EEA1, an Early Endosome-Associated Protein.

doi:10.1074/jbc.270.22.13503

EEA1, an Early Endosome-Associated Protein.

EEA1 IS A CONSERVED α-HELICAL PERIPHERAL MEMBRANE PROTEIN FLANKED BY CYSTEINE “FINGERS” AND CONTAINS A CALMODULIN-BINDING IQ MOTIF (*)

From the (1) Department of Pathology, National University of Singapore, Singapore 0511, Singapore, the
(2) European Molecular Biology Laboratory, Heidelberg 69021, Federal Republic of Germany, the
(3) Centre for Transfusion Medicine and Immunology, Flinders Medical Centre, Adelaide 5042, South Australia, and the Department of Pathology and Immunology, Monash Medical School, Melbourne 3181, Australia

^¶ To whom correspondence should be addressed:
Dept. of Pathology and Immunology, Monash Medical School, Commercial Rd., Prahran, Victoria 3181, Australia.
Tel.: 613-276-2713; Fax: 613-529-6484.

Abstract

Early endosomes are cellular compartments receiving endocytosed material and sorting them for vesicular transport to late endosomes and lysosomes or for recycling to the plasma membrane. We have cloned a human cDNA encoding an evolutionarily conserved 180-kDa protein on early endosomes named EEA1 (Early Endosome Antigen1). EEA1 is associated with early endosomes since it co-localizes by immunofluorescence with the transferrin receptor and with Rab5 but not with Rab7. Immunoelectron microscopy shows that it is associated with tubulovesicular early endosomes containing internalized bovine serum albumin-gold. EEA1 is a hydrophilic peripheral membrane protein present in cytosol and membrane fractions. It partitions in the aqueous phase after Triton X-114 solubilization and is extracted from membranes by 0.3 M NaCl. It is a predominantly α-helical protein sharing 17-20% sequence identity with the myosins and contains a calmodulin-binding IQ motif. It is flanked by metal-binding, cysteine “finger” motifs. The COOH-terminal fingers, Cys-X²-Cys-X-Cys-X²-Cys and Cys-X²-Cys-X-Cys-X²-Cys, are present within a region that is strikingly homologous with Saccharomyces cerevisiae FAB1 protein required for endocytosis and with Caenorhabditis elegans ZK632. These fingers also show limited conservation with S. cerevisiae VAC1, Vps11, and Vps18p proteins implicated in vacuolar transport. We propose that EEA1 is required for vesicular transport of proteins through early endosomes and that its finger motifs are required for this activity.

Footnotes

↵^§ Supported by a National Health and Medical Research Council Australian Postdoctoral Fellowship.
↵* This study was supported in part by grants from the National University of Singapore, the Mary Whight Lupus Fellowship and the National Health and Medical Research Council of Australia. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) L40157.
↵¹ The abbreviations used are:

PBS

phosphate-buffered saline

BSA

bovine serum albumin

PAGE

polyacrylamide gel electrophoresis

bp

base pair(s).
↵²H. P. Seelig, unpublished results.