The association of annexin I with early endosomes is regulated by Ca2+ and requires an intact N-terminal domain.
Annexin I is a member of a multigene family of Ca2+/phospholipid-binding proteins and a major substrate for the epidermal growth factor (EGF) receptor kinase, which has been implicated in membrane-related events along the endocytotic pathway, in particular in the sorting of internalized EGF receptors occurring in the multivesicular body. We analyzed in detail the intracellular distribution of this annexin by cell fractionation and immunoelectron microscopy. These studies used polyclonal as well as a set of species-specific monoclonal antibodies, whose epitopes were mapped to the lateral surface of the molecule next to a region thought to be involved in vesicle aggregation. Unexpectedly, the majority of annexin I was identified on early and not on multivesicular endosomes in a form that required micromolar levels of Ca2+ for the association. The specific cofractionation with early endosomes was also observed in transfected baby hamster kidney cells when the intracellular fate of ectopically expressed porcine annexin I was analyzed by using the species-specific monoclonal antibodies in Western blots of subcellular fractions. Interestingly, a truncation of the N-terminal 26, but not the N-terminal 13 residues of annexin I altered its intracellular distribution, shifting it from fractions containing early to those containing late and multivesicular endosomes. These findings underscore the regulatory importance of the N-terminal domain and provide evidence for an involvement of annexin I in early endocytotic processes.
Seemann
J
J
Department of Biochemistry, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
Weber
K
K
Osborn
M
M
Parton
R G
RG
Gerke
V
V
D000595:Amino Acid Sequence; D000818:Animals; D017305:Annexin A1 / Q000235:genetics* / Q000276:immunology / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D010856:Columbidae; D006224:Cricetinae; D011992:Endosomes / Q000378:metabolism*; D018604:Epitope Mapping; D006801:Humans; D007668:Kidney / Q000737:chemistry / Q000166:cytology / Q000378:metabolism; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009154:Mutation; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions; D013552:Swine
D017305:Annexin A1; D002118:Calcium