CALCIUM-DEPENDENT FUSION AMONG ENDOSOMES

Fusion among endosomes is an important step for transport and sorting of internalized macromolecules. Working in a cell-free system, we have previously reported that, in the absence of externally added calcium, endosome fusion requires cytosol, ATP, and is sensitive to N-ethylmal eimide (NEM) and to anti-NEM-sensitive factor (NSF) antibody. This cyt osol-dependent fusion is regulated by monomeric and heterotrimeric GTP -binding proteins. Further studies have revealed, however, that in the presence of micromolar concentrations of free calcium, fusion is obse rved even in the absence of cytosol and ATP. At the electron microscop e level, Ca2+-dependent endosome aggregation and fusion were similar t o that observed for cytosol-dependent fusion. Calcium-dependent fusion was not affected by nonhydrolyzable analogs of GTP or GDP nor by NEM or anti-NSF antibody. However, Ca2+-dependent fusion was abrogated by trypsin treatment of the vesicles or by a membrane wash with 60 mM EDT A indicating that peripheral proteins are required. An anti-annexin II antibody and an annexin II peptide blocked Ca2+-dependent fusion by 5 0%. After the EDTA wash, Ca2+-dependent fusion was reconstituted by ad dition of purified annexin II and arachidonic acid. We conclude that e ndosomes can fuse by two mechanisms, one that has an absolute requirem ent for calcium and is probably mediated by annexins, and another that does not require calcium.