PLEKHM1 directly binds to Arl8b via its N-terminal RUN domain–containing region. (a) Domain architecture of PLEKHM1 and SKIP/PLEKHM2. (b) Yeast two-hybrid assay. Cotransformants were spotted on -Leu-Trp and -Leu-Trp-His media to confirm viability and interactions, respectively. (c) FLAG-PLEKHM1 was cotransfected with different forms of Arl8b-HA into HEK293T cells; lysates were immunoprecipitated (IP) with anti–HA antibody resin, and the precipitates were immunoblotted (IB) with the indicated antibodies. (d and e) GST and GST-PLEKHM1 (1–300) proteins were immobilized on glutathione (GSH) resin and incubated either with His-Arl8b in the presence of GTPγS or GDPβS or with HEK293T cell lysates expressing either Arl8b WT-HA or Arl8b T34N-HA. The precipitates were immunoblotted with anti–His (d) or anti–HA (e) antibodies. Ponceau S stain was done to visualize purified protein. LIR, LC3/GABARAP interaction; PH, pleckstrin homology; WD/WE, tryptophan-acidic.