Essential role of Ca2+/calmodulin in Early Endosome Antigen-1 localization.
Ca2+ is an essential requirement in membrane fusion, acting through binding proteins such as calmodulin (CaM). Ca2+/CaM is required for early endosome fusion in vitro, however, the molecular basis for this requirement is unknown. An additional requirement for endosome fusion is the protein Early Endosome Antigen 1 (EEA1), and its recruitment to the endosome depends on phosphatidylinositol 3-phosphate [PI(3)P] and the Rab5 GTPase. Herein, we demonstrate that inhibition of Ca2+/CaM, by using either chemical inhibitors or specific antibodies directed to CaM, results in a profound inhibition of EEA1 binding to endosomal membranes both in live cells and in vitro. The concentration of Ca2+/CaM inhibitors required for a full dissociation of EEA1 from endosomal membranes had no effect on the activity of phosphatidylinositol 3-kinases or on endogenous levels of PI(3)P. However, the interaction of EEA1 with liposomes containing PI(3)P was decreased by Ca2+/CaM inhibitors. Thus, Ca2+/CaM seems to be required for the stable interaction of EEA1 with endosomal PI(3)P, perhaps by directly or indirectly stabilizing the quaternary organization of the C-terminal FYVE domain of EEA1. This requirement is likely to underlie at least in part the essential role of Ca2+/CaM in endosome fusion.
Lawe
Deirdre C
DC
Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 10615, USA.
Sitouah
Nachida
N
Hayes
Susan
S
Chawla
Anil
A
Virbasius
Joseph V
JV
Tuft
Richard
R
Fogarty
Kevin
K
Lifshitz
Lawrence
L
Lambright
David
D
Corvera
Silvia
S
D020816:Amino Acid Motifs; D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000378:metabolism; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology; D002522:Chlorocebus aethiops; D011992:Endosomes / Q000378:metabolism / Q000502:physiology; D008081:Liposomes; D008561:Membrane Fusion / Q000502:physiology; D008565:Membrane Proteins / Q000378:metabolism* / Q000502:physiology; D008856:Microscopy, Fluorescence; D009154:Mutation; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D020836:Protein Structure, Quaternary / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology; D033921:Vesicular Transport Proteins; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism
D002118:Calcium; D002147:Calmodulin; D008081:Liposomes; D008565:Membrane Proteins; D019869:Phosphatidylinositol 3-Kinases; D011994:Recombinant Proteins; D013449:Sulfonamides; D033921:Vesicular Transport Proteins; D020696:rab5 GTP-Binding Proteins; C093846:early endosome antigen 1; C034700:N-(6-aminohexyl)-1-naphthalenesulfonamide