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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.
The outermost cell layer of plants, the epidermis, and its outer (lateral) membrane domain facing the environment are continuously challenged by biotic and abiotic stresses. Therefore, the epidermis and the outer membrane domain provide important selective and protective barriers. However, only a small number of specifically outer membrane-localized proteins are known. Similarly, molecular mechanisms underlying the trafficking and the polar placement of outer membrane domain proteins require further exploration. Here, we demonstrate that ACTIN7 (ACT7) mediates trafficking of the PENETRATION3 (PEN3) outer membrane protein from the trans-Golgi network (TGN) to the plasma membrane in the root epidermis of Arabidopsis (Arabidopsis thaliana) and that actin function contributes to PEN3 endocytic recycling. In contrast to such generic ACT7-dependent trafficking from the TGN, the EXOCYST84b (EXO84b) tethering factor mediates PEN3 outer-membrane polarity. Moreover, precise EXO84b placement at the outer membrane domain itself requires ACT7 function. Hence, our results uncover spatially and mechanistically distinct requirements for ACT7 function during outer lateral membrane cargo trafficking and polarity establishment. They further identify an exocyst tethering complex mediator of outer lateral membrane cargo polarity.