Abstract
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 approximately 45 microM and approximately 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'-gamma-thiophosphate (GTPgammaS), 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 delta1, 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 GTPgammaS-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.
Highlights
Phatidic acid (PA) in a manner dependent on phosphatidylinositol 4,5-bisphosphate (PIP2) [3, 5,6,7]
Neomycin has been used in studies of an ADP-ribosylation factor (ARF)-regulated phospholipase D (PLD) activity that appears to be involved in neutrophil activation [22] and an ARF-regulated PLD activity that enhances release of secretory vesicles from the trans-Golgi network [10]; a second aminoglycoside, Geneticin, has been shown to block secretory vesicle formation stimulated by exogenous PLD [11]
Many investigators have attributed the nephrotoxicity of aminoglycosides to arise from their inhibitory effects on lysosomal phospholipase activity [23, 24], an attractive model is that these antibiotics may interfere with elements of endocytic membrane traffic involving the activation of PLD by ARF
Summary
Aminoglycosides have been widely studied because of the nephrotoxicity associated with their use as antibiotics and are well known to cause a vacuolarization of the apical endosomal compartment of renal proximal tubules [23, 24]. Our data demonstrate that these aminoglycosides block steps of endosome fusion temporally related to steps affected by GTP␥S-bound ARFs; the fact that these antibiotics act synergistically with GTP␥S to inhibit fusion suggests that additional endosomal functions are affected This concept is supported by the observations that a PIP2independent PLD does not suppress the effects of neomycin on endosome fusion and that this in vitro activity is unaffected by the pleckstrin homology (PH) domain of phosphoinositide-specific phospholipase C-␦1 (PI-PLC␦1), which is known to bind PIP2 with high affinity [26, 27]. These results argue that there are additional mechanistic roles for acidic phospholipids in the endosomal pathway
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
