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.