Identification of a domain that mediates vesicle aggregation reveals functional diversity of annexin repeats.
Annexins are structurally related proteins that bind phospholipids in a Ca2(+)-dependent manner and possess at least four conserved 70-amino acid repeat domains. The ability of certain annexins to promote contact between vesicle membranes in vitro has prompted the suggestion that these proteins regulate membrane traffic in exocytosis. We have previously found that annexins I and II promote contact between vesicles whereas annexin V does not. In order to understand the mechanism of annexin I-mediated vesicle-vesicle contact, we prepared a monoclonal antibody that specifically inhibits annexin I-mediated vesicle aggregation. We identified the domain of annexin I recognized by this monoclonal antibody by using it to screen an expression library containing random fragments of annexin I cDNA. The antibody identified a fragment encoding amino acids 41-118 (the first repeat plus 8 residues of the amino-terminal tail). We constructed a chimeric protein containing these amino acids of annexin I fused to the second, third, and fourth repeats of annexin V. Transfer of this domain conferred the ability to promote vesicle aggregation, confirming that this domain participates directly in mediating contact between vesicle membranes.
Ernst
J D
JD
Department of Medicine, University of California, San Francisco 94143-0868.
Hoye
E
E
Blackwood
R A
RA
Mok
T L
TL
D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D002678:Chimera; D003001:Cloning, Molecular; D000939:Epitopes / Q000032:analysis; D015723:Gene Library; D014644:Genetic Variation; D006801:Humans; D008081:Liposomes; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010718:Phosphatidylserines; D016133:Polymerase Chain Reaction; D012091:Repetitive Sequences, Nucleic Acid
D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D000939:Epitopes; D008081:Liposomes; D015345:Oligonucleotide Probes; D010718:Phosphatidylserines; D002118:Calcium