Endosomal localization of the autoantigen EEA1 is mediated by a zinc-binding FYVE finger.
EEA1, a 162-kDa autoantigen associated with subacute cutaneous systemic lupus erythematosus, is a coiled-coil protein localized to early endosomes and cytosol. At its C terminus, the protein contains a cysteine-rich motif, which is shared with Vps27, Fab1, and Vac1, yeast proteins implicated in membrane traffic (Mu, F. T., Callaghan, J. M., Steele-Mortimer, O., Stenmark, H., Parton, R. G., Campbell, P. L., McCluskey, J., Yeo, J. P., Tock, E. P., and Toh, B. H. (1995) J. Biol. Chem. 270, 13503-13511). Here we show that this motif constitutes a genuine zinc binding domain, which we term the FYVE finger (based on the first letters of four proteins containing this motif). Profile-based data base searches identified the FYVE finger in 11 distinct proteins. The FYVE finger-containing C terminus of EEA1 was found to bind 2 mol equivalents of Zn2+. Mutations of conserved histidine and cysteine residues in the FYVE motif independently reduced zinc binding to 1 mol equivalent. Confocal immunofluorescence microscopy of transfected HEp2 cells revealed that the C-terminal part (residues 1277-1411) of EEA1 colocalizes extensively with a GTPase-deficient mutant of the early endosomal GTPase Rab5, while deletion of the FYVE finger or mutations that interfere with zinc binding cause a cytosolic localization. These results implicate the FYVE finger in the specific localization of EEA1 to endosomes.
Stenmark
H
H
Department of Biochemistry, the Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway.
Aasland
R
R
Toh
B H
BH
D'Arrigo
A
A
D000595:Amino Acid Sequence; D001324:Autoantigens / Q000737:chemistry / Q000378:metabolism*; D002451:Cell Compartmentation; D002460:Cell Line; D011992:Endosomes / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008180:Lupus Erythematosus, Systemic; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D033921:Vesicular Transport Proteins; D015032:Zinc / Q000378:metabolism*; D016335:Zinc Fingers
D001324:Autoantigens; D008565:Membrane Proteins; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; D015032:Zinc