Fig. 6. The trimeric Ccz1-Mon1-C18orf8 (MCC) complex activates mammalian Rab7.

a The MCC complex binds an inactive Rab7 (T22N). Immune precipitations of 2xHA-tagged wild-type, T22N or Q67L Rab7 from C18orf8-3xMyc knock-in cells, were analysed by immunoblot using Myc-, Ccz1- and Mon1B-specific antibodies (2 independent experimental replicates, see also Supplementary Fig. 6a). b, c C18orf8-, Ccz1- and Mon1A/B-deficient cells lack activation-dependent recruitment of Rab7 effectors. b Immune precipitations of 3xFLAG-RILP from wild-type, C18orf8-, Ccz1- or Mon1A/B-deficient cells were analysed by immunoblotting for endogenous Rab7 (two independent experimental replicates). c Wild-type and C18orf8-deficient cells were transfected with HA-RILP or ORP1L (Supplementary Fig. 7a) and stained intracellularly for HA (green) and LAMP1 (magenta). Mander’s correlation was determined for n = 5 fields per condition from two independent experiments. Error bars reflect standard deviation (two-sided unpaired t-test, p = 2.1 × 10⁻⁵, ***p < 0.001). d–f Cholesterol and trafficking defects in C18orf8-deficient cells can be rescued by knockdown of Rab7GAPs or expression of a constitutively active Rab7 (Q67L). d HMGCS1-Clover expression was determined at day 8 after transduction of C18orf8-deficient cells with shRNAs against TBC1D5 (green), TBC1D15 (blue) or both (red). e-f C18orf8-deficient cells were transduced with 2xHA-tagged Rab7-T22N (green), -Q67L (red) or an empty vector (black) and either (e) analysed at day 10 by flow cytometry for HMGCS1-Clover expression (two independent experimental replicates, see also Supplementary Fig 6b), or f pulse-labelled with Dil-LDL (red), incubated for 3 h and stained intracellularly for LAMP1 (green) and EEA1 (blue). Representative images are shown from 5 fields per condition. Scale bars = 10 µm.