Differential effects of PE17 expression on early, late and recycling endosomes and lysosomes
As PE17 expression affected the Golgi stack, TGN, and mitochondria, but not the ER, we investigated whether PE17 has a broad effect on most organelles or these were specifically targeted by PE17 activity. Inhibition of trafficking through early endosomal compartments by bafilomycin A - or late endosomal compartments by wortmannin – results in the accumulation of enlarged spherical endosomes (Reaves & Banting, 1994, Reaves et al. , 1996). As the PE17 positive structures were often distinctly spherical and perinuclear, we labelled cells transfected with PE17-myc with antibodies to markers of early (EEA1) and recycling endosomes (Tfr, transferrin receptor), respectively. There was no colocalization between PE17 and EEA1 or Tfr (Figure 4A,C). No changes in the morphology of the recycling or early endosomes were apparent. Upon quantifying the mass, we found no difference between cells expressing PE17 and those that were not (p = 0.4033, p = 0.0576, respectively) (Figure 4B,D).
We next investigated effects on late endosomes and lysosomes following transfection with PE17 using antibodies against CD63 and LAMP1, respectively. In both instances, we again saw no colocalization of these markers with PE17 and saw no obvious changes in organelle morphology (Figure 4E,G). However, we measured a significant reduction in late endosomes (p < 0.0001) and a significant, but milder, effect on lysosomal mass (p < 0.0001) (Figure 4F,H). These results support a specific effect on a subset of organelles and preclude a general ablation of organelle integrity.