigma-Aldrich) and kinesin (Chemicon). Supernates (S3) were spun again at 30,000 ϫ g for 20 min to obtain a crude fraction and the pellet (P4) was resuspended in solution B (0.32 M sucrose, 1 mM NaHCO3) This homogenate was layered on top of a three-step sucrose gradient (0.85 M, 1.0 M, 1.2 M) and centrifuged at 82,500 ϫ g for 2 h. Synaptosomal fractions prepared from rat and null Myo5a mutant mouse brains were treated with 0.2 mg/ml complete protease inhibitor mixture and 1% Triton X-100, filtered through a syringe filter unit (Millipore) and applied to the GST-Myo5a tail affinity column. Triton X-100 treated synaptosomes were incubated in the presence of GDP, GTP, and GTP␥S and protein kinase/phosphatase inhibitors for 1 h at room temperature and were applied to GSTMyo5a tail or GST-RabGDI affinity columns. The resulting individual, normalized signal intensities were averaged for each experiment and analyzed by a two-tailed t test

RESULTS

Theoretical mass kDa

DISCUSSION