Mito-VPS41 recruits the core HOPS subunit VPS18 via its RING domain. (A) Schematic representation of the yeast HOPS complex (Shvarev et al., 2022). (B) Schematic representation of mito-VPS41^WT-V5 and mito-VPS41^R662*-V5 showing constituent WD40/TPR–like, CHCR, and RING domains at the C-terminal and their association with the outer mitochondrial membrane via TOM70. (C) Confocal images showing the distribution of VPS18-GFP co-expressed with mito-V5, mito-VPS41^WT-V5, or mito-VPS41^R662*-V5 (labeled for V5) in HeLa^VPS41KO cells. (D) Quantification of recruitment of VPS18-GFP to mitochondria in HeLa^VPS41KO cells transfected as in C, showing that the VPS41 RING domain (absent from R662*) is required for VPS18 recruitment. Fluorescence intensity of the VPS18-GFP signal within a mask of the mito-construct was normalized to the mito-V5 control condition and expressed as a fold change. Statistical significance was calculated using Tukey’s multiple comparisons test, ****P < 0.0001. n > 40. Data are presented as the mean ± SEM. Scale bars: 20 and 5 µm (inset). See also Fig. S1 C.