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 prev iously reported that endosome fusion requires cytosol and ATP, and is sensitive to N-ethylmaleimide. Fusion is regulated by monomeric and he terotrimeric GTP-binding proteins. We now report that fusion can proce ed 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 mu M ionomycin or A23187) in the p resence of calcium chelators (5 mM EGTA or 60 mM EDTA). The results in dicate that fusion can proceed at extremely low concentrations of intr avesicular and extravesicular Ca2+ However, BAPTA [1,2-bis-(o-aminophe noxy)ethane-N,N,N',N tetra-acetic acid], a relatively specific Ca2+ ch elator, inhibits fusion. BAPTA binds other metals besides Ca2+. We pre sent evidence that BAPTA inhibition is due not to Ca2+ chelation but t o Zn2+ depletion. TPEN [N,N,N',N'-tetrakis-(2-pyridylmethyl) ethylened iamine], another metal-ion chelator with low affinity for Ca2+, also i nhibited fusion. TPEN- and BAPTA-inhibited fusions were restored by ad dition of Zn2+. Zn2+-dependent fusion presents the same characteristic s as control fusion. In intact cells, TPEN inhibited transport along t he endocytic pathway. The results indicate that Zn2+ depletion blocks endosome fusion, suggesting that this ion is necessary for the functio n of one or more factors involved in the fusion process.