Fig. 6: PNPLA7 is a MAM protein and interacts with Parkin to facilitate mitochondrial translocation of Parkin.
a Representative Immunoblot results of PNPLA7 protein levels in the indicated organelle fractions isolated from 3T3-L1 cells. H: homogenate, Mp: pure mitochondrial fraction, Mc: crude mitochondrial fraction, ER: the endoplasmic reticulum, MAM: mitochondria-associated membrane, Cyto: cytosol. Organelle fractions were verified by testing the presence of specific markers: Calnexin for ER and MAMs, FACL-4 for MAMs, COX4 for mitochondrial, tubulin for cytosol. Representative results from 3 independent experiments were shown. b Representative immunofluorescence image of EGFP-PNPLA7 colocalization with ER in 3T3-L1 cells. EGFP-PNPLA7 localization to ER was analyzed by confocal microscopy. Scale bar = 20 μm. (n = 3 biological replicates). c Representative immunofluorescence image of mCherry-PNPLA7 colocalization with mitochondria-associated membrane in 3T3-L1 cells. Mitochondria-associated membranes were stained for FACL-4 (green), mCherry-PNPLA7 localization to mitochondria-associated membrane was analyzed by confocal microscopy. Scale bar = 20 μm. (n = 3 biological replicates). d Representative immunofluorescence image of EGFP-PNPLA7 colocalization with mitochondria in 3T3-L1 cells. EGFP-PNPLA7 localization to mitochondria was analyzed by confocal microscopy. Scale bar = 20 μm. (n = 3 biological replicates). e, f Representative co-immunoprecipitation Immunoblot data of endogenous Parkin with PNPLA7 in iWAT. Pnpla7Tg mice iWAT total lysates were immunoprecipitated with anti-Parkin (e) or anti-Flag (f) antibodies, respectively. Calnexin was used as a protein loading control. IgG, immunoglobulin G. (n = 3 biological replicates). g Schematic diagram of construction of PNPLA7 truncations. Full length of PNPLA7(1-1352aa), N-terminal region (1-941aa), Transmembrane domain (36-1352aa), and C-terminal region (942-1352aa). All truncations carry HA-tag at the carboxyl terminus. h, i Representative co-immunoprecipitation Immunoblot data of Flag-Parkin with different truncations of HA-PNPLA7 in HEK293T cells. HEK293T cells were transfected with Flag-Parkin and different truncations of HA-PNPLA7 for 48 h. Cell total lysates were immunoprecipitated with anti-HA (h) or anti-Flag (i) antibodies, respectively. HA-vector was used as a negative control of immunoprecipitation. Calnexin was used as a protein loading control. (n = 3 biological replicates). j Mitochondrial translocation of Parkin in PNPLA7-depleted mature adipocytes overexpressing different PNPLA7 truncations. SVF from iWAT of PNPLA7 KO mice was differentiated into adipocytes ex vitro. Immunofluorescence images of EGFP-Parkin (green) and mCherry-PNPLA7 (red) were analyzed by confocal microscopy. Scale bar = 20 μm. (n = 3 biological replicates). Source data are provided as a Source Data file.
