The traditionally accepted role for ERManI in ERAD is that it functions as a mannosidase in the ER, contributing to the generation of degradation signals in response to the persistent retention of misfolded glycoproteins in the early secretory pathway of mammalian cells (Wu et al., 2003
; Lederkremer, 2009
). However, our recent discovery of Golgi-localized ERManI, and the fact that a portion of misfolded glycoproteins can escape the ER (this study; Sifers et al., 1989
; Hosokawa et al., 2007
) and are recycled through the Golgi complex prior to proteasome-mediated intracellular disposal in both yeast and mammalian cells (Hammond and Helenius, 1994
; Caldwell et al., 2001
; Vashist et al., 2001
; Kincaid and Cooper, 2007b
), demanded a revision of the functional mechanism in which ERManI operates in the Golgi complex as a component of ERAD.
Currently, COPI-mediated vesicle trafficking is the only known route for the recycling of Golgi-situated proteins back to the ER. Theoretically, this routing system would also serve as the prominent mechanism by which ERAD substrates are retrieved from post-ER compartments. The identification of a direct interaction between ERManI and γ-COP supports this idea, and served to validate the notion that γ-COP plays a functional role in the intracellular disposal of the classical ERAD substrate, NHK. The identification of γ-COP–binding sites in the ERManI cytoplasmic tail, plus the consequences of mutating them, provided additional validation for the functional partnership. Our study provides, for the first time, experimental evidence to support this hypothesis.
The incorporation of protein cargo into COPI vesicles is a selective process. The selection of luminal cargo is mediated by groups of transmembrane proteins designated as cargo receptors (Nickel et al., 1998
). In this study, we discovered that the ERManI/γ-COP complex associates with the luminal ERAD substrate NHK. Such an association likely brings ERAD substrates in close proximity to the COPI coatomers, thus allowing their selective recruitment into COPI vesicles. In this regard, ERManI functions similar to a cargo receptor that retains escaped proteins in the Golgi complex prior to facilitating their loading into COPI vesicles. In support of this notion, in the absence of ERManI, the retention of NHK is impaired, allowing its enhanced secretion. The interconnected and transient natures of the aforementioned events were further validated by the enhanced secretion of NHK in response to the mutation of γ-COP-binding sites in the ERManI cytoplasmic tail. Also, RNAi-mediated knockdown of endogenous γ-COP enhanced the association between NHK and ERManI, implying that their dissociation either precedes vesicle loading or is coupled to delivery of the associated ERAD substrate to the ER. Enhanced secretion, possibly caused by saturation of the retention process in the absence of dissociated ERManI, implies that the freed mannosidase is recycled to the Golgi complex where it can functionally support the retention process. Unlike classic cargo receptors recruited with their cargo into COPI vesicles, the movement of ERManI from the Golgi complex has not been detected in our studies. On the basis of these findings, we propose a model in which ERManI contributes to a Golgi-based quality control module that captures ERAD substrates that have escaped from the ER and facilitates their loading into COPI vesicles via a dynamic process driven by an association with γ-COP (). However, considering the detection limits of our methodology, especially after proteins are diluted upon recycling back to the ER, our findings do not exclude the possibility that a trace amount of ERManI actually recycles in COPI vesicles. In fact, a previous proteomics study has indicated that ERManI is present in COPI vesicles (Gilchrist et al., 2006
), supporting this notion.
The absence of obvious interaction domains in the crystallized three-dimensional structure of the mannosidase (Dole et al., 1997
) suggests that the association between ERManI and luminal ERAD substrates likely takes place within the context of a much larger complex. In this regard, Cormier et al. (2009
) reported the existence of a glycan-independent interaction between EDEM1, an evolutionary relative of ERManI, and NHK. Because the human orthologues of ERManI and EDEM1 share 33% sequence identity and 49% similarity (Kanehara et al., 2007
), it is possible that a similar interaction might exist between ERManI and NHK. Currently, intensive immunoaffinity purification/proteomic investigations are underway to test the validity of this hypothesis and/or to both identify and characterize the components of a much larger complex. An additional prediction, which must be revisited as the subject of future investigations, is that degradation signals initiated by the enzymatic removal of terminal α-1, 2–linked mannose units diverts the recycled molecules away from additional rounds of folding events in response to their recognition by ER-situated lectins (Kanehara et al., 2007
; Yoshida and Tanaka, 2010
), all of which precedes their dislocation into the cytoplasm for proteasomal degradation.
Currently, we are not aware of the precise mechanism responsible for regulating the concentration of Golgi-situated ERManI, although previously published experiments have indicated that a significant fraction of the newly synthesized molecules are eliminated by lysosomal proteases (Wu et al., 2007
). It is apparent, however, that the intracellular residence of the mannosidase does not rely on its direct binding to γ-COP, because ERManIγ-COP-def
continued to localize to the Golgi complex (Figure S4A). Also, unlike the capacity of γ-COP knockdown to diminish the steady-state level of the wild-type mannosidase, the ERManIγ-COP-def
did not exhibit a diminished intracellular concentration (), implying that the capacity of γ-COP to regulate the concentration of the mannosidase does not rely on a direct interaction. It should be noted, however, that the RNAi-mediated knockdown of γ-COP did result in the dispersal of endogenous ERManI throughout the cytoplasm (Figure S4B). In support of the notion that this phenomenon reflected fragmentation of the Golgi complex (Lippincott-Schwartz et al., 1990
; Beller et al., 2008
), we observed that incubating cells in media that contained brefeldin A (BFA), an antibiotic that fragments the Golgi complex by interfering with COPI complex assembly (Peyroche et al., 1999
; Mossessova et al., 2003
; Renault et al., 2003
), led to a significantly diminished steady-state level of ERManI (Figure S4C). However, in contrast to that notion, incubation with nocodazole, a potent drug that is able to induce Golgi disassembly via the depolymerization of microtubules (Lippincott-Schwartz et al., 1990
; Dinter and Berger, 1998
), did not alter the steady-state level of ERManI (Figure S4C). Altogether, our observations support the idea that the steady-state level of ERManI is likely regulated by protein trafficking in the secretory pathway, rather than by the structural integrity of the Golgi apparatus.
In summary, our study indicates that Golgi-localized ERManI apparently serves as a linchpin in an expanded mammalian ERAD network in which functional modules are linked by the vesicular transport of cargo. This arrangement provides the temporospatial distancing of essential proteostasis events that would otherwise directly compete with one another if performed in the same compartment. In this regard, ERManI contributes to the establishment of a multifunctional gatekeeper.