Translation in Proximity to Forming Autophagosomes During Sustained Autophagy
Autophagy is an evolutionarily conserved catabolic process. In a process requiring a cascade of over 35 autophagy-related genes (Atg), a cupped phagophore membrane expands to surround cytoplasmic material, and seals itself to form an autophagosome, which finally fuses with lysosomes. Large numbers of autophagosomes form during stress responses, while simultaneously cells drastically reduce translation to conserve energy. Here, using proximity-labeling and Fluorescence in situ Hybridization we demonstrate that multiple mRNAs encoding proteins required for autophagy preferentially localize in proximity to forming autophagosomes. Polysome fractionation and proteomics of nascent proteins in proximity to forming autophagosomes provides evidence for the local translation of these mRNAs. Translation and the ribosome-binding protein RACK1 were required for the localization of these mRNAs to forming autophagosomes. Inhibition of translation or knockdown of RACK1 caused depletion of several proteins required for autophagy and a reduction in the number of autophagosomes. Local translation may enable a rapid, energy-efficient supply of proteins for autophagy to enable cells to massively induce autophagy while conserving energy during cell stress. Overall design: Human cells expressing APEX2 fused to defined subcellular markers were used to identify RNAs proximal to forming autophagosomes. Samples include APEX2-DFCP1 (phagophore), APEX2-LC3B (autophagosome), and control APEX2 constructs (diffuse cytosolic V5 tag or Perilipin). Autophagy was induced by mTOR inhibition (Torin1). Untreated cells serve as non-autophagy controls. Proximity labeling was activated with biotin-phenol and H2O2, with -H2O2 samples as labeling controls. Biotinylated RNAs were affinity-purified and sequenced. Primary comparisons are DFCP1-APEX2 or LC3B-APEX2 (+H2O2) versus matched  -H2O2 controls to define proximal RNAs, and DFCP1-APEX2 versus cytosolic or Perilipin APEX2 to identify phagophore-specific enrichment.