Figure 4

Mitochondrial localization of PAQR10. (A) Mitochondrial localization assay. Total (T), cytoplasmic (C), or mitochondria-enriched (M) αTC1 (a) and βTC3 (β) lysates were analyzed for PAQR10 expression by Western blot (left panel). Mitochondrial enrichment was confirmed by Western blot analysis of prohibitin expression in the same C and M lysates (right panel). (B) Confocal microscopy. CHO cells were transfected with plasmid constructs encoding versions of PAQR10 tagged at the C-terminus with myc (PAQR10-myc) or enhanced green fluorescent protein (PAQR10-GFP), at the N-terminus with flag peptide (Flag-PAQR10) or GFP (GFP-PAQR10), or at both ends Flag-PAQR10-myc. Mitochondria were detected by red fluorescence after staining with Mitotracker Red (left panels). PAQR10 or protein tags were detected with the indicated specific antibodies and FITC-labeled secondary antibodies. GFP was visualized under FITC channels (center panels), and the images were merged after confocal microscopy (right panels). These data demonstrate that PAQR10 localizes to mitochondria and that N-terminal tagging of PAQR10 prevents localization and changes its intracellular distribution to perinuclear sites.