Calcium-Dependent Fusion Among Endosomes

MAYORGA, LS; BERON, W; SARROUF, MN; COLOMBO, MI; CREUTZ, C; STAHL, PD

JOURNAL OF BIOLOGICAL CHEMISTRY

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Lugar: Bethesda, Maryland; Año: 1994 vol. 269 p. 30927 - 30934

0021-9258

Fusion among endosomes is an important step for transport and sorting of internalized macromolecules. Working in a cell-free system, we have previously re- ported that, in the absence of externally added calcium, endosome fusion requires cytosol, ATP, and is sensitive to N-ethylmaleimide (NEM) and to anti-NEM-sensitive factor (NSF) antibody. This cytosol-dependent fusion is regulated by monomeric and heterotrimeric GTP-bind- ing proteins. Further studies have revealed, however, that in the presence of micromolar concentrations of free calcium, fusion is observed even in the absence of cytosol and ATP. At the electron microscope level, Ca2?- dependent endosome aggregation and fusion were sim- ilar to that observed for cytosol-dependent fusion. Cal- cium-dependent fusion was not affected by non- hydrolyzable 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 m~ EDTA indicating that pe- ripheral proteins are required. An anti-annexin I1 anti- body and an annexin I1 peptide blocked Ca2?-dependent fusion by 60%. After the EDTA wash, Cazi-dependent fu- sion was reconstituted by addition of purified annexin I1 and arachidonic acid. We conclude that endosomes can fuse by two mechanisms, one that has an absolute re- quirement for calcium and is probably mediated by an- nexins, and another that does not require calcium