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© The Rockefeller University Press, 0021-9525/1998//751 $5.00
The Journal of Cell Biology, Volume 140, Number 4, , 1998 751-765

Article

gp25L/emp24/p24 Protein Family Members of the cis-Golgi Network Bind Both COP I and II Coatomer



Michel Dominguez*, Kurt Dejgaard{ddagger}, Joachim Füllekrug{ddagger}, Sophie Dahan*, Ali Fazel*, Jean-Pierre Paccaud§, David Y. Thomas||, John J. M. Bergeron*, and Tommy Nilsson{ddagger}

* Department of Anatomy and Cell Biology, McGill University, Montreal, PQ, H3A2B2, Canada; {ddagger} Cell Biology Programme, European Molecular Biology Laboratory, 69012 Heidelberg, Germany; || Genetics Group, Biotechnology Research Institute, National Research Council of Canada, H4P2R2 Montreal, Canada; and § Department of Morphology, University of Geneva School of Medicine, 4CH-1211 Geneva, Switzerland

Abstract. Five mammalian members of the gp25L/ emp24/p24 family have been identified as major constituents of the cis-Golgi network of rat liver and HeLa cells. Two of these were also found in membranes of higher density (corresponding to the ER), and this correlated with their ability to bind COP I in vitro. This binding was mediated by a K(X)KXX-like retrieval motif present in the cytoplasmic domain of these two members. A second motif, double phenylalanine (FF), present in the cytoplasmic domain of all five members, was shown to participate in the binding of Sec23 (COP II). This motif is part of a larger one, similar to the F/YXXXXF/Y strong endocytosis and putative AP2 binding motif. In vivo mutational analysis confirmed the roles of both motifs so that when COP I binding was expected to be impaired, cell surface expression was observed, whereas mutation of the Sec23 binding motif resulted in a redistribution to the ER. Surprisingly, upon expression of mutated members, steady-state distribution of unmutated ones shifted as well, presumably as a consequence of their observed oligomeric properties.

We thank the following colleagues for kindly sharing reagents, even before their publication: J. Rothman (Sloan-Kettering, NY) and F. Wieland (University of Heidelberg, Heidelberg, Germany) for gifts of p24 and p23 antibodies; T. Kreis (University of Geneva, Geneva, Switzerland) and C. Harter (University of Heidelberg ) for COP I reagents; H.-P. Hauri (University of Basel, Basel, Switzerland) and J. Saraste (University of Bergen, Bergen, Norway) for sharing p53 and p58 antibodies; H.-D. Söling (University of Göttingen, Göttingen, Germany), for the KDEL receptor antibody; N. Hui (Imperial Cancer Research Fund, London, England) for syntaxin 5 antibodies and J. Ostermann (Vanderbilt University School of Medicine, Nashville, TN) for purified coatomer I. Also, we wish to acknowledge F. Parlati (Genetics Group, Biotechnology Research Institute, Montreal, Canada) for help in cloning; B. Storrie (Blacksburg) and M. Otter (Cell Biology Programme, European Molecular Biology Laboratory [EMBL], Heidelberg, Germany), for critically reading the manuscript. EST's were obtained from the Institute for Genomic Research database TIGR and from GenBank. We would also like to thank the support facilities at EMBL along with A. Bell, P. Cameron, and the Sheldon Biotechnology center at McGill for antibody production, peptide sequencing, and DNA sequencing.

This work was supported by a McGill major Hydro-Québec fellowship to M. Dominguez, a Deutsche Forschungsgemeinschaft to J. Füllekrug, a Swiss National Science Fundation grant (31-43366-95) and Jules Thorn Charitable Overseas Trust to J.-P. Paccaud, and the Medical Research Council of Canada and Glaxo Wellcome Inc. to J.J.M. Bergeron.

M. Dominguez and K. Dejgaard contributed equally to this work.

Address all correspondence to T. Nilsson, Cell Biology Programme, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69012 Heidelberg, Germany. Tel.: (49) 622-138-7294. Fax: (49) 622-138-7512. E-mail: nilsson{at}embl-heidelberg.de

1. Abbreviations used in this paper: CGN, cis-Golgi network; COP, coat proteins; ECL, enhanced chemiluminescence; endo H, endo glycosidase H; ERGIC, ER-to-GOLGI intermediate compartment; GalNAc-T1, N-acetylgalactosylaminyltransferase I; GalT, β1,4-galactosyltransferase; Man II, {alpha}1,2 mannosidase II; MLP, multiple large platform; NAGT I, N-acetylglucosaminyltransferase I; RT, reverse transcriptase; Sialyl, {alpha}2,6-sialytransferase; UDP-GT, UDP-glucuronyltransferase.


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