Fig 2: Localisation of endogenous Rab34 Maximum intensity projection images of confocal Z-stacks showing LAMP1 and endogenous Rab34 localisation in HeLa cells under growth and starvation conditions. Boxes highlight regions in zoom panels in Fig 4B. Scale bar, 10 µm.Maximum intensity projection images of confocal Z-stacks under starvation conditions showing LAMP1 and endogenous Rab34 localisation in cells transfected with a non-targeting siRNA or when FLCN is depleted. Scale bar, 10 µm.

Fig 3: In vitro, the FLCN DENN domain does not possess Rab34 GEF activity Graphs showing results of Rab34 GEF assays; 300 nM Rab34 loaded with mant-GDP and incubated with various combinations and concentrations of FLCN-DENN and RILP. GTP was added at a concentration of 0.3 mM or EDTA at a concentration of 10 mM at the 2 min time point. Data were acquired at 10-s intervals for 25 min. Curves are mean of duplicate samples and are smoothened using a 6-point rolling average to reduce instrument noise. The same EDTA curve is reproduced across all three graphs for comparison.Coomassie stained SDS–PAGE gel showing samples of His-tagged Rab34 WT, Q111L, T66N and FLCN-DENN domain proteins used in this study.

Fig 4: FLCN directly promotes the association of Rab34 and RILP Confocal immunofluorescence images of HeLa cells transfected with Rab34-dsRED-Mito, FLCN-GFP and HA-FNIP2. White arrows highlight FLCN-GFP/HA-FNIP2 co-localisation distinct from Rab34-mitochondria/FLCN-GFP localisation. Scale bar, 10 µm.Confocal immunofluorescence images of Rab34-dsRED-Mito (WT) and FLCN-GFP DENN only or ?DENN transfected HeLa cells showing DENN domain-dependent recruitment to mitochondria. Scale bar, 10 µm.Western blot analysis of GST-RILP pull down showing association of endogenous FLCN, Rab34 and Rab7 with RILP. Input lane shows 1% input cell extract.Western blot analysis of GST-RILP pull down from UOK257 cells showing DENN domain-dependent association of FLCN with RILP N-terminal, Rab binding domain (RBD) and C-terminal fragments.Western blot showing binding of His-tagged FLCN-DENN domain in E. coli extract to GST-RILP.Western blot analysis of GST-RILP pull down showing direct interaction between RILP and GTP hydrolysis-deficient (Q111L) Rab34 (at 200 nM, in the presence of 1 mM GTP?S) and FLCN-DENN domain (1 µM), but not low nucleotide affinity (T66N) Rab34. FLCN-DENN enhances association of RILP with Q111L Rab34. Graph shows quantification of relative RILP/Rab34 Q111L binding. Error bars show ± SEM. **P < 0.01 (two-tailed t-test).Western blot analysis of GST-RILP/Rab34 Q111L pull down (Rab34 at 200 nM) showing that enhanced association of Rab34 with RILP is achieved with low concentrations of FLCN. Western blot analysis of GST-RILP pull-down experiments with GTP?S loaded (in the presence of 1 mM GTP?S) and GDP loaded (in the presence of 1 mM GDP) wild-type Rab34, showing FLCN-DENN (500 nM)-induced interaction with RILP.Western blot analysis of a GFP-Trap immunoprecipitation experiment from HeLa cells showing that expression of HA-RILP promotes the association of GFP-Rab34 and HA-Rab7.

Fig 5: Targeting of Rab34 and folliculin to mitochondria Confocal immunofluorescence images of Rab34/Rab35-dsRED-Mito transfected HeLa cells showing expected targeting of Rab34-dsRED-Mito and Rab35-dsRED-Mito to mitochondria (labelled with anti-mitochondria antibody). Scale bar, 10 µm.Confocal Immunofluorescence images of HeLa cells transfected with Rab35-dsRED-Mito FLCN-GFP and HA-FNIP2. White arrows highlight FLCN-GFP/HA-FNIP2 co-localisation.Confocal immunofluorescence images of Rab34-dsRED-Mito (WT or Q111L) and FLCN-GFP (without HA-FNIP2) transfected HeLa cells showing recruitment to mitochondria. Scale bar, 10 µm.SIM super-resolution image showing a single plane of a region of a Rab34 (Q111L)-dsRED-Mito/FLCN-GFP/HA-FNIP-2 transfected cell stained with LAMP1 antibodies. Scale bar, 2 µm.