Independent groups have reported possible recruitment of proteasome subunits to the ER (Kruger et al., 2001
; Lee et al., 2004
; Kalies et al., 2005
; Okuda-Shimizu and Hendershot, 2007
) and suggested that such recruitment may be required for efficient degradation of ER-exported ubiquitinated proteins. Here we demonstrate that JAMP is part of a complex that contains components that facilitate ERAD, in addition to specific proteasome subunits, which is required for efficient clearance of ER-residing misfolded proteins in mammalian cells. JAMP contribution to ERAD may not be global, because initial studies reveal that it does not affect degradation of ER-luminal substrates (ERAD-L) or cytosol-localized misfolded proteins. The effect on clearance of CFTRΔ508, CD3δ, and TCRα shown here implies that JAMP activity may be limited to substrates of ubiquitin ligases/chaperones with which it associates or to proteins positioned proximal to JAMP's location in the ER (i.e., ERAD-M; Carvalho et al., 2006
The notion that JAMP does not serve all ERAD processes is consistent with the finding that JAMP associates with different channel proteins implicated in ERAD, including Sec61 and Derlin1 (Scott and Schekman, 2008
). However, association with Derlin1 but not with Sec61 was seen only after expression of misfolded protein. These observations suggest that JAMP may serve diverse functions in the ER-stress response.
Originally we identified JAMP as an RNF5/RMA1-associated protein. RNF5/RMA1 is an ER membrane-anchored RING finger ubiquitin ligase regulating protein trafficking and degradation (Broday et al., 2004
; Didier et al., 2003
; our unpublished data). RNF5/RMA1 was also shown, together with the ubiquitin ligases CHIP or gp78, to mediate CFTRΔ508 ubiquitination, resulting in its degradation (Younger et al., 2006
; Morito et al., 2008
). JAMP associates with both RNF5/RMA1 and gp78 (data not shown), consistent with the cooperation reported for these ligases in ubiquitination of misfolded CFTR protein (Younger et al., 2006
; Morito et al., 2008
). Association of RNF5 with JAMP results in its ubiquitination but not degradation, suggesting regulation of JAMP's contribution to ERAD by RNF5 (our unpublished data).
We provide important support for the role of JAMP in ERAD by using a C. elegans
strain harboring deletion of most of the jamp-1
. Exposure of these mutants to ER stress promotes developmental arrest at low concentrations of tunicamycin, suggesting that JAMP depletion causes endogenous ER stress during normal growth conditions that is exacerbated by drug treatment. The role of JAMP in causing stress and cell death is likely to also depend on cellular factors including type and degree of stress and its effect on proteaseome recruitment and/or control of JNK activity. This finding is further supported by observation of high basal levels of the UPR-inducible hsp-4
promoter::gfp reporter in jamp-1(ok756)
mutant, findings consistent with observation of worms depleted of derlin, Sel1
, or Ero1
(Ye et al., 2004
Elevated ER stress and induction of UPR markers seen in C. elegans JAMP mutant support the notion that JAMP constitutes an important regulatory component of the ERAD system as part of the UPR. The finding that loss of JAMP increases ER stress and causes accumulation of misfolded and labile proteins seen in both human and worm systems indicates a critical role for JAMP in maintaining efficient clearance of misfolded proteins under normal physiological conditions. Thus, we conclude that JAMP is important to prevent ER stress, a function amplified in response to ER stress.
JNK, a JAMP-associated protein, is also an important regulatory component of ER stress and UPR (Urano et al., 2002
). We have demonstrated that JAMP association with JNK prolongs JNK kinase activity (Kadoya et al., 2005
). The possibility that JNK may phosphorylate JAMP or associated proteins at the ER is currently being investigated. It is expected that the type and degree of stress will dictate JAMP function in coordinated recruitment of proteasomes and/or effect on JNK-mediated apoptosis.
Given the growing evidence supporting redistribution of mammalian proteasomes during specific phases of cell cycle and also in response to cytokine stimuli (Amsterdam et al., 1993
; Wilkinson et al., 1998
; Brooks et al., 2000
; Isono et al., 2007
), it is possible that receptors for proteasome localization exist in mammalian cells and facilitate spatial organization of proteasomes to support timely cellular processes. We propose that JAMP contributes to proteasome localization at the ER as a mechanism for normal maintenance under nonstress conditions, and even more so after ER stress response, when JAMP levels temporarily increase to support the need for proper processing of ERAD.
In summary, this study identifies JAMP as a novel component of the ERAD machinery, which facilitates degradation of ERAD substrates by organizing proteasomes in the vicinity of the ER. JAMP thus emerges as part of the ERAD system, which marks, escorts, and exports ER-resident proteins destined for degradation. Collectively, our findings offer a new paradigm for regulation of proteasome localization and consequently ERAD efficiency.