ERQC substrates continue to be degraded in the absence of a functional HRD/DER pathway using a newly identified alternative mechanism that requires ER-Golgi vesicular transport of substrates and the ubiquitin ligase Rsp5p before proteasomal degradation. This alternative system is distinct from the HRD/DER pathway, but together the two comprise the ERQC degradative capacity because only by disabling both pathways is degradation completely blocked. We propose the acronym HIP for the alternative mechanism. Overexpression of ERQC substrates appears to saturate the capacity of the HRD/DER pathway, resulting in the degradation of ERQC substrates to be completely dependent on the HIP pathway.
The ubiquitin ligase Rsp5p is a component of the HIP pathway and together with Hrd1p is responsible for the ubiquitination of CPY*. Only by inactivating both Rsp5p and Hrd1p does CPY* fail to be ubiquitinated and remains completely stable. Rsp5p-dependent ubiquitination involves the ubiquitin-conjugating enzymes Ubc4p and Ubc5p (Gitan and Eide, 2000
), and these enzymes were also required by the HIP pathway to degrade overexpressed CPY*. In addition to its role in ERQC, Rsp5p is responsible for mediating ubiquitin-dependent protein sorting and trafficking within the secretory pathway. The ubiquitination of plasma membrane proteins by Rsp5p results in their internalization and subsequent transport to the vacuole for degradation (Galan et al., 1996
; Hicke and Riezman, 1996
). Rsp5p, acting in concert with Bul1p and Bul2p, can also ubiquitinate proteins such as Gap1p to direct them from the trans-Golgi compartment to the vacuole for degradation (Helliwell et al., 2001
). However, the role of Rsp5p in ERQC is distinct from that involving vacuolar sorting, as the HIP pathway is independent of vacuolar proteases and instead utilizes the proteasome. Furthermore, the disruption of both BUL1
has no effect on the degradation rate of CPY* (unpublished data).
Examination of the mechanisms used by mammalian cells to degrade ERQC substrates suggests extensive overlap with the HIP pathway described here. This similarity includes the observation of the following ERQC substrates in post-ER compartments (ER-Golgi intermediate compartment and/or Golgi subcompartments): unassembled MHC class I molecules (Hsu et al., 1991
), misfolded G protein of vesicular stomatitis virus (Hammond and Helenius, 1994
), mutant forms of sucrase-isomaltase and lysosomal α-glucosidase (Moolenaar et al., 1997
), and precursors of human asialoglycoprotein receptor H2a (Kamhi-Nesher et al., 2001
). The soluble secretory form of IgM molecules in differentiated B lymphocytes is degraded intracellularly in a manner completely dependent on transport to a post-ER compartment (Winitz et al., 1996
), and truncated versions of CFTR reach the Golgi apparatus before degradation by the proteasome (Benharouga et al., 2001
). Further evidence suggesting the involvement of post-ER compartment(s) is that some ERQC components are themselves found in post-ER locations: UDP-glucose:glycoprotein glucosyltransferase (UGGT), an important ERQC glycoprotein folding sensor, is found in post-ER vesicles (Zuber et al., 2001
), whereas endo-α-mannosidase has been implicated in quality control and was localized to the cis/medial-Golgi apparatus (Zuber et al., 2000
How far through the secretory pathway do ERQC substrates such as CPY* progress and what function would delivery of ERQC substrates to the Golgi apparatus serve? CPY* in either DER1
-deficient cells (Knop et al., 1996
) or those lacking an ER-localized proposed ERQC lectin (mnl1
Δ) (Jakob et al., 2001
; Nakatsukasa et al., 2001
) received α1,6-mannose addition but not α1,3-mannose addition, indicating that CPY* reached the cis-Golgi but not the trans-Golgi compartment (Brigance et al., 2000
). Similarly, we have also found that CPY* is delivered to the cis-Golgi compartment when overexpressed in hrd1
Δ cells as evidenced by the addition of α1,6-mannose. A recent report has shown that a heterologously expressed fusion ERQC substrate Kar2 hemagglutinin neuraminidase receives O-linked glycosylation indicative of it reaching the yeast cis/medial-Golgi apparatus, whereas both KHN and CPY* can be found in in vitro ER-derived COPII vesicles (Vashist et al., 2001
). Vashist et al. (2001)
concluded that all the KHN is delivered to the Golgi apparatus, and then is returned to the ER where it is exported to the cytosol and likely ubiquitinated by Hrd1p. However, we suggest another possibility wherein KHN (much like CPY*) is degraded via two distinct pathways (HIP and HRD
), in which a portion of KHN is retained in the ER and is exported and ubiquitinated by Hrd1p while the remaining KHN is transported to the Golgi apparatus and eventually ubiquitinated by Rsp5p.
After transportation to the Golgi apparatus, CPY* must then be exported from within the secretory pathway to the cytosol to be accessible to the cytosolically located Rsp5p. Therefore CPY* is likely returned from the Golgi apparatus to the ER where it is exported to the cytosol, presumably through the translocon, and subsequently ubiquitinated by Rsp5p before proteasomal degradation. A highly speculative alternative model involves the export of CPY* directly from the cis-Golgi compartment to the cytosol by an unidentified mechanism before ubiquitination by Rsp5p. These two possibilities are presented in the model shown in . Unfortunately, the broad subcellular distribution of Rsp5p provides no indication as to which model is correct (Gajewska et al., 2001
). Rsp5p can presumably associate with both the Golgi apparatus to mediate Gap1p sorting (Helliwell et al., 2001
) and the ER, as evidenced by its ubiquitination of the nuclear membrane/ER-restrained transcription factor Spt23p (Hoppe et al., 2000
). Although ERQC substrates are likely returned to the ER from the Golgi apparatus, such a requirement has yet to be definitely demonstrated. The use of Golgi to ER retrograde trafficking mutants is problematic as those mutants tested also show defects in forward transport over the time course required for the degradation experiments (unpublished data). The rationale for delivering ERQC substrates to the Golgi apparatus may be either to (a) access a mechanism responsible for exporting it directly from the Golgi apparatus to the cytosol, or (b) to receive a Golgi apparatus-based modification that signals either its efficient translocation to the cytosol on its return to the ER or its efficient ubiquitination by Rsp5p on its retrotranslocation from the ER to the cytosol. We favor an alternative model in which ERQC substrates are not actively targeted to the Golgi apparatus, but instead are inadequately retained in the ER by the relevant components of the HRD
pathway and are transported to the Golgi apparatus. The insufficient ER retention may be because certain individual ERQC substrates interact poorly with the HRD
components or due to saturation of this pathway. Therefore the cis-Golgi apparatus may act as a quality control catchment system to capture ERQC substrates that have “escaped” the ER before returning them by retrograde transport to the ER.
Figure 9. Summary: ERQC consists of two degradative mechanisms, the HIP and the HRD/DER pathway. Substrates destined for the HRD/DER pathway are recognized as being misfolded or unassembled, and are retained in the ER before being retrotranslocated through the (more ...)
The existence of an alternative degradative system has previously been proposed by us (Hill and Cooper, 2000
) and others (Friedlander et al., 2000
) to explain either the continued degradation of ERQC substrates in HRD
-deficient cells or the surprising finding that cells deficient for the HRD
pathway display no growth defects even when expressing misfolded proteins in the ER. However, double mutants lacking both the UPR and HRD
pathways display significantly impaired growth phenotypes that are further exacerbated by ER stress (Friedlander et al., 2000
; unpublished data). These observations are consistent with our findings that the HIP pathway is likely under UPR control such that the hrd1
Δ mutant is deprived of both of its options in dealing with ER stress. Consistent with this model of UPR-regulated HIP is the identification of a number of genes under UPR transcriptional regulation that are involved in ER to Golgi vesicular transport and therefore likely necessary for the HIP pathway (Travers et al., 2000
The importance of preventing both the aggregation of misfolded proteins in the ER and the transport of these proteins to the cell surface is underscored by the fact that the cell uses at least two distinct mechanisms to effect their degradation. However, this apparent redundancy raises the question of why one would observe any stabilization of ERQC substrates in HRD/DER-deficient cells while the HIP pathway is functional. It is possible that the two systems complement each other, with the HRD/DER pathway comprising a low capacity system for contending with the nominal loads of misfolded proteins accruing in the ER under optimal growth conditions. The HIP pathway might act as a high capacity mechanism that is up-regulated, potentially by the UPR, to accommodate increased levels of ERQC substrates.
In summary, we have identified an HRD/DER-independent degradation mechanism in which ER-Golgi trafficking and Rsp5p-dependent ubiquitination is required before degradation by the proteasome. Further work will identify the purpose of delivering ERQC substrates to the Golgi apparatus, where in the cell ERQC substrate ubiquitination by Rsp5p occurs, and how such substrates gain access to the Rsp5p in the cytosol.