Abstract
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.
Highlights
endosome antigen 1 (EEA1), a 162-kDa autoantigen associated with subacute cutaneous systemic lupus erythematosus, is a coiled-coil protein localized to early endosomes and cytosol
In order to investigate if this motif does bind zinc, we first expressed the C-terminal 155 residues of EEA1 as a fusion protein with maltosebinding protein (MBP) in E. coli and purified it on an amylose column
Whereas essentially no Zn2ϩ was found associated with albumin and MBP used as negative controls, the fusion protein was found to contain 1.9 mol equivalents of Zn2ϩ (Fig. 1B), indicating that the cysteine-rich motif coordinates two Zn2ϩ ions
Summary
Materials—Hydroxyurea, isopropyl -D-thiogalactopyranoside, 4-(2pyridylazo)resorcinol (PAR), and p-hydroxymercuriphenyl sulfonate were from Sigma. Cells were first infected for 30 min at room temperature with T7 RNA polymerase recombinant vaccinia virus, and transfected at 37 °C with the appropriate plasmid using DOTAP, as described previously [10], in the presence of 10 mM hydroxyurea. Plasmids—For expression in mammalian cells using the T7 RNA polymerase recombinant vaccinia virus system, the EEA1 sequence [5] was placed in frame behind the 9E10 myc epitope [9], under the T7 promotor of pGEM-1 (Promega). Confocal Immunofluorescence Microscopy—Cells on 11-mm round glass coverslips were fixed with 3% paraformaldehyde and permeabilized with 0.1% Triton X-100, as described previously [10] They were stained with 9E10, anti-Rab, or anti-EEA1 antibodies, followed by rhodamine- or FITC-conjugated secondary antibodies against mouse, rabbit, or human IgG [14]. The secondary structure prediction was obtained with PHD [21]
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