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

Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 03/2012; 109(15):5862-7. DOI: 10.1073/pnas.1113819109
Source: PubMed


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.

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    • "During neuronal development, Munc18-1/syntaxin-1 and syn- taxin-1 are transported by FEZ1–KIF5C (Chua et al., 2012) and syntabulin–KIF5B (Su et al., 2004; Cai et al., 2007) transport complexes. Our FRAP experiments in axons (Fig. 3) showed identical recovery of Munc18-1-Venus and Stx-YFP and increased Munc18-1-Venus recovery after cleavage of syntaxin-1, which indicates that lateral diffusion via membrane-bound syn- taxin-1 rather than active transport of organelles is the main mode of delivering Munc18-1 to release sites in mature neurons . "
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    ABSTRACT: Munc18-1 is a soluble protein essential for synaptic transmission. To investigate the dynamics of endogenous Munc18-1 in neurons, we created a mouse model expressing fluorescently tagged Munc18-1 from the endogenous munc18-1 locus. We show using fluorescence recovery after photobleaching in hippocampal neurons that the majority of Munc18-1 trafficked through axons and targeted to synapses via lateral diffusion together with syntaxin-1. Munc18-1 was strongly expressed at presynaptic terminals, with individual synapses showing a large variation in expression. Axon-synapse exchange rates of Munc18-1 were high: during stimulation, Munc18-1 rapidly dispersed from synapses and reclustered within minutes. Munc18-1 reclustering was independent of syntaxin-1, but required calcium influx and protein kinase C (PKC) activity. Importantly, a PKC-insensitive Munc18-1 mutant did not recluster. We show that synaptic Munc18-1 levels correlate with synaptic strength, and that synapses that recruit more Munc18-1 after stimulation have a larger releasable vesicle pool. Hence, PKC-dependent dynamic control of Munc18-1 levels enables individual synapses to tune their output during periods of activity.
    The Journal of Cell Biology 03/2014; 204(5):759-75. DOI:10.1083/jcb.201308026 · 9.83 Impact Factor
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    • "FEZ1 acts as an adaptor in kinesin-1 mediated axonal transport to nerve terminals by binding to both the heavy chain of the motor protein kinesin-1 [7,8] and its cargo, for example as recently shown for Syntaxin 1a and Munc18 containing transport vesicles [9]. Phosphorylation of FEZ1 regulates cargo [10] and kinesin binding [9]. "
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    ABSTRACT: The short coiled coil protein (SCOC) forms a complex with fasciculation and elongation protein zeta 1 (FEZ1). This complex is involved in autophagy regulation. We determined the crystal structure of the coiled coil domain of human SCOC at 2.7 Å resolution. SCOC forms a parallel left handed coiled coil dimer. We observed two distinct dimers in the crystal structure, which shows that SCOC is conformationally flexible. This plasticity is due to the high incidence of polar and charged residues at the core a/d-heptad positions. We prepared two double mutants, where these core residues were mutated to either leucines or valines (E93V/K97L and N125L/N132V). These mutations led to a dramatic increase in stability and change of oligomerisation state. The oligomerisation state of the mutants was characterized by multi-angle laser light scattering and native mass spectrometry measurements. The E93V/K97 mutant forms a trimer and the N125L/N132V mutant is a tetramer. We further demonstrate that SCOC forms a stable homogeneous complex with the coiled coil domain of FEZ1. SCOC dimerization and the SCOC surface residue R117 are important for this interaction.
    PLoS ONE 10/2013; 8(10):e76355. DOI:10.1371/journal.pone.0076355 · 3.23 Impact Factor
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    • "As an adapter molecule, the protein FEZ1 recruits both effector and regulatory elements to the processes of formation and transport of vesicles. In fact, in drosophila, UNC-76 protein is involved in kinesin-1 based transport of synaptic precursor vesicle, interacting with synaptotagmin when UNC-76 is phosphorylated by UNC-51 [16], as so in humans, interacting with syntaxin 1 [41]. Futhermore, FEZ1-SCOCO complex is involved in axon outgrowth and vesicle transport [10] and this complex is a likely target for ULK1 (which interacts directly with FEZ1 [14]) regulating the autophagy process [40]. "
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    ABSTRACT: Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in C. elegans), SCOCO (short coiled-coil protein / UNC-69) and kinesins (e.g. kinesin heavy chain / UNC116) are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth), we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance), cross-linking coupled with mass spectrometry (MS), SAXS (Small Angle X-ray Scattering) and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance) studies of the region involved in this process, corresponding to FEZ1 (92-194). Through studies involving the protein in its monomeric configuration (reduced) and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth.
    PLoS ONE 10/2013; 8(10):e76602. DOI:10.1371/journal.pone.0076602 · 3.23 Impact Factor
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