To analyze interactions of a luminal misfolded protein, we employed a shortened version of the well-characterized ERAD-L substrate CPY* (sCPY*). This protein contains the last 180 amino acids, including the N-glycosylation site that is part of the degradation signal. This protein is degraded more rapidly than the original CPY* but requires the same ERAD-L components [9
]. We also used a version of sCPY* in which dihydrofolate reductase (DHFR) is fused to the C-terminus (sCPY*-DHFR), which slows the degradation of the protein [9
]. Both sCPY* and sCPY*-DHFR also contain three hemagglutinin (HA)-tags at the C-terminus for detection with HA antibodies.
To analyze direct interactions of sCPY*-HA or sCPY*-DHFR-HA with potential recognition components, we employed a site-specific photocrosslinking method in intact yeast cells [23
]. Single amber stop codons were introduced at various positions of the polypeptide chain (; positions upstream and downstream of the glycosylation site are given negative and positive numbers, respectively). These constructs were expressed together with a suppressor tRNA and a modified tRNA synthetase that charges the tRNA with a phenylalanine derivative containing a photoreactive benzophenone (Bpa). The photoreactive amino acid is then incorporated at the position specified by the amber codon. Irradiation of intact cells leads to crosslinks between the ERAD-L substrate and neighboring proteins (see scheme in ).
Experimental design of in vivo site-specific photocrosslinking experiments
We first tested interactions of sCPY*-HA and sCPY*-DHFR-HA with the soluble luminal domain of Hrd3p (1-767) fused to 13 Myc-tags, a construct that fully complements a HRD3
]. After irradiation, cell extracts were subjected to immunoprecipitation with HA antibodies, followed by SDS-PAGE and immunoblotting with HA- or Myc- antibodies. The HA blot demonstrates that sCPY*-DHFR-HA is degraded less efficiently than the protein lacking the DHFR moiety, resulting in elevated steady-state levels. The Myc-blot shows that the most prominent crosslinks occurred with position +7 near the N-glycosylation site, although crosslinks were also observed with other positions. As expected, without irradiation, no crosslinks occurred ([9
] and Supplementary Fig. S1A
). When similar experiments were performed with a Hrd1p construct containing 13 Myc tags at its C-terminus, crosslinks were only observed with sCPY*-DHFR-HA, and not with sCPY* (). As reported before [9
], the crosslinks with sCPY*-DHFR-HA were observed primarily at positions +38 to +42. Given that the presence of the DHFR moiety increases the substrate population contacting Hrd1p but not that contacting Hrd3p, it appears that the rate-limiting step in the degradation of this substrate occurs after Hrd3p- function.
Consistent with the idea that substrate-Hrd3p interaction occurs early in ERAD-L, the crosslinking efficiency seen with sCPY* containing a probe at position +7 was not affected by deletion of ERAD components (). Even the simultaneous deletion of several ERAD components had no effect (). The glycosidase Htm1p was also not required, indicating that Hrd3p interaction is independent of an α1,6-mannose signal in the carbohydrate chain. Indeed, sCPY* lacking a N-glycosylation site crosslinked with the same efficiency as the glycosylated protein (). Substrate interaction requires residues 1-392 of Hrd3p (Fig. S1B
), consistent with previous suggestions [25
]; the putative Yos9p-/Hrd1p-binding domain can be deleted without affecting crosslinking to substrate. Collectively, these data suggest that Hrd3p is involved in the early recognition of unstructured polypeptide segments. Such a chaperone-like function of Hrd3p is supported by the observation that Hrd3p interacts with many positions in sCPY* when substrate accumulates in the ER lumen. For example, positions that interacted only weakly with Hrd3p in wild type cells, crosslinked significantly stronger in ERAD-deletion mutants () or in a Hrd1p mutant with a defective RING finger domain (C399S) (Fig. S2
). Thus, weaker binding sites for Hrd3p appear to exist throughout the unfolded polypeptide chain and become more prominent at elevated substrate levels.
Photocrosslinking to Hrd3p in different ERAD mutants
To investigate whether Yos9p contributes to polypeptide binding or is exclusively involved in sugar recognition, we performed similar crosslinking experiments in a strain expressing Yos9p containing 13 Myc-tags and an HDEL ER-retention signal at its C-terminus. As with Hrd3p, the tagged protein complements a deletion strain (data not shown). Using sCPY*-DHFR-HA, crosslinks were only observed with position −69 (); no crosslinks were seen around the carbohydrate attachment site. The crosslinks at position −69 were significantly reduced when the substrate was non-glycosylated or when Hrd3p was absent (). When both effects were combined, essentially no crosslinking to Yos9p was observed (). Deletion of HTM1
had only a moderate effect on Yos9p crosslinking, while deletion of HRD1
did not affect it at all (). The specificity for position −69 was not altered in a hrd1Δ
strain (Supplementary Fig. S3
). These results are consistent with the idea that Yos9p does not interact with the polypeptide segment around the carbohydrate attachment site that signals degradation. Rather, it appears that a distant segment comes in contact with Yos9p once Yos9p binds to the α1,6-signal in the carbohydrate chain. It should be noted that sCPY* without the attached DHFR domain did not give crosslinks at all (data not shown), consistent with the assumption that Yos9p acts downstream of Hrd3p.
Photocrosslinking of sCPY*-DHFR-HA to Yos9p
Finally, we studied the interaction of sCPY*-DHFR-HA with Der1p containing 13 myc-tags (Der1p-13myc), which retains partial activity [9
]. Previous work has shown that position +24 of the substrate gives the most prominent crosslinks [9
]. These crosslinks were still present in yos9Δ
cells, but disappeared when USA1
was deleted (). The interaction could be restored when fragments of Usa1p were expressed that are defective in either Usa1p-Hrd1p or Usa1p-Usa1p interaction (). All constructs retain the C-terminal segment (residues 584-838) that has been proposed to interact with Der1p [11
]. These data therefore support the idea that there are parallel pathways of substrate recruitment, one involving Hrd3p/Yos9p and another involving Der1p. In addition, it appears that Der1p does not need to be recruited to the Hrd1p complex to interact with substrate. The role of Usa1p may be in stabilizing Der1p, as the levels of Der1p are reduced in a usa1Δ