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J Biol Chem, Vol. 274, Issue 15, 10413-10420, April 9, 1999

A Different Intracellular Distribution of a Single Reporter Protein Is Determined at Steady State by KKXX or KDEL Retrieval Signals

Lavinia V. Lotti, Giovanna Mottola^§, Maria R. Torrisi^¶, and Stefano Bonatti^§

From the  Istituto Nazionale Ricerca sul Cancro di Genova, Sezione di Biotecnologie, Viale Regina Elena 324, 00161, Rome, the ^¶ Dipartimento di Medicina Sperimentale e Patologia, Università di Roma "La Sapienza," Viale Regina Elena 324, 00161, Rome, the  Istituto Dermatologico San Gallicano, Via San Gallicano, 00100, Rome, and the ^§ Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli "Federico II," via S. Pansini 5, 80131 Naples, Italy

To establish the specific contribution to protein topology of KKXX and KDEL retrieval motifs, we have determined by immunogold electron microscopy and cell fractionation the intracellular distribution at steady state of the transmembrane and anchorless versions of human CD8 protein, tagged with KKXX (CD8-E19) and KDEL (CD8-K), respectively, and stably expressed in epithelial rat cells (Martire, G., Mottola, G., Pascale, M. C., Malagolini, N., Turrini, I., Serafini-Cessi, F., Jackson, M. R., and Bonatti, S. (1996) J. Biol. Chem. 271, 3541-3547). The CD8-E19 protein is represented by a single form, initially O-glycosylated: only about half of it is located in the endoplasmic reticulum, whereas more than 30% of the total is present in the intermediate compartment and cis-Golgi complex. In the latter compartments, CD8-E19 colocalizes with -coat protein (COP) (COPI component) and shows the higher density of labeling. Conversely, about 90% of the total CD8-KDEL protein is localized in clusters on the endoplasmic reticulum, where significant co-localization with Sec-23p (COPII component) is observed, and unglycosylated and initially O-glycosylated forms apparently constitute a single pool. Altogether, these results suggest that KKXX and KDEL retrieval motifs have different topological effects on theirs own at steady state: the first results in a specific enrichment in the intermediate compartment and cis-Golgi complex, and the latter dictates residency in the endoplasmic reticulum.

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