HRD1 negatively regulates autolysosome formation by inhibiting liquid-liquid phase separation of SNAP29

Autophagy is a highly conserved cellular process in which cytoplasmic contents are sequestrated by autophagosomes and delivered to lysosomes for degradation. Generation of degradative autolysosomes mediated by SNARE proteins is essential; however, the regulatory mechanisms governing this process remain underexplored. This study aimed to demonstrate that E3 ubiquitin ligase HRD1 regulates liquid-liquid phase separation (LLPS) of SNAP29, thereby modulating SNARE assembly. We found that HRD1 deficiency enhances autophagy activity and promotes autolysosome formation in a SNAP29-dependent manner. We also determined that SNAP29 forms highly dynamic condensates in in vivo and in vitro, which are crucial for the assembly of the SNARE complex. Mechanistically, HRD1 interacts with SNAP29 to suppress its condensation, whereas HRD1 depletion accelerates both SNAP29 condensate formation and SNARE complex assembly. Our findings reveal that HRD1 acts as a negative regulator in autolysosome formation by interacting with SNAP29, inhibiting its LLPS process, thereby modulating the binding affinity among SNARE components.