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Vol. 19, Issue 4, 1328-1336, April 2008

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Gp78 Cooperates with RMA1 in Endoplasmic Reticulum-associated Degradation of CFTRF508

Daisuke Morito^*, Kazuyoshi Hirao^*, Yukako Oda, Nobuko Hosokawa^*, Fuminori Tokunaga, Douglas M. Cyr, Keiji Tanaka^||, Kazuhiro Iwai, and Kazuhiro Nagata^*

*Department of Molecular and Cellular Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8397, Japan and Core Research for Evolution Science and Technology, Japan Science and Technology Agency, Japan; Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan; Department of Molecular Cell Biology, Graduate School of Medicine, Osaka City University, Osaka 545-8585, Japan; Department of Cell and Developmental Biology, UNC Cystic Fibrosis Center, School of Medicine, University of North Carolina, Chapel Hill, NC 27599; and ^||Tokyo Metropolitan Institute of Medical Science, Bunkyo-ku, Tokyo 113-8613, Japan

Submitted June 25, 2007; Revised January 4, 2008; Accepted January 16, 2008
Monitoring Editor: Thomas Sommer

Misfolded or improperly assembled proteins in the endoplasmic reticulum (ER) are exported into the cytosol and degraded via the ubiquitin–proteasome pathway, a process termed ER-associated degradation (ERAD). Saccharomyces cerevisiae Hrd1p/Der3p is an ER membrane-spanning ubiquitin ligase that participates in ERAD of the cystic fibrosis transmembrane conductance regulator (CFTR) when CFTR is exogenously expressed in yeast cells. Two mammalian orthologues of yeast Hrd1p/Der3p, gp78 and HRD1, have been reported. Here, we demonstrate that gp78, but not HRD1, participates in ERAD of the CFTR mutant CFTRF508, by specifically promoting ubiquitylation of CFTRF508. Domain swapping experiments and deletion analysis revealed that gp78 binds to CFTRF508 through its ubiquitin binding region, the so-called coupling of ubiquitin to ER degradation (CUE) domain. Gp78 polyubiquitylated in vitro an N-terminal ubiquitin-glutathione-S-transferase (GST)-fusion protein, but not GST alone. This suggests that gp78 recognizes the ubiquitin that is already conjugated to CFTRF508 and catalyzes further polyubiquitylation of CFTRF508 in a manner similar to that of a multiubiquitin chain assembly factor (E4). Furthermore, we revealed by small interfering RNA methods that the ubiquitin ligase RMA1 functioned as an E3 enzyme upstream of gp78. Our data demonstrates that gp78 cooperates with RMA1 with E4-like activity in the ERAD of CFTRF508.

Address correspondence to: Kazuhiro Nagata (nagata{at}frontier.kyoto-u.ac.jp).

Abbreviations used: CFTR, cystic fibrosis transmembrane conductance regulator; CUE, coupling of ubiquitin to ER degradation; ER, endoplasmic reticulum; ERAD, endoplasmic reticulum-associated degradation; GFP, green fluorescent protein; HA, hemagglutinin; siRNA, small interfering RNA.