Par17 is targeted to mitochondria in human cells. A. Endogenous Par17 in sub-cellular fractions. HeLa cells were separated into cytoplasmic (Cyt), nucleic (Nuc), membrane (Me) and mitochondrial (Mito) fractions. Equal protein amounts were separated by SDS-PAGE, blotted onto nitrocellulose membranes and incubated with antibodies against the Par17 N-terminus or marker proteins. Par17 was detected as 17 kDa protein species within the membrane and mitochondrial fractions. Par17 without fusion tags expressed in E. coli BL21 from a pET-41 vector was used as positive control (+). B. Cellular localization of parvulin EGFP fusions. Par14, Par17-QR and -RS C-terminally fused to EGFP as well as EGFP without fusions were transfected into HeLa cells grown on cover slips, stained with MitoTracker and analyzed by fluorescence microscopy and 3D reconstitution. EGFP and MitoTracker fluorescence are shown as well as an overlay of these channels. White bar corresponds to 10 μm. The position of the Z stack image (3–4 μm in height) is indicated within the respective overlay image. A pronounced overlap between EGFP and MitoTracker signals is visible with the Par17-EGFP constructs that is not observed with Par14-EGFP or EGFP alone. C. Expression of the transfected constructs within HeLa cells as full length proteins. Anti-GFP Western blot and part of the Coomassie stained gel showing equal loading. All constructs were detected as full length protein species: Par17-EGFP (44.1 kDa, arrow head), Par14-EGFP (44.1 kDa, arrow) and EGFP (26.9 kDa, asterisk). D. Co-localization values of different EGFP fusion constructs. The fraction of EGFP fluorescence overlapping with the MitoTracker signal (in percent) was determined using the Cell^P co-localization tool for the fusion constructs shown in B. In addition, co-localization values for EGFP fusions of prepeptide and prepeptide coupled to the basic domain are displayed. All constructs containing the prepeptide were both tested as QR and RS variants. Co-localization values are expressed in percent ± standard deviation for more than 10 cells.