The active zone protein CLA-1 (Clarinet) bridges two subsynaptic domains to regulate presynaptic sorting of ATG-9

Abstract

In neuronal synapses, autophagosome biogenesis is coupled with the activity-dependent synaptic vesicle cycle via ATG-9. How vesicles containing ATG-9 are sorted at the presynapse is unknown. We performed forward genetic screens at single synapses of C. elegans neurons for mutants that disrupt ATG-9 presynaptic localization, and identified the long isoform of the active zone protein CLA-1 (Clarinet; CLA-1 L). We find that disrupting CLA-1 L results in abnormal accumulation of ATG-9-containing vesicles enriched with clathrin. The adaptor protein complexes and proteins at the periactive zone genetically interact with CLA-1 L in ATG-9 sorting. Moreover, the phenotype of the ATG-9 protein in cla-1(L) mutants was not observed for integral synaptic vesicle proteins, suggesting distinct mechanisms that regulate sorting of ATG-9-containing vesicles and synaptic vesicles. Our findings reveal novel roles for active zone proteins in the sorting of ATG-9 and in presynaptic macroautophagy/autophagy.

Keywords: ATG-9; CLA-1; active zone; adaptor protein complexes; periactive zone; synaptic vesicle cycle; syndapin 1.

Figure 1.

Schematic diagrams of presynaptic sorting of ATG-9 and genetic regulations. (A) Schematic of the presynaptic compartment. The active zone and the adjacent periactive zone cooperate in regulating the sorting of synaptic vesicles and ATG-9 containing vesicles. The large active zone protein CLA-1 (long isoform; CLA-1 L) is anchored, via its C terminus, to the active zone, whereas its N terminus interacts with periactive zone proteins EHS-1/EPS15 and ITSN-1/intersectin 1 and regulates presynaptic events such as ATG-9 sorting. (B) Genetic model of presynaptic sorting of ATG-9. We hypothesize based on our data that the adaptor complex AP-1 and SDPN-1/syndapin 1 mediate trafficking of ATG-9 to a transient sorting station from which AP2-AP180 complexes facilitate clathrin-mediated ATG-9 vesicle budding. CLA-1 L, together with presynaptic endocytic proteins that reside in the periactive zone, facilitates the AP2-AP180-mediated ATG-9 vesicle budding. Mutations in the active zone gene cla-1 L result in abnormal accumulation of ATG-9 into endocytic intermediates, and defects in activity-dependent autophagosome formation. The italicized words adjacent to arrows refer to the genes encoding proteins that are necessary for the processes indicated by the arrows.