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J. Biol. Chem., Vol. 279, Issue 17, 17376-17383, April 23, 2004

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Sorting Competition with Membrane-permeable Peptides in Intact Epithelial Cells Revealed Discrimination of Transmembrane Proteins Not Only at the trans-Golgi Network but Also at Pre-Golgi Stages^*

Andrea Soza, Andrés Norambuena, Jorge Cancino, Erwin de la Fuente, Peter Henklein, and Alfonso González¶

From the Departamento de Inmunología Clínica y Reumatología, Facultad de Medicina, and Centro de Regulación Celular y Patología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, 6510260 Santiago, Chile, and Millennium Institute for Fundamental and Applied Biology, 7780344 Santiago, Chile, and Institute of Biochemistry Faculty of Medicine, Humboldt University, 10117 Berlin, Germany

Transmembrane proteins destined to the basolateral cell surface of epithelial cells contain in their cytosolic domain at least two classes of sorting signals: one class promotes exit from the endoplasmic reticulum (ER) and transport to the Golgi complex, and the other class operates at the trans-Golgi network (TGN) specifying segregation into basolateral exocytic pathways. Both kinds of addressing motifs are quite diverse among different proteins. It is unclear to what extent this feature reflects alternative decoding mechanisms or variations in motifs recognized by the same sorting factor. Here we applied a novel strategy based on permeable peptide technology and temperature-sensitive model proteins to study competition between cytosolic sorting motifs in the context of mammalian living cells. We used the transduction domain of HIV-1 Tat protein to make a membrane-permeable peptide of the cytosolic tail of GtsO45, which contains a well characterized ER exit di-acidic (DIE) motif and a tyrosine-based basolateral sorting signal (YTDI). This peptide added to the media inhibited transport of GtsO45 from both ER-to-Golgi and TGN-to-basolateral cell surface in transfected Madin-Darby canine kidney cells. Instead, it did not affect the exocytic trafficking of a GtsO45-derived chimeric protein bearing 30 juxtamembrane residues from the cytosolic domain of the epidermal growth factor receptor that contains a variant ER exit motif (ERE) and an unconventional proline-based basolateral sorting signal. These results not only proved the feasibility of competing for sorting events in intact cells but also showed that distinct plasma membrane proteins can be discriminated at pre-TGN stages, and that basolateral sorting involves different recognition elements for tyrosine-based motifs and an unconventional basolateral motif.

Received for publication, December 3, 2003

^* This work was supported by Fondo Nacional de Areas Prioritarias Grant 13980001 and Fondo Nacional de Ciencia y Tecnologia Grant 3990016. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Depto. Inmunología Clínica and Reumatología, Facultad de Medicina, Pontificia Universidad Católica de Chile, 6510260 Santiago, Chile. Tel.: 56-2-6862713; Fax: 56-2-2229995; E-mail: agonzara{at}med.puc.cl.

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