UNC-10/SYD-2 complex is sufficient to link kinesin-3 to RAB-3 containing synaptic vesicles in the absence of the motor’s PH domain

Abstract

ABSTRACTKinesin-3 KIF1A (UNC-104 in C. elegans) is the major fast axonal transporter of STVs (synaptic vesicle protein transport vesicles) containing synaptic precursors such as RAB3A (RAB-3) or VAMP2 (SNB-1). Heritable mutations in neuronal motor proteins (and their adaptors) lead to numerous neurodegenerative diseases. The C-terminal PH (pleckstrin homolog) domain of kinesin-3 UNC-104 directly binds to phosphatidylinositol 4,5-bisphosphates that form the lipid bilayers of STVs. We hypothesized that RAB-3-bound STVs employ a dual linker UNC-10/SYD-2 (RIMS1/liprin-α in mammals) acting as a UNC-104 receptor. This tripartite RAB-3/UNC-10/SYD-2 complex would also act as an additional linker to strengthen the motor-lipid interaction. RT-PCR and Western blot experiments favor a genetic relation between SYD-2, UNC-10 and RAB-3. Co-immunoprecipitation assays revealed changes in binding affinities between SYD-2 and UNC-104 depending on the presence or absence of UNC-10 and RAB-3. Bimolecular fluorescence complementation (BiFC) assays revealed in situ interaction changes between SYD-2 and UNC-104 in either unc-10 or rab-3 mutants. Neuronal expression of UNC-104 appears to be more diffused and is restricted to travel short distances with significantly reduced speeds in these mutants. Though both SNB-1 and RAB-3 are actively transported by UNC-104, the movement of RAB-3 is generally enhanced and largely depending on the presence of the dual linker. Strikingly, the deletion of UNC-104’s PH domain did not affect UNC-104/RAB-3 colocalization but did affect UNC-104/SNB-1 colocalization. These findings solidly support the model of a dual UNC-10/SYD-2 linker acting as a sufficient buttress to connect the motor to RAB-3-containing STVs to enhance their transport.SCIENTIFIC STATEMENTThe interaction between molecular motors and their membranous vesicular cargoes is generally specific. However, for the major axonal transporter kinesin-3 UNC-104, only its weak and non-specific interaction via phosphatidylinositol 4,5-bisphosphates (forming the lipid bilayers of synaptic vesicles) has been characterized. Here, we present a novel, more specific way for UNC-104 to interact with synaptic vesicles - specifically with RAB-3 bound vesicles - via the dual linker complex UNC-10/SYD-2. Because many neurological diseases are linked to defects in axonal trafficking (often with protein accumulation phenotypes in neurons), understanding the molecular basis of motor/vesicle interaction might lead to the design of new drugs that may cure or prevent such diseases.