Comobility of GABARAP and Phosphatidylinositol 4‑Kinase 2A on Cytoplasmic Vesicles

We previously reported that recruitment of the type IIA phosphatidylinositol 4-kinase (PI4K2A) to autophagosomes by GABARAP, a member of the Atg8 family of autophagy-related proteins, is important for autophagosome–lysosome fusion. Because both PI4K2A and GABARAP have also been implicated in the intracellular trafficking of plasma membrane receptors in the secretory/endocytic pathway, we characterized their interaction in cells under nonautophagic conditions. Fluorescence fluctuation spectroscopy measurements revealed that GABARAP exists predominantly as a cytosolic monomer in live cells, but is recruited to small cytoplasmic vesicles upon overexpression of PI4K2A. C-Terminal lipidation of GABARAP, which is essential for its autophagic activities, is not necessary for its recruitment to these PI4K2A-containing transport vesicles. However, a GABARAP truncation mutant lacking C-terminal residues 103–117 fails to bind to PI4K2A, is not recruited to cytoplasmic vesicles, and does not codistribute with PI4K2A on subcellular organelles. These observations suggest that the PI4K2A–GABARAP interaction plays a role in membrane trafficking both under autophagic and nonautophagic conditions.