Accumulated evidence suggests that the ERAD and ER exits of misfolded proteins compete with each other. However, the mechanism of ER retention of ERAD substrates remained unclear because ER retention and later steps in the ERAD process are usually coupled with each other. In the present study, we assessed ER retention of misfolded CPY/CPY* fusion proteins, especially CPY*-CPY, by monitoring their vacuolar traffic, which competes with ER retention, independent of the later steps in ERAD. We thus found that the two ERAD factors Yos9p and Hrd1p play essential roles in ER retention of misfolded proteins. The ER retention depends on Yos9p and Hrd1p and correlates well with the interactions of Yos9p, but not BiP, with substrate proteins. The interaction between CPY*-CPY and Hrd1p, as well as ER retention of CPY*-CPY, is impaired in yeast cells lacking Yos9p (, and ). However in the absence of Yos9p, overexpression of Hrd1p can still recover the ER retention of CPY*-CPY (), suggesting that Hrd1p itself has an activity to interact with misfolded proteins and retain them in the ER. Therefore, efficient ER retention of misfolded proteins requires the recognition of misfolded proteins and their delivery to Hrd1p by Yos9p (), although Yos9p is not essential but increases efficiency of this process mediated by Hrd1p. Because inhibition of degradation in ERAD in general does not increase vacuolar traffic of CPY*-CPY, degradation and ER retention of misfolded proteins are separable processes, and ER retention becomes irreversible past a certain step in ERAD. ER retention is likely committed by Yos9p recognition and delivery of misfolded proteins to Hrd1p.
FIGURE 9: A model for ER retention of CPY*-CPY by ERAD factors. (A) Yos9p likely functions in the committed step of ER retention of CPY*-CPY in addition to its role in the glycan-dependent degradation step. (B) CPY*-CPY is missorted to ER exit in the absence of (more ...)
Although CPY*-CPY was preferentially sorted to the ERAD pathway, CPY-CPY* was transported to the vacuole as efficiently as CPY-CPY in spite of the presence of the misfolded CPY* unit. Yos9p recognizes CPY-CPY* less efficiently than CPY*-CPY, indicating that not only the presence of a misfolded domain, but also the position of the misfolded domain is important for recognition by Yos9p. In the present case, the N-terminally placed misfolded CPY* unit was recognized by Yos9p more efficiently than the C-terminally placed one. This may perhaps connected with the fact that secretory proteins enter the ER lumen from their N-termini. In this scenario, recognition of misfolded domains starts as early as the protein translocation process. Further study will be needed to test this possibility.
Although BiP is required for ERAD and can interact with proteins with misfolded domains (Nishikawa et al., 2001
), BiP binding is not sufficient for sorting of proteins to degradation by ERAD. Only proteins efficiently interacting with Yos9p are efficiently retained in the ER and degraded by ERAD. Because BiP contributes to maintaining solubility of misfolded proteins in the ER (Nishikawa et al., 2001
), misfolded proteins interacting with both BiP and Yos9p are likely sorted to the ERAD pathway. Depletion of Lhs1p, a nucleotide exchange factor for BiP, did not affect the fate of CPY*-CPY (Supplemental Figure S8), which also supports the interpretation that BiP does not function in the sorting process.
PDI is another ER-resident chaperone required for ERAD of misfolded proteins, including CPY*. However, overexpression of PDI but not PDIΔ252-277 inhibited both ERAD and vacuolar transport of CPY*-CPY, which was accompanied by significant increase in the interaction of CPY*-CPY with PDI (). Preferential binding of chaperone PDI to CPY*-CPY could simply render it unavailable for further sorting to ERAD or ER exit pathway. Because PDI functions in oxidative protein folding, interaction between PDI and CPY*-CPY might take place in the early step of the ER quality control. CPY*-CPY trapped by PDI was probably retained in the ER by the action of the C-terminal ER retention signal of PDI.
Recent cross-link experiments suggested that Hrd3p functions upstream of Yos9p (Stanley et al., 2011
). Consistent with this, we found that overexpression of Hrd3p suppressed vacuolar transport of CPY*-CPY by enhancing interaction of CPY*-CPY with Yos9p and Hrd1p (). However, depletion of Hrd3p did not decrease the amount of Yos9p coprecipitated with CPY*-CPY (). Because depletion of Hrd3p causes decrease in the Hrd1p level as well (Plemper et al., 1999
; Gardner et al., 2000
; Gauss et al., 2006
; unpublished data), the observed enhancement of the vacuolar transport of CPY*-CPY in the hrd3
Δ mutant could have merely arisen from decreased Hrd1p (). Perhaps the decreased interaction between CPY*-CPY and Yos9p caused by Hrd3p depletion may be compensated by the opposite effect due to decrease in the Hrd1p level.
It was unexpected that, although the N-linked glycan attached to Asn479 of CPY*-CPY () functions as a degradation signal as in the case of CPY* (Kostova and Wolf, 2005
; Spear and Ng, 2005
), efficient ER retention of CPY*-CPY did not require this degradation signal (). Coimmunoprecipitation experiments showed that CPY*(N479Q)-CPY interacted with both Yos9p and Hrd1p as efficiently as CPY*-CPY ( and Supplemental Figure S7A). How can we reconcile those divergent results on the role of the degradation signal in ER retention and ERAD? Perhaps, whereas Yos9p can target CPY*-CPY to Hrd1p for ER retention in a glycan ERAD signal–independent manner, Yos9p may be also involved in the later step at the level of the Hrd1p complex for decoding the glycan signal for degradation. Although the glycan degradation signal is dispensable for ER retention of CPY*-CPY, Yos9p-R200A containing a mutation in the MRH domain, which was suggested to mediate the glycan signal recognition, is defective in this process (). Yos9p-R200A efficiently interacted with CPY*-CPY (Supplemental Figure S7B), but the interaction between CPY*-CPY and Hrd1p was significantly impaired in the yos9
-R200A mutant cells (Supplemental Figure S7C). The MRH domain of Yos9p may be required for targeting of misfolded proteins to Hrd1p by, for example, interacting with components of the Hrd1p complex.
OS-9 is a mammalian orthologue of Yos9p, and involvement of OS-9 variants OS-9.1 and OS-9.2 in ER retention of misfolded proteins was reported (Bernasconi et al., 2008
). However, mechanisms of Yos9p and the OS-9 variants functioning in the ER retention of misfolded protein seem to be different. Whereas overexpression of OS-9 variants enhances ER retention of misfolded proteins (Bernasconi et al., 2008
), overproduction of Yos9p itself does not increase retention efficiency of CPY*-CPY in the ER (). The MRH domain of the OS-9 variants but not of Yos9p is dispensable for the ER retention of misfolded proteins. It will be interesting to ask whether Hrd1p orthologues are involved in the OS-9 variants–mediated ER retention process in mammalian cells.
In summary, by using CPY* fusion proteins as new model ERAD substrates, we dissected the process mediated by Yos9p in ERAD and revealed a novel role of Yos9p in the early steps of ERAD, that is, ER retention of misfolded proteins by delivering them to Hrd1p. Revealing the precise role of Yos9p in the later step of ERAD, most likely in cooperation with the Hrd1p complex, is open to future studies.