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Vol. 15, Issue 10, 4444-4456, October 2004

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Over-Expression of Rififylin, a New RING Finger and FYVE-like Domain-containing Protein, Inhibits Recycling from the Endocytic Recycling Compartment

Franck Coumailleau ^*, Vincent Das , Andres Alcover , Graça Raposo , Sandrine Vandormael-Pournin ^*, Stéphanie Le Bras ^* , Patricia Baldacci ^* ||, Alice Dautry-Varsat , Charles Babinet ^*, and Michel Cohen-Tannoudji ^* ¶

^* Unité Biologie du Développement, CNRS URA 2578, Institut Pasteur, 75724 Paris Cedex 15, France; Electron Microscopy of Intracellular Compartments, CNRS UMR 144, Institut Curie, 75248 Paris Cedex 05, France; and Unité de Biologie des Interactions Cellulaires, Centré National de la Recherche Scientifique URA 2582, Institut Pasteur, 75724 Paris Cedex 15, France

Submitted March 31, 2004; Accepted June 4, 2004
Monitoring Editor: Keith Mostov

Endocytosed membrane components are recycled to the cell surface either directly from early/sorting endosomes or after going through the endocytic recycling compartment (ERC). Studying recycling mechanisms is difficult, in part due to the fact that specific tools to inhibit this process are scarce. In this study, we have characterized a novel widely expressed protein, named Rififylin (Rffl) for RING Finger and FYVE-like domain-containing protein, that, when overexpressed in HeLa cells, induced the condensation of transferrin receptor-, Rab5-, and Rab11-positive recycling tubulovesicular membranes in the perinuclear region. Internalized transferrin was able to access these condensed endosomes but its exit from this compartment was delayed. Using deletion mutants, we show that the carboxy-terminal RING finger of Rffl is dispensable for its action. In contrast, the amino-terminal domain of Rffl, which shows similarities with the phosphatidylinositol-3-phosphate–binding FYVE finger, is critical for the recruitment of Rffl to recycling endocytic membranes and for the inhibition of recycling, albeit in a manner that is independent of PtdIns(3)-kinase activity. Rffl overexpression represents a novel means to inhibit recycling that will help to understand the mechanisms involved in recycling from the ERC to the plasma membrane.

Abbreviations used: DAPI, 4',6-diamidino-2-phenylindole; ERC, endocytic recycling compartment; MVB, multivesicular bodies; PAG, protein A gold conjugate; PtdIns(3)P, phosphatidyl-inositol-3-phosphate; RACE, rapid amplification of cDNA ends; Rffl, Rififylin; Tf, transferrin; TfR, transferrin receptor; TGN, trans-Golgi network.

Present address: Department of Biology, The Johns Hopkins University, Baltimore, MD 21218

^|| Present address: Biologie et Génétique du Paludisme, Département de Parasitologie, Institut Pasteur, Paris Cedex 15, France.

^¶ Corresponding author. E-mail address: m-cohen{at}pasteur.fr.

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