Fig. 5. The V-ATPase V₁ domain reversibly associates with the cytosolic chaperonin TRiC.

a, b Cartoon summarizing SILAC quantification data of proteins in Co-IPs from MEFs with a Flag-Voa3, b HA-V1B2. c, d Changes in protein abundance in SILAC Co-IPs with c Flag-Voa3, d HA-V1B2 for 1 h aa starvation (−aa) versus full medium, or for 1 h aa starvation + 30 min aa restimulation versus 1 h aa starvation (+aa). e Changes in V-ATPase subunit abundance in Cct1 and Cct2-deficient MEFs, analysed by western blot. f Western blot quantification of V-ATPase subunit abundance changes in Cct1 and Cct2-deficient cells as in e. Data are normalized replicate mean ± SEM. The dashed line indicates protein levels in control cells. g Fluorescence quenching of lysosomally loaded FITC-dextran in Cct2-deficient MEFs ± 1 h torin 1 [400 nM]. h Quantification of FITC fluorescence quenching of Cct2-deficient cells ± torin 1 as in g. Data are normalized replicate mean ± SEM (closed circles) and fields of view (open circles; ≥10 per replicate). i Fluorescence quenching of lysosomally loaded FITC-dextran in Cct1 and Cct2-deficient MEFs + 1 h torin 1 [400 nM] after removal of the V-ATPase inhibitor bafilomycin A1 [20 nM]. Scale bars = 20 µm. j Quantification of FITC fluorescence quenching over time in cells treated as in i. Data are normalized replicate mean ± SEM (12 fields of view per replicate). k DQ BSA degradation in Cct1 and Cct2-deficient MEFs after 6 h DQ BSA uptake + torin 1 [400 nM]. Scale bars = 20 µm. l Quantification of DQ BSA fluorescence of Cct1 and Cct2-deficient cells treated as in k. Data are normalized replicate mean ± SEM (closed circles) and fields of view (open circles; ≥10 per replicate). a–d n = 4, f n = 8 (Cct1 KO) or n = 10 (Cct2 KO), h n = 4, j n = 3, l n = 3 biologically independent experiments. In f, p-values were calculated using a one sample t-test with a hypothetical mean of 1. In h, l, p-values were calculated using a two-tailed unpaired t-test with Welch correction. Source data are provided as a Source data file.