Figure 3A. Western blot of primary fibroblasts derived from patient 2 (VPS41S285P/R662*), his mother (VPS41WT/R662*), father (VPS41WT/S285P), patient 3 (VPS41c.1423-2A>G /R662*) and an unrelated, healthy control (VPS41WT/WT) (western blot of longer exposure time shown in Appendix Fig S3A). Full-length VPS41 (FL VPS41), representing VPS41WT and VPS41S285P, is observed in all cells. Truncated VPS41R662* in patients and maternal fibroblasts is not detectable. VPS41R662* is also not visible in fibroblasts treated with 50µM MG132 (4 hours) to inhibit proteasomal degradation, indicating that the mRNA encoding for this mutant is degraded. (A') Quantification of VPS41 protein levels show a reduction in both patients compared to VPS41WT/WT, with only 10% remaining VPS41 levels in VPS41c.1423-2A>G /R662* (n=2). B. Immunoprecipitation (IP) on HeLa cells co-expressing GFP-empty vector (EV), GFP-VPS41WT, GFP-VPS41S285P or GFP-VPS41R662* and FLAG-VPS18. GFP-VPS41WT and GFP-VPS41S285P both interact with FLAG-VPS18 and as shown in Appendix Fig S4A with HA-VPS33A. Interaction between GFP-VPS41R662* and FLAG-VPS18 is strongly reduced. Note that the stop codon in VPS41R662* leads to a truncated protein of lower MW (n=3). C. IP on Hela cells co-expressing VPS41WT-APEX2-V5, VPS41S285P-APEX2-V5 or VPS41R662*-APEX2-V5 and FLAG-RAB7 or GFP-Arl8b. The IP was performed on FLAG or GFP respectively. All VPS41 variants interact with Rab7 and Arl8b (n=3). D. HeLaVPS41KO cells transfected with VPS41WT-APEX2-V5, VPS41S285P-APEX2-V5 or VPS41R662*-APEX2-V5 constructs labeled for LAMP-1 and V5 immunofluorescence microscopy. All VPS41 variants co-localize with LAMP-1, indicating that VPS41S285P and VPS41R662* are recruited to late endosomes/lysosomes. Scale bars 10µm; zoom of squared area, 1µm. E. Quantification of endocytosis-rescue experiments in HeLa cells. HeLaVPS41KO cells transfected with VPS41WT-APEX2-V5 show a significant increase in colocalization of endocytosed Dextran-568 and SiR-Lysosome Cathepsin D (SiR-Lyso), indicating rescue of the endocytosis phenotype. Neither VPS41 variant rescues this HOPS complex functionality. >13 Cells per cell line per experiment were quantified (n=3). F. Quantification of lysosomal delivery of endocytosed cargo in fibroblasts, based on fluorescent data. VPS41WT/WT, VPS41WT/S285P, VPS41WT/R662* and VPS41S285P/R662* primary fibroblasts were incubated with Dextran-568 and SiR-Lysosome Cathepsin D (SiR-Lyso) for 2 and 3 hours, respectively. Colocalization representing delivery of Dextran to enzymatically active lysosomes is reduced in VPS41WT/R662* and VPS41S285P/R662* cells. >11 Cells per cell line per experiment were quantified (n=3). G. Quantification of lysosomal delivery of endocytosed cargo in fibroblasts, based on EM data. VPS41WT/WT, VPS41WT/S285P, VPS41WT/R662* and VPS41S285P/R662* fibroblasts were incubated with BSA-Au5 for 2 hours and labeled for LAMP-1 (10 nm gold particles) immuno-EM (Fig 2D). LAMP-1-positive lysosomes were scored for presence of BSA-Au5 and the ratio between BSA+ and BSA- lysosomes was calculated. >46 Lysosomes per condition were quantified. Both VPS41WT/R662* and VPS41S285P/R662* show a strong decrease in BSA-positive lysosomes indicating a fusion defect between late endosomes and lysosomes. H. VPS41WT/WT, VPS41WT/R662* and VPS41S285P/R662* primary fibroblasts incubated with Dextran-568 for 0.5, 2, 5, 8 and 24 hours. Colocalization of Dextran-568 and SiR-Lysosome Cathepsin D (SiR-Lyso) reveals a delay in lysosomal delivery in maternal (VPS41WT/R662*) and patient (VPS41S285P/R662*) cells at 2 hours of Dextran uptake. After 5 hours, maternal cells show similar to control colocalization levels. Patient cells show only after 24 hours colocalization levels similar to control, indicating a delay rather than a block in late endosome - lysosome fusion. >10 Cells per cell line were quantified.