Conserved basic residues within the RUN domain of PLEKHM1 are important for its interaction with Arl8b. (a) Yeast two-hybrid assay. Cotransformants were spotted on -Leu-Trp and -Leu-Trp-His media to confirm viability and interactions, respectively. AD, GAL4 activation domain; BD, GAL4-DNA binding domain. (b) Dot-blot assay: GST alone or GST-PLEKHM1 (1–300) or indicated point mutants were spotted on nitrocellulose membrane and incubated with His-Arl8b or His-Rab7. The interaction was analyzed by immunoblotting (IB) with anti–His antibody. Proteins were visualized by Coomassie staining. (c and d) Representative confocal images showing HeLa cells transfected with GFP-PLEKHM1 or GFP-PLEKHM1 (HRR→A) and immunostained for Arl8. Yellow arrowheads mark colocalized pixels, and white arrowheads mark peripheral Arl8b⁺-lysosomes. (e) PC quantification of WT or mutant PLEKHM1 with LAMP1 and Rab7 (n = 3; 30 cells analyzed per experiment). (f) Arl8b-HA was cotransfected with FLAG-PLEKHM1 (WT) or HRR→A mutant in HEK293T cells. The lysates were immunoprecipitated (IP) using anti–HA antibody resin and immunoblotted using the indicated antibodies. (g) Immunoblot of a GST pulldown assay using HEK293T cell lysates expressing FLAG-PLEKHM1 (WT) or -Arl8b-binding–defective mutants of PLEKHM1 incubated with GST-Rab7 bound to GSH resin. GST-Rab7 protein was visualized by Ponceau S staining. (h and i) Representative confocal panels showing LAMP1 staining in HeLa cells cotransfected with Arl8b-GFP and FLAG-PLEKHM1 (WT) or HRR→A mutant. LAMP1 staining is shown in insets. (j) Mean size of LAMP1⁺ compartments in HeLa cells cotransfected with indicated PLEKHM1 plasmid and Arl8b-GFP (n = 3; 25 cells analyzed per experiment). Data represent mean ± SEM (n.s., not significant; *, P < 0.05; ****, P < 0.0001; Student’s t test). Bars: (main) 10 µm; (insets) 2 µm.