Inhibition of in Vitro Endosomal Vesicle Fusion Activity by Aminoglycoside Antibiotics

Abstract

The effects of two aminoglycoside antibiotics, neomycin and Geneticin, on the endocytic pathway were studied using a cell-free assay that reconstitutes endosomeendosome fusion. Both drugs inhibit the rate and extent of endosome fusion in a dose-dependent manner with IC 50 values of ϳ45 M and ϳ1 mM, respectively. Because the IC 50 for neomycin falls within the range of affinities reported for its binding to acidic phospholipids, notably phosphatidylinositol 4,5-bisphosphate (PIP 2 ), 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 PIP 2 -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 (GTP␥S), these inhibitors appear to act in a synergistic manner. This idea is confirmed by the fact that addition of a PIP 2 -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 PIP 2 , with high affinity. Thus, although aminoglycoside-sensitive elements of endosome fusion are required at mechanistic stages that are also blocked by GTP␥S-bound ARF, these effects are unrelated to inhibition of the PIP 2 -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.

Prominin‐1 (CD133) modulates the architecture and dynamics of microvilli

“…Note that the microvillar morphology did not change when cells were cultured in medium with 0.5 or 1 mM geneticin or without (data not shown). These data rule out a direct effect of the drug on polyphosphoinositides …”

Identification of Discrete Classes of Endosome-derived Small Vesicles as a Major Cellular Pool for Recycling Membrane Proteins

“…To investigate the potential role of PLD in ESV formation, we assayed the effects of neomycin on ESV formation. Neomycin, an aminoglycoside, has been shown to inhibit PIP2-dependent PLD indirectly by binding PIP2 (Liscovitch et al, 1994;West et al, 1997;Jones and Wessling-Resnick, 1998). When varying concentrations of neomycin are added to labeled endosomal membranes, we observe a dose-dependent inhibition of ESV formation (Figure 6C).…”

“…Phospholipase D (PLD) has been postulated to be a downstream effector of ARF (Kahn et al, 1993;Cockcroft, 1996) mediating budding of Golgi vesicles (Ktistakis et al, 1996), adaptor recruitment to endosomes (West et al, 1997), and endosome-endosome fusion (Jones and Wessling-Resnick, 1998). PLD has been proposed to facilitate coat recruitment to donor membranes by increasing the local production of phosphatidic acid and phosphatidylinositol 4,5-biphosphate (PIP2; Ktistakis et al, 1996).…”

“…When varying concentrations of neomycin are added to labeled endosomal membranes, we observe a dose-dependent inhibition of ESV formation (Figure 6C). Poly-l-aspartic acid, which binds to PIP2-containing membranes and displaces aminoglycosides (Kishore et al, 1990), has been shown to reverse the inhibition of PLD by neomycin (Jones and Wessling-Resnick, 1998). Including poly-l-aspartic acid in the reaction mixture ( Figure 6D) prevents inhibition of ESV formation by neomycin.…”

“…Consistent with the latter possibility, addition of dominant active (Q71L) or dominant negative (T31N) mutant forms of recombinant ARF1 (Randazzo et al, 1992) to the reaction mixtures did not significantly alter levels of ESV production (Lim, Bonzelius, Low, Wille, Weimbs, and Herman, unpublished observations). Thus, ESV formation appears to be mediated by one or more BFA-insensitive GTPases.Phospholipase D (PLD) has been postulated to be a downstream effector of ARF (Kahn et al, 1993;Cockcroft, 1996) mediating budding of Golgi vesicles (Ktistakis et al, 1996), adaptor recruitment to endosomes (West et al, 1997), and endosome-endosome fusion (Jones and Wessling-Resnick, 1998). PLD has been proposed to facilitate coat recruitment to donor membranes by increasing the local production of phosphatidic acid and phosphatidylinositol 4,5-biphosphate (PIP2; Ktistakis et al, 1996).…”