Although
cell-penetrating peptides (
CPP) facilitate endocytic uptake of
proteins, little is known regarding the extent to which CPPs facilitate
protein cargo exit from endocytic vesicles for targeting to other intracellular sites. Since the plasma membrane and less so intracellular membranes contain
cholesterol, the fluorescent
sterol analogues
dansyl-
cholestanol (DChol) and
dehydroergosterol (DHE) were used to monitor the uptake and intracellular distribution of fluorescent-tagged
acyl coenzyme A binding protein (ACBP) into COS-7 cells and rat
hepatoma cells. Confocal microscopy colocalized DChol and
Texas Red-ACBP (TR-ACBP) with markers for the major endocytosis pathways, especially fluorescent-labeled
cholera toxin (marker of
ganglioside GM1 in plasma membrane
lipid rafts) and
dextran (macropinocytosis marker), but less so with
transferrin (
clathrin-mediated endocytosis marker). These findings were confirmed by multiphoton
laser scanning microscopy colocalization of TR-ACBP with DHE (naturally-fluorescent
sterol) and by double immunofluorescence labeling of native endogenous ACBP. Serum greatly and Pep-1 further 2.4-fold facilitated uptake of TR-ACBP, but neither altered the relative proportion of TR-ACBP colocalized with membranes/organelles (nearly 80%) vs cytoplasm and/or nucleoplasm (20%). Interestingly, Pep-1 selectively increased TR-ACBP associated with mitochondria while concomitantly decreasing that in endoplasmic reticulum. In summary, fluorescent
sterols (DChol, DHE) were useful markers for comparing the distributions of both transported and endogenous
proteins. Pep-1 modestly enhanced the translocation and altered the intracellular targeting of exogenous-delivered (TR-ACBP) in living cells.