Multiple overlapping SNARE complexes drive endosome maturation in Drosophila nephrocytes

Abstract

Endosomal maturation determines whether internalized cargo is recycled or degraded, yet the molecular logic governing early endosomal fusion remains poorly defined. This process is often depicted as a linear Rab5-to-Rab7 transition mediated by a single, ordered SNARE pathway, but extensive redundancy in mammalian systems has obscured pathway architecture. Here, using Drosophila nephrocytes as a genetically tractable in vivo model with minimal SNARE redundancy, we show that early endosome maturation is driven by multiple parallel, non-interchangeable SNARE-dependent pathways. We first resolve a long-standing discrepancy in Syntaxin 7 family orthology, demonstrating that the Drosophila protein previously termed Syx7/Avl is functionally analogous to mammalian STX12 rather than STX7, while late endosomal and lysosomal fusion is mediated by a distinct Syntaxin 7 homolog (Syx13). Based on this reclassification, we define a Syx12L-Snap29-Ykt6 complex that drives canonical homotypic early endosomal fusion. In addition, we identify two related SNARE assemblies - Syx7L-Snap29-Ykt6 and Syx7L-Snap29-Vamp7-that promote later stages of endosomal and lysosomal fusion with distinct Rab GTPase requirements. These partially compensatory complexes remain active when the canonical pathway is disrupted, producing divergent morphological outcomes, including the formation of aberrant endolysosomal swirls. We establish Snap29 as a central Qbc-SNARE integrating all endosomal fusion routes and uncover a dual role for Ykt6 in promoting maturation while also participating in endosomal recycling. Together, our findings revise the prevailing model of endosome maturation, revealing a network of parallel, regulated fusion pathways that confer robustness and plasticity to the endolysosomal system.