Effect of co-overexpression of Arf1 and Arf6 variants on peroxisomal and mitochondrial protein import in PtK2 cells. (A) Ptk2 cells were transiently transfected with plasmids coding for no protein (/), Arf1WT-HA (1), Arf1T31N-HA (1-), or Arf1Q71L-HA (1+) and/or a bicistronic plasmid encoding EGFP-PTS1 together with no other protein (-), non-tagged Arf6WT (6), Arf6T27N (6-), or Arf6Q67L (6+). After 36 hours, the cells were fixed and processed for fluorescence analysis. The subcellular localization of EGFP-PTS1 was determined by its punctate (peroxisomal) or diffuse staining pattern in at least 250 cells, and the results were quantified. (B) Representative images of the subcellular distribution pattern of EGFP-PTS1, HsPMP34-Myc-His, and HsLK2-Myc-His in Ptk2 cells co-overexpressing Arf1T31N and Arf6T27N. Note that the latter protein is encoded by the bicistronic expression vector coding for EGFP-PTS1, and a mislocalization of the reporter proteins is only observed in double-transfected cells. As (i) in mammalian cells the fluorescence intensity of EGFP-PTS1 is significantly higher upon mislocalization to the cytosol , and (ii) this increase may mask the (partial) association of this reporter protein with peroxisomes, insets are included in which the outlined regions are shown with moderately less intense green fluorescence signals. Scale bar: 20 μm.