Abstract
ATP production requires the establishment of an electrochemical proton gradient across the inner mitochondrial membrane. Mitochondrial uncouplers dissipate this proton gradient and disrupt numerous cellular processes, including vesicular trafficking, mainly through energy depletion. Here we show that Endosidin9 (ES9), a novel mitochondrial uncoupler, is a potent inhibitor of clathrin-mediated endocytosis (CME) in different systems and that ES9 induces inhibition of CME not because of its effect on cellular ATP, but rather due to its protonophore activity that leads to cytoplasm acidification. We show that the known tyrosine kinase inhibitor tyrphostinA23, which is routinely used to block CME, displays similar properties, thus questioning its use as a specific inhibitor of cargo recognition by the AP-2 adaptor complex via tyrosine motif-based endocytosis signals. Furthermore, we show that cytoplasm acidification dramatically affects the dynamics and recruitment of clathrin and associated adaptors, and leads to reduction of phosphatidylinositol 4,5-biphosphate from the plasma membrane.
Highlights
ATP production requires the establishment of an electrochemical proton gradient across the inner mitochondrial membrane
As a model synapse we used the Drosophila third instar neuromuscular junction (NMJ), where nerves were stimulated for 5 min with 90 mM KCl to enhance endocytosis and exocytosis[19,20] in the presence of FM1-43, a dye that becomes internalized in newly formed vesicles upon nerve stimulation
To investigate whether the ES9- and TyrA23-induced clathrin-mediated endocytosis (CME) inhibition was triggered by ATP depletion, we evaluated the effect of carbonyl cyanide m-chlorophenyl hydrazone (CCCP), antimycin A (AA), and oligomycin on endocytosis in Arabidopsis, given that the CME inhibitors ES9 and TyrA23 depleted cellular ATP
Summary
ATP production requires the establishment of an electrochemical proton gradient across the inner mitochondrial membrane. We show that Endosidin[9] (ES9), a novel mitochondrial uncoupler, is a potent inhibitor of clathrin-mediated endocytosis (CME) in different systems and that ES9 induces inhibition of CME not because of its effect on cellular ATP, but rather due to its protonophore activity that leads to cytoplasm acidification. Chemical genetics has the potential to facilitate studies of CME by providing small molecule effectors that can interfere with CME in a conditional manner[4] An example of such a CME inhibitor is tyrphostinA23 (TyrA23). We identified and characterized Endosidin[9] (ES9), a small molecule inhibitor of CME in Arabidopsis thaliana and Drosophila melanogaster We reveal that this molecule uncouples mitochondrial oxidative phosphorylation, a mode of action that is shared with TyrA23. Acidification, but not plasma membrane depolarization, caused a dramatic increase in the lifetimes of clathrin and associated adaptors and led to a reduction of the phosphatidylinositol 4,5-biphosphate (PI(4,5)P2), thereby most likely inhibiting clathrin-coated pits formation
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