Journal: bioRxiv

Article Title: Geranylgeranylated-SCF FBXO10 Regulates Selective Outer Mitochondrial Membrane Proteostasis and Function

doi: 10.1101/2024.04.16.589745

Figure Lengend Snippet: Geranylgeranylation of CaaX-cysteine953 is indispensable for the distribution of FBXO10 at outer mitochondrial membrane. (A) Inhibition of geranylgeranylation re-distributes GFP-FBXO1O away from mitochondria into cytosol. HeLa cells expressing GFP-FBXO10 were treated with geranylgeranylation inhibitor (GGTi-2418, 50 µM) farnesylation inhibitor (FTi-lonafarnib, 10 µM), clinical drug statin (lovastatin, 15 µM) and vehicle for 16 hours. MitoTracker was added to decorate mitochondrial for ∼30 minutes prior to live cell confocal imaging using ZEISS-LSM700 microscope. Representative images depict the changes in mitochondrial distribution of GFP-FBXO10 (panel 1) upon inhibition of farnesylation (panel 2), geranylgeranylation (panel 3) and mevalonate pathway (panel 4). Pearson correlation coefficients (PCC) for the colocalization of GFP-FBXO10 and mitochondria were analyzed in different cells [vehicle (n=70), GGTi (n=72), Fti (n=73) and lovastatin (n= 77)]. Statistical analysis of plotted PPCs in scatter plots is shown (right). p-values were calculated by student-T-test. Scale bar 10µM. (B) Inhibition of geranylgeranylation results in loss of FBXO10 from subcellular mitochondrial fractions. HEK-293T cells expressing STREP-tagged FBXO10 cells were treated with geranylgeranylation inhibitor (GGTi-2418, 50 µM) or vehicle DMSO for 16 hours. Post-harvesting cell lysates were subjected to subcellular fractionation using stepwise ultracentrifugation (see Methods). Enriched mitochondrial and cytosolic fractions were assayed by SDS-PAGE and immunoblotted as indicated. Shown is representative of two independent experiments. (C) GGTi-2418 delocalizes FBXO10 from mitochondria. HeLa cells expressing ptd-Tomato-FBXO10 were treated with geranylgeranylation inhibitor (GGTi-2418, 50 µM) or vehicle DMSO for 16 hours. Enriched mitochondrial fractions were isolated by stepwise centrifugation. Isolated mitochondrial fractions were incubated with MitoView green facilitating mitochondrial tracking during flowcytometry analysis (FACS) for the quantification of mitochondria-associated ptd-Tomato-FBXO10 signal. Representative FACS histogram confirms localization of FBXO10 at mitochondria (middle panel) shown as rightward shifted peak from the baseline non-expressing controls (top panel) and delocalization of FBXO10 away from mitochondria upon GGTi-2418 treatment shown as peak closer/matching baseline non-expressing controls (bottom panel). Shown is representative of three independent experiments. (D) Metabolic labelling with tritiated mevalonate shows Cystine953 acceptor site for the FBXO10 prenylation. HEK293T cells expressing FLAG-FBXO10 and FLAG-FBXO10(C953S) were metabolically labeled with tritiated (H 3 )-mevalonolactone according to the protocol described in the methods section. Post-labeling, cells were harvested, and whole cell lysates were processed to carry out anti-FLAG immunoprecipitations. Tritium (H 3 ) labeled immunocomplexes and lysates were subjected to SDS-PAGE using pre-cast gradient Bolt TM 4-12% Bis-Tris Plus gels and transferred to a PVDF membrane. Subsequently, the PVDF membrane was exposed to a Phosphor-screen and H 3 -labeled proteins were detected with the Phosphor-imager. The autoradiograph (top), developed after exposure to Phosphor-screen, shows that FLAG-FBXO10, but not FLAG-FBXO10(C953S) is prenylated. (*) indicates non-specific. Bottom: PVDF membrane was probed with anti-FLAG immunoblotting. (E) FBXO10 along with SCF-subunits associate with outer mitochondrial membrane (OMM). Enriched intact mitochondrial fractions were isolated from HEK293T cells expressing STREP-FBXO1O by stepwise centrifugation. Isolated mitochondrial fractions were subjected to Trypsin protease protection (TPP) assay protocol (see methods). Mitochondrial lysates prepared from samples that underwent TPP-assay and untreated controls were subjected to SDS-PAGE followed by immunoblotting for the SCF-FBXO10 components and sub-mitochondrial membrane compartment specific markers, as indicated. Representative of three independent experiments is shown. (F) FBXO10 localizes at outer mitochondrial membrane (OMM). Enriched intact mitochondrial fractions were isolated from HEK293T cells expressing GFP-FBXO1O by stepwise centrifugation. Isolated mitochondrial fractions were subjected to Trypsin protease protection (TPP) as in (H). Samples that underwent TPP-assay and untreated controls were subjected to flowcytometry analysis (FACS) to quantify mitochondria-associated GFP-FBXO10 signal. Representative FACS histogram confirms FBXO10 at OMM (middle panel) shown as rightward shifted peak in untreated samples from the baseline non-expressing controls (top panel) and its loss from OMM due to Trypsin protease access at OMM shaving off GFP-FBXO10 in TPP-samples shown as leftward shifted peak closer/matching baseline non-expressing controls (bottom panel). Representative of three independent experiments is shown. (G) Dynamic subcellular distribution of FBXO10 at mitochondria. Fluorescence recovery after photobleaching (FRAP) analysis was carried out to monitor mobility of FBXO10 in HeLa cells expressing GFP-FBXO10. After focal photodestruction of the GFP signal with 488nm laser, the recovery of the GFP-FBXO10 signal in the bleached area was recorded over indicated time period using ZEISS-LSM700 microscope. In parallel an unbleached focal area was also monitored as control. Shown are the representative images of 18 independent cells analyzed for FRAP. The plot shows averaged GFP-FBXO10 signal recovery of 18 independent bleached areas from separate cells. GFP-signal before photodestruction was set as 100%. Calculated Recovery Rate: 0.23±0.04(%/µm2/sec). Error bar: SEM

Article Snippet: Samples were lysed in SDS based lysis buffer (50 mM Tris Ph7.4, 150 mM NaCl, 1% SDS, 1mM EDTA and 50mM NaF) containing protease inhibitors (Roche) and sonicated.

Techniques: Membrane, Inhibition, Expressing, Imaging, Microscopy, Fractionation, SDS Page, Isolation, Centrifugation, Incubation, Metabolic Labelling, Labeling, Autoradiography, Western Blot, Fluorescence