Zn²⁺ depletion blocks endosome fusion

Fusion among endosomes is an important step for transport and sorting of internalized macromolecules. Working in a cell-free system, we previously reported that endosome fusion requires cytosol and ATP, and is sensitive to N-ethylmaleimide. Fusion is regulated by monomeric and heterotrimeric GTP-binding proteins. We now report that fusion can proceed at very low Ca²⁺ concentrations, i.e. < 30 nM, Moreover, fusion is not affected when intravesicular Ca²⁺ is depleted by preincubation of vesicles with calcium ionophores (5 μM ionomycin or A23187) in the presence of calcium chelators (5 mM EGTA or 60 mM EDTA). The results indicate that fusion can proceed at extremely low concentrations of intravesicular and extravesicular Ca²⁺. However, BAPTA [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid], a relatively specific Ca²⁺ chelator, inhibits fusion. BAPTA binds other metals besides Ca²⁺. We present evidence that BAPTA inhibition is due not to Ca²⁺ chelation but to Zn²⁺ depletion. TPEN [N,N,N',N'-tetrakis-(2-pyridylmethyl) ethylenediamine], another metal-ion chelator with low affinity for Ca²⁺, also inhibited fusion. TPEN- and BAPTA-inhibited fusions were restored by addition of Zn²⁺. Zn²⁺-dependent fusion presents the same characteristics as control fusion. In intact cells, TPEN inhibited transport along the endocytic pathway. The results indicate that Zn²⁺ depletion blocks endosome fusion, suggesting that this ion is necessary for the function of one or more factors involved in the fusion process.