Phosphorylation-regulated axonal dependent transport of syntaxin 1 is mediated by a Kinesin-1 adapter

Abstract

Presynaptic nerve terminals are formed from preassembled vesicles that are delivered to the prospective synapse by kinesin-mediated axonal transport. However, precisely how the various cargoes are linked to the motor proteins remains unclear. Here, we report a transport complex linking syntaxin 1a (Stx) and Munc18, two proteins functioning in synaptic vesicle exocytosis at the presynaptic plasma membrane, to the motor protein Kinesin-1 via the kinesin adaptor FEZ1. Mutation of the FEZ1 ortholog UNC-76 in Caenorhabditis elegans causes defects in the axonal transport of Stx. We also show that binding of FEZ1 to Kinesin-1 and Munc18 is regulated by phosphorylation, with a conserved site (serine 58) being essential for binding. When expressed in C. elegans, wild-type but not phosphorylation-deficient FEZ1 (S58A) restored axonal transport of Stx. We conclude that FEZ1 operates as a kinesin adaptor for the transport of Stx, with cargo loading and unloading being regulated by protein kinases.

Fig. 1.

FEZ1 interacts with Munc18 and Stx. (A) Identification of FEZ1 as an interactor of Munc18 and Stx using a Y2H assay. Bait constructs containing fragments of Munc18 and Stx (without its transmembrane domain) were used to screen preys containing different FEZ1 fragments [amino acid residues (aa) given in parentheses]. (B and C) Validation of FEZ1 interactions by coimmunoprecipitation. HEK 293 cells expressing tagged FEZ1 with either Stx or Munc18 (black circles on top of the lanes) were immunoprecipitated (IP) using tag-specific antibodies (anti-GFP) and analyzed by immunoblotting (IB) for the presence of GFP-FEZ1, Myc-Stx, and FLAG-Munc18, respectively. FEZ1 interacts with both Munc18 and Stx. Input corresponds to 1% of the starting material used for immunoprecipitation. Molecular mass markers indicated are in kilodaltons.

Fig. 2.

Protein complexes formed by FEZ1 reveal its function as cargo adaptor for Kinesin-1 (KIF5C). (A and B) HEK 293 cells expressing combinations of tagged versions of FEZ1, Munc18, KIF5C, or Stx were immunoprecipitated (IP) using anti-GFP antibodies and immunoblotted (IB) using tag-specific antibodies (anti-FLAG, anti-GFP, anti-Myc, or anti-V5). Molecular mass markers are indicated are in kilodaltons. *Ig heavy chain. (A) FEZ1 forms a trimeric complex with Munc18 and Stx. Full-length and FEZ1 (amino acids 1–220) efficiently immunoprecipitate Stx and Munc18 indicating that direct binding of Munc18 to FEZ1 is not necessary for trimeric complex formation. (B) KIF5C, FEZ1, Stx, and Munc18 can be concurrently isolated as a complex. In addition, binding of Stx to KIF5C can occur without Munc18 but is dependent on the presence of the coiled-coil domain of FEZ1. (C) The FEZ1/Kinesin-1 transport complex comprising FEZ1, Stx, Munc18, and Kinesin-1 can be immunoisolated from rat brain postnuclear supernatants using anti-FEZ1 or anti-Kinesin-1 antibodies.

Fig. 3.

FEZ1 colocalizes with Stx, Munc18, and α-tubulin in neuronal growth cones. Two to three DIV neurons were fixed and stained for endogenous FEZ1, Stx, Munc18, or α-tubulin. (A) Numerous FEZ1 puncta are observed in neuronal growth cones that are strongly microtubule-associated (e.g., arrowheads). (B) Stx and Munc18 colocalizes in growth cones as expected. FEZ1 colocalizes with Stx (D) and Munc18 (E) in growth cones. Line scans of regions of interest indicated are shown in C and F. (Scale bars, 10 μm.) Correlation coefficients for each colocalization pair are 0.88 ± 0.02 (Munc18+Stx), 0.802 ± 0.02 (FEZ1+Stx), and 0.75 ± 0.02 (FEZ1+Munc18), respectively. Eight growth cones were taken for each set of analysis. Images of the entire growth cones are shown in Fig. S4.

Fig. 4.

Mutation of FEZ1 (unc-76) affects trafficking of syntaxin (UNC-64) in ventral nerve cords of C. elegans. (A) Axonal distribution of GFP-tagged UNC-18 and UNC-64 in ventral nerve cords (VNC) in wild-type and mutant C. elegans. (a–c) Distribution of GFP-UNC-64 differs between wild-type (a), unc-76 (e911) mutants (b), and unc-116 (e2310) mutants (c). (c) Unc-116 mutants exhibit greater axonal clustering of GFP-UNC-64 than unc-76 mutants (b). Expression of mCherry-UNC-76 rescues GFP-UNC-64 clustering in unc-76 mutants (d). Unc-76;unc-116 double mutants exhibit both axonal clustering and significantly more GFP-UNC-64 accumulation in cell bodies (e). Expression patterns of GFP-UNC-18 in wild-type (f), unc-76 (g), and unc-116 (h) mutants show no significant differences. (Scale bar, 10 μm.) Diagram in i shows the schematic organization of C. elegans ventral nerve chord (VNC) as a continuous row of neuronal cell bodies (somata) with axonal bundles running adjacent to the ventral hypodermis. En passant synapses between motor neurons and muscle arms are regularly spaced along the entire length of the VNC. (B) Quantification of GFP-UNC-64 and GFP-UNC-18 clustering using intensity variations along the nerve (line scan analyses). SDs of pixel intensities obtained from the line scans were used to compute an index to compare the extent of clustering (SI Text). Eight to nine worms were taken for each analysis. Error bars represent SEM. (C) Abnormal membranous structures and autophagosomes (arrows) were found within neurites of the ventral cord neurons in unc-116 and unc-76 mutants but not in wild-type animals. (Scale bar, 200 nm.)

Fig. 5.

Phosphorylation of FEZ1 regulates binding to its interaction partners. HEK 293 cell lysates expressing combinations of tagged versions of FEZ1, Munc18, KIF5C, or Stx treated with or without AP were immunoprecipitated using tag-specific antibody (anti-GFP) and immunoblotted using tag-specific antibodies (anti-FLAG, anti-GFP, anti-Myc, or anti-V5). The amount of immunoprecipitated protein was determined by densitometry, with the untreated sample serving as reference (100%). A representative Western blot is shown in A. Data in A–C were obtained from three independent experiments. Error bars represent SDs. (A) Treatment of cell lysates with AP significantly reduces Munc18 binding to FEZ1. (B) Phosphorylation regulates assembly of the trimeric FEZ1-Munc18-Kinesin-1 complex. Dephosphorylation of FEZ1 reduced its binding to KIF5C (left chart) and dissociates the ternary complex of FEZ1, Munc18, and KIF5C (right chart). (C) Dephosphorylation reduces the amount of KIF5C bound to FEZ1 but significant amounts of Stx remain bound to FEZ1. (D) Significant amounts of FEZ1-Stx-Munc18 complexes remain isolatable even after AP treatment. HEK 293 cell lysates expressing tagged versions of Munc18, Stx, and FEZ1 treated with or without AP were immunoprecipitated with tag-specific (anti-FLAG) antibody and immunoblotted using tag-specific antibodies (anti-FLAG, anti-Myc, or anti-GFP). Data were obtained from the average of two independent experiments.

Fig. 6.

S58A is essential for phosphorylation-dependent transport of Stx (UNC-64) in axons. Localization of GFP-UNC-64 in VNC of unc-76(e911) mutants coexpressing mCherry-FEZ1 (wild-type) or mCherry-FEZ1(S58A). Irregular distribution of GFP-UNC-64 in unc-76 mutants was rescued by expression of wild-type FEZ1, whereas expression of FEZ1(S58A) aggravated the clustering phenotype of GFP-UNC-64 in these worms. Quantifications of GFP-UNC-64 clustering for both transgenic worms are shown (Right). Eight worms were taken for each analysis. Error bars represent SEM. (Scale bar, 10 μm.)