HOPS-dependent vesicle tethering lock inhibits endolysosomal fusions and autophagosome secretion upon the loss of Syntaxin17

The autophagosomal SNARE (soluble <i>N</i>-ethylmaleimide-sensitive factor attachment protein) Syntaxin17 (Syx17) plays a pivotal role in autophagosome-lysosome fusion, yet the broader impact of its loss remains elusive. Our investigation of Syx17 function in <i>Drosophila</i> nephrocytes and salivary gland cells revealed unexpected effects. We find that Syx17 loss induces the formation of autophagosome-lysosome clusters in a HOPS (homotypic fusion and vacuole protein sorting)-dependent manner, entrapping this tether, autophagosomes, and lysosomes. While locked in clusters, these organelles cannot participate in other vesicle fusions, impeding endosomal progression and autophagosome secretion. Therefore, the absence of Syx17 not only inhibits autophagosome-lysosome fusion but also prevents HOPS release from autophagosome-lysosome tethering sites causing a "tethering lock." Preventing autophagosome formation or removing the HOPS adaptor Plekhm1 (pleckstrin homology domain-containing family M member 1) leads to release of HOPS and lysosomes from these clusters, thus rescuing secondary effects of Syx17 loss. Our findings show that a tethering lock can disrupt multiple vesicle trafficking routes.