Dual linker UNC-10/SYD-2 is sufficient to bind kinesin-3 UNC-104 to RAB-3 containing synaptic vesicles in the absence of the motor’s PH domain

Kinesin-3 KIF1A (UNC-104 in C. elegans) is the major axonal transporter of synaptic vesicles and mutations in this molecular motor are linked to KIF1A-associated neurological disorders (KAND) including Charcot-Marie-Tooth disease, amyotrophic lateral sclerosis and hereditary spastic paraplegia. UNC-104 binds via its PH (pleckstrin homology) domain to the lipid bilayers of membranous vesicles which is considered a weak interaction. RT-PCR and Western blot experiments reveal genetic relations between SYD-2, UNC-10 and RAB-3. Co-immunoprecipitation assays reveal functional relations and bimolecular fluorescence complementation (BiFC) assays expose in situ interactions between these proteins. 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 SYD-2/UNC-10. Deletion of UNC-104s PH domain did not affect UNC-104/RAB-3 colocalization but did affect UNC-104/SNB-1 colocalization. Similarly, motility of RAB-3-labeled vesicles is unaltered in nematodes carrying a point mutation in the PH domain while movement of SNB-1 is significantly reduced in anterograde directions. These findings suggest a dual UNC-10/SYD-2 linker acting as a sufficient buttress to connect the motor to RAB-3-containing vesicles to enhance their transport. This additional linker will also strengthen the rather weak motor-lipid interaction. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/723247v4_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@5e41c0org.highwire.dtl.DTLVardef@2ed9c8org.highwire.dtl.DTLVardef@1dbdb9dorg.highwire.dtl.DTLVardef@12f30c2_HPS_FORMAT_FIGEXP M_FIG C_FIG