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J Biol Chem, Vol. 273, Issue 39, 25301-25309, September 25, 1998

Inhibition of in Vitro Endosomal Vesicle Fusion Activity by Aminoglycoside Antibiotics

From the Department of Nutrition, Harvard School of Public Health, Boston, Massachusetts 02115

The effects of two aminoglycoside antibiotics, neomycin and Geneticin, on the endocytic pathway were studied using a cell-free assay that reconstitutes endosome-endosome fusion. Both drugs inhibit the rate and extent of endosome fusion in a dose-dependent manner with IC₅₀ values of ~45 µM and ~1 mM, respectively. Because the IC₅₀ for neomycin falls within the range of affinities reported for its binding to acidic phospholipids, notably phosphatidylinositol 4,5-bisphosphate (PIP₂), these data suggest that negatively charged lipids are required for endosome fusion. A role for negatively charged lipids in membrane traffic has been postulated to involve the activity of a PIP₂-dependent phospholipase D (PLD) stimulated by the GTP-binding protein ADP-ribosylation factor (ARF). Although neomycin blocks endosome fusion at a stage of the in vitro reaction that is temporally related to steps inhibited by cytosolic ARFs when they bind guanosine-5'--thiophosphate (GTPS), these inhibitors appear to act in a synergistic manner. This idea is confirmed by the fact that addition of a PIP₂-independent PLD does not suppress neomycin inhibition of endosome fusion; moreover, in vitro fusion activity is not affected by the pleckstrin homology domain of phosphoinositide-specific phospholipase C 1, which binds to acidic phospholipids, particularly PIP₂, with high affinity. Thus, although aminoglycoside-sensitive elements of endosome fusion are required at mechanistic stages that are also blocked by GTPS-bound ARF, these effects are unrelated to inhibition of the PIP₂-dependent PLD activity stimulated by this GTP-binding protein. These results argue that there are additional mechanistic roles for acidic phospholipids in the endosomal pathway.

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