Skip to main content
. 2023 Sep 29;14:6099. doi: 10.1038/s41467-023-41849-8

Fig. 6. TMEM135 translocates from peroxisomes to mitochondria in response to acute β-adrenergic stimulation and interacts with AKAP1 to mediate PKA-dependent phosphorylation of Drp1.

Fig. 6

a Confocal microscopy in control (n = 3) or 1 h NE-treated brown adipocytes (n = 3) expressing peroxisome-targeted GFP (GFP-PTS1) and labelled with TMRE to mark mitochondrial networks. Quantification of peroxisome-mitochondria colocalization using ImageJ. Quantification was performed with the investigator blinded to the identity of the samples. Scale bar: 5 μm. b Two-photon confocal microscopy in control (n = 4) or 1 h NE-treated brown adipocytes (n = 4) expressing GFP-PTS1 and BFP-TMEM135 and stained with TMRE. Quantification of BFP-TMEM135 localization with mitochondria using ImageJ is shown.Scale bar: 5 μm. c Western blot analysis of TMEM135 protein levels in peroxisomal and mitochondrial fractions of NE-treated brown adipocytes. Ponceau S staining as loading control. The blots are representative of three independent experiments. d Western blot analysis of Drp1 (Ser600) phosphorylation in NE-treated brown adipocytes with or without CRISPR/Cas9-mediated knockout of TMEM135; n = 3. e Western blot analysis of Drp1 (Ser600) phosphorylation in brown adipocytes transduced with lenti-GFP (n = 2) or lenti-TMEM135 (n = 3). f Immunofluorescence analysis using an antibody against Drp1 in mito-GFP expressing brown adipocytes transduced with empty or TMEM135 overexpression lentivirus. Colocalization was quantified using ImageJ. Scale bar: 10 μm. The images are representative of three independent experiments and the quantification is based on a total of 16 empty vector and 23 TMEM135 OE cells cells per group. g TMEM135 interaction network based on search of a publicly available dataset of global protein–protein interactions using a proximity-dependent biotinylation approach. h Co-immunoprecipitation of endogenous AKAP1 with FLAG-TMEM135 in HEK293 cells. The blots are representative of three independent experiments. i Pulldown assay showing that GST-TMEM135 interacts with endogenous AKAP1 in brown adipocyte protein lysates. The blots are representative of three independent experiments. Data are presented as mean ± SEM; statistical significance was determined by two-tailed unpaired Student’s t test (a, b, f).