MYO5A-mediated stabilization promotes the acquisition of fusion competence in sealed autophagosomes.

Autophagy requires precise regulation of autophagosome-lysosome fusion, yet the molecular details of this process remain incompletely understood. Here, we identify the class V myosin MYO5A as a critical regulator of autophagic flux. The genetic or pharmacological inhibition of MYO5A in Saccharomyces cerevisiae, mammalian cells, or Caenorhabditis elegans blocked autophagic flux by preventing autophagosome-lysosome fusion. MYO5A facilitates the maturation of autophagosomes into fusion-competent intermediates as its loss altered the localization of fusion machinery on autophagosomes and reduced the pool of stationary autophagosomes, a step that proved critical for subsequent fusion with lysosomes. Domain mapping and targeted mutagenesis revealed that two LIR motifs (PAYRVL and QAYIGL) within the coiled-coil and globular tail domains of MYO5A mediate its direct interaction with LC3 on autophagosomes. Live imaging in mammalian cells and C. elegans added support for this role, revealing how MYO5A regulates autophagic flux to ensure fusion. Together, these findings establish MYO5A as a regulator of autophagy and highlight its potential as a target for fine-tuning autophagic flux.