Proteins misfolded in the ER are degraded by the proteasome in the cytosol via a process called ERAD, in which misfolded proteins are recognized inside the ER lumen, targeted to a channel, extracted from the ER membrane, and delivered to the proteasome (Wilhovsky et al., 2000
; Tsai et al., 2002
). Misfolded glycoproteins are recognized by presentation of an N-linked oligosaccharide processing intermediates, such as Man8GlcNAc2 (Jakob et al., 1998
), which acts as a motif that triggers binding to EDEM (Hosokawa et al., 2001
). In this way, glycoproteins that are unable to fold properly are transferred from calnexin, a glucoprotein-specific molecular chaperone, to EDEM (Molinari et al., 2003
; Oda et al., 2003
). Subsequently, the cytosolic multifunctional protein p97 acts to extract the glycoprotein from the ER membrane (Dai and Li, 2001
; Ye et al., 2001
; Braun et al., 2002
; Kobayashi et al., 2002
; Meyer et al., 2002
). However, the events that link these two steps have remained undefined.
The recent identification of Derlin-1 has brought new insights into the molecular mechanism of the US11-mediated retrotranslocation of class I HC, providing a missing link between events in the ER and those in the cytosol (Lilley and Ploegh, 2004
; Ye et al., 2004
). Further, the findings that Derlin-1 binds to misfolded and ubiquitinated proteins and that RNA interference of Derlin-1 in Caenorhabditis elegans
evokes ER stress suggest a more generalized role of Derlin-1 in ERAD (Ye et al., 2004
). Recent studies demonstrated that Derlin-1 is associated with ubiquitin ligases such as HRD1 and gp78 via binding to p97 and vasolin-containing protein (VCP)–interacting membrane protein and that Derlin-2 also forms such a multiprotein complex, consisting of p97, VCP-interacting membrane protein, HRD1, and HRD3/SEL1L (Lilley and Ploegh, 2005
; Ye et al., 2005
). However, it remains to be determined whether Derlin-2 is required for ERAD.
We show that the properties of Derlin-2 and -3 are exactly those expected for proteins able to provide the missing link between EDEM and p97 in the process of degrading glycoproteins misfolded in the ER. Derlin-2 and -3 are transmembrane proteins that span the ER membrane multiple times ( and ) and are required for the ERAD of misfolded glycoprotein ( and ). They are associated with the degradation substrate, p97, and EDEM ( and ). p97 and EDEM form a complex only in the presence of Derlin-2 (), and Derlin-2 mediates the association of p97 with the degradation substrate (). In contrast, Derlin-1 does not mediate the association of p97 with either EDEM or the degradation substrate (). This specificity might be attributable to differences in amino acid sequences between Derlin-1 and -2 () or, as Derlin-1 and -2 or -3 appear to be associated with different cellular proteins (), to differences in accessory proteins.
Interestingly, MEFs express only Derlin-2 mRNA, whereas human cells express both Derlin-2 and -3 mRNAs ( and and not depicted). In addition, Derlin-2 and -3 mRNAs are distributed differently in various human tissues (). The finding that the knockdown of Derlin-2 or -3 alone effectively blocked the degradation of a misfolded glycoprotein () suggested that they may form heterooligomers to function when expressed simultaneously, and this was indeed shown to be the case (). Similarly, they may form homooligomers when expressed singularly. It is of great interest whether Derlin-2 and -3 themselves can form a channel through which misfolded glycoproteins move from the ER lumen to the cytosol, as has been proposed for Derlin-1 (Lilley and Ploegh, 2004
; Ye et al., 2004
). We are also aware of a recent study showing that the proteasome binds directly to Sec61 (Kalies et al., 2005
), supporting the idea that misfolded proteins are retrotranslocated from the ER to the cytosol through the translocon as originally proposed (Wiertz et al., 1996b
). Thus, it appears that we are a far way from any conclusive determination on this issue.
The UPR consists of transcriptional control only in yeast, and the Ire1p-mediated program covers transcriptional induction of not only ER chaperones but also components of ERAD in response to ER stress (Kaufman, 1999
; Mori, 2000
; Patil and Walter, 2001
). On this basis, the UPR plays little role in determining the fate of unfolded or misfolded proteins in yeast ER. The activated refolding system and degradation system may deal with unfolded proteins in a competitive manner. In contrast, metazoan cells have developed the ability to attenuate translation via activation by PERK in response to ER stress, thereby decreasing the burden on the ER (Ron, 2002
). Mammals have further evolved two signaling pathways, namely the ATF6 and IRE1–XBP1 pathways, for transcriptional induction of ER chaperones and components of ERAD in response to ER stress (Mori, 2003
). The transcription factor ATF6 is activated by a posttranslational mechanism, whereas the transcription factor XBP1 is activated by a posttranscriptional mechanism, causing the active form of ATF6 to be detected in cell extracts earlier than the active form of XBP1. In addition, XBP1 has broader target specificity than ATF6. Thus, the transcription of genes unaffected by the active form of ATF6 can be later induced by the active form of XBP1, allowing a time-dependent decision (Yoshida et al., 2001
Accumulating evidence indicates that the ATF6 pathway mainly controls the expression of ER chaperones, whereas the IRE1–XBP1 pathway regulates the expression of not only ER chaperones but also components of ERAD such as EDEM, EDEM2, and HRD1 (Kaneko et al., 2002
; Okada et al., 2002
; Lee et al., 2003
; Yoshida et al., 2003
; Olivari et al., 2005
). Our present analysis adds Derlin-1, -2, and probably -3 to the list of targets of the IRE1–XBP1 pathway ( and ). We have also found that the misfolded glycoprotein NHK was degraded more slowly in IRE1α−/− cells than in IRE1α+/+ cells (Yoshida et al., 2003
), whereas the misfolded nonglycoprotein NHK(QQQ) was degraded at similar rates in both cell types (unpublished data). These results strongly support our time-dependent phase-transition model for determining the fate of proteins that are unfolded or misfolded in the mammalian ER, at least as far as glycoproteins are concerned, in which the ATF6-mediated unidirectional phase (refolding only) is shifted to the XBP1-mediated bidirectional phase (refolding plus degradation) depending on the quality, quantity, or both of unfolded or misfolded proteins in the ER (Yoshida et al., 2003
). The availability of multiple pathways for transcriptional control confers diversity for mammalian cells to adjust to the accumulation of unfolded proteins in the ER.
List of mammalian ERAD components regulated by the IRE1–XBP1 pathway