Figure 2.

Loss of TRIM37 impairs import of peroxisomal matrix proteins. (A) TRIM37 detection in HepG2 control and TRIM37 KD cells. (B) Immunostaining of peroxisomal proteins in HepG2 control and TRIM37 KD cells. Cells were costained with antibodies against PMP70 and PTS1 proteins. Bars: 10 µm. (C and D) Localization of peroxisomal matrix and membrane proteins in HepG2 control and TRIM37 KD cells. The tripeptide SKL was fused to the C terminus of GFP (GFP-SKL). Bars: 5 µm. The cDNAs encoding the PTS1-containing proteins (ACOX1, acyl-CoA oxidase 1; PECR, peroxisomal trans-2-enoyl-CoA reductase) were fused to the C terminus of GFP (GFP-ACOX1 and GFP-PECR). Thiolase and PMP22 were fused to the N terminus of GFP (thiolase-GFP and PMP22-GFP). These GFP constructs were transfected into HepG2 control and TRIM37 KD cells. Images were taken with the same exposure time for each construct (24 h after transfection). Nuclei were stained with DAPI. The expression levels of these constructs are equal in control and TRIM37 KD cells, as shown in Fig. S1 A. The number of punctate peroxisomes per cell was calculated and presented as mean ± SEM; ***, P < 0.001 (Student’s t test). The results are representative of three independent experiments. (E) Subcellular distribution of peroxisomal proteins in HepG2 control, TRIM37 KD, and PEX5 KD cells. Cytosolic (S) and organelle (P) fractions were separated by 100,000 g centrifugation of PNSs ECH1 (enoyl-CoA hydratase 1), ACOT1/2 (acyl-CoA thioesterase 1/2), GSTK1, GSTκ1, and GAPDH.