Endosomal Localization of the Autoantigen EEA1 Is Mediated by a Zinc-binding FYVE Finger

doi:10.1074/jbc.271.39.24048

Endosomal Localization of the Autoantigen EEA1 Is Mediated by a Zinc-binding FYVE Finger*

From the ^‡ Department of Biochemistry, the Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway,
^¶Laboratory of Biotechnology, HiB, N-5020, Bergen, Norway, and
^″Department of Pathology and Immunology, Monash Medical School, Prahran, Victoria 3181, Australia

^§Recipient of a Fellowship from the Norwegian Cancer Society. To whom correspondence should be addressed:
Dept. of Biochemistry, the Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway.
Tel.: 47-22934951; Fax: 47-22508692; E-mail: stenmark{at}ulrik.uio.no

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.

Footnotes

↵^∥ Supported by the Norwegian Research Council and the L. Meltzer Legat.
↵^′ Supported by a grant from the National Health and Medical Council of Australia.
↵* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

EEA1

early endosome antigen 1

DOTAP

N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium methylsulfate

FITC

fluorescein isothiocyanate

MBP

maltose-binding protein

PAR

4-(2-pyridylazo)resorcinol.
↵²H. Stenmark, unpublished results.
- Received March 19, 1996.
- Revision received June 24, 1996.