Degradation of RTA in various yeast mutants. (A) Expressed RTA proteins exist in both g1 and g0 forms in yeast. Flag-tagged RTA was overexpressed in wild-type or sec65-1 mutant yeast cells and recovered by immunoprecipitation. RTA immunoprecipitates were mock treated (−) or digested (+) with EndoH, resolved by SDS-PAGE, and visualized by immunoblotting. The arrows indicate RTA proteins modified with one (g1) or no (g0) glycan. In sec65-1 cells (lanes 3 and 4), untranslocated RTA containing its ER signal sequence was also detected and is indicated by an asterisk. (B) Involvement of known ERAD components in RTA degradation. Wild-type (BY4741) and mutant cells containing a GAL1 promoter–regulated Flag-RTA were first grown in raffinose-containing media. Expression of RTA was induced by the addition of galactose. Samples were taken after promoter shutoff at intervals and analyzed by anti-Flag Western blots. Equal amounts of protein extracts were used and confirmed by blotting with anti-Rpt5 antibody in all the promoter shutoff experiments (not depicted). (C) Efficient degradation of glycosylated RTA requires Rad23 and Png1. Pulse-chase analysis of RTA in wild-type and mutant cells was performed as described in Material and methods. (D–F) Quantitation of the data in C for glycosylated (g1) RTA, g0 RTA, and the combined intensity of both g1 and g0 RTA. The amount of proteins was determined by phosphorimager analysis.

Mutation L276Q in Rad23 specifically affects its binding to Png1 and its function in glycosylated RTA degradation. (A) Sequence alignment of the XPCB domain of Rad23 from yeast, mouse, and human. Conserved residues are indicated by the gray boxes. Mutations constructed are indicated above the sequences. Four α helices are indicated below the sequences. (B) Two-hybrid analysis of interactions between Rad23 derivatives and the Rad23 binding proteins Png1, Rad4, Rpn1, Ufd2, Ub, and Rad23. The baits are indicated on the left of the panels. The experiments were performed as in Fig. 1 D. (C) Purified Rad23^L276Q mutant does not bind Png1. Lanes 1 and 2 contain 5% input extracts without or with tagged Png1, respectively. Flag-tagged, wild-type, and mutant Rad23 were expressed in E. coli and purified to homogeneity (not depicted) by previously described procedures (Kim et al., 2004). The mixtures containing Flag beads, equal amounts of Rad23 or Rad23^L276Q proteins, and yeast extracts with His6-tagged Png1 were incubated at 4°C for 2 h. The bound proteins were eluted, fractioned by SDS-PAGE, and immunoblotted with antibody to His6 (lanes 3–5). (D) The Rad23 mutant maintains a fully functional nucleotide excision repair pathway. Yeast cultures were grown to an OD A600 of ∼0.5 and were spotted onto YPD media. Cells were left untreated or exposed to various doses of UV radiation from 20 to 150 J/m². The plates were incubated at 30°C for 2–4 d. rad23Δ cells were transformed with a low-copy plasmid with or without expressing RAD23 derivatives from the RAD23 promoter as indicated. The RAD23 alleles on the plasmids are labeled above the panel. No difference was observed between wild-type and mutant Rad23 in these experiments. The plate exposed to 20 J/m² UV radiation is shown. (E) Temperature sensitivity of cells lacking RAD23 and DSK2 is restored by the L276Q mutant. Yeast strains were grown to an OD A600 of ∼1.2 and were spotted onto YPD media. The plates were incubated at 30 and 37°C for 2–5 d. The plasmids transformed are labeled above the panel. (F) The UFD pathway is not affected by the L276Q mutation. Levels of β-gal activity in rad23Δ cells carried a plasmid bearing Ub^V76-V-βgal, a UFD substrate, and a low-copy plasmid with or without expressing RAD23 derivatives as indicated. The experiments were done as previously described (Kim et al., 2004), and the average values of three experiments with standard deviation are shown. (G) Degradation of glycosylated RTA is impaired in rad23 mutant cells. Pulse-chase analysis was performed with yeast rad23Δ cells coexpressing Flag-RTA with either vector alone or Rad23 derivatives as indicated.

Schematic model for Rad23-mediated substrate proteolysis. Rad23 uses different cofactors to escort distinct substrates to the proteasome. Domains of Rad23 are colored as follows: blue for the UBL motif, brown for the XPCB element, and black for the COOH-terminal UBA domain. Depicted in the top pathway, glycosylated ER proteins (e.g., RTA) are ubiquitylated and transported back to the cytosol. Png1 removes N-linked glycan. The XPCB domain of Rad23 binds Png1, which in turn facilitates the substrate recognition of Rad23. Through interactions with Ub chains and the proteasome mediated by the UBA and UBL domains in Rad23, Rad23 facilitates substrate transfer to the proteasome. The red dots depict Ub, and the brown diamonds depict sugar moiety. In the bottom pathway, Ufd2 (E4) catalyzes Ub chain elongation to substrates attached with one or two Ub (e.g., Spt23^p90 and Ub^V76-V-βgal, possibly Deg1-Sec62). Through interactions mediated by the UBL and UBA domains in Rad23 with Ufd2 and Ub chains, Rad23 recognizes the substrate and facilitates substrate transfer to the proteasome. See Discussion for details.

Interactions between Rad23 and Png1. (A) Domain organization of Rad23. Proteins that bind to each domain are indicated. (B) Png1 is stable in wild-type and rad23Δ cells. Png1 is fused to the Flag epitope, which does not interfere with its function. The stability of Png1 was determined by promoter shutoff assay. Equal sample loading was confirmed by blotting the extracts with anti-Rpt5 antibody (not depicted). The identity of Png1 is indicated on the left. As a negative control, the extract containing no Png1-Flag was processed similarly (lane 1). (C) GST pull-down assays. The mixtures containing glutathione beads, various GST fusion proteins, and yeast extracts with overexpressed His6-tagged Png1 were incubated at 4°C for 2 h. Equal amounts of GST fusion proteins were used (Rao and Sastry, 2002). The bound proteins were eluted, fractioned by SDS-PAGE, and immunoblotted with antibody to His6. Lanes 1 and 2 contain 10% input extracts without or with tagged Png1, respectively. (D) Two-hybrid interactions. Yeast cells were cotransformed with plasmids encoding Gal4 activation domain fused to Png1 indicated on the left with Gal4 DNA binding domain linked to Rad23 or portions of Rad23, as indicated above the panel. Growth on this plate is indicative of protein–protein interaction.

The abilities to remove glycans and bind Ub chains are required for the functioning of the Png1–Rad23 complex. (A) The Png1^H218Y mutant binds Rad23. This shows a two-hybrid analysis of interactions between Png1 derivatives and Rad23. The experiments were performed as in Fig. 1 D. (B) Degradation of glycosylated RTA is compromised in the Png1^H218Y mutant. We cotransformed the plasmids harboring the PNG1 wild type or H218Y mutation with a plasmid expressing Flag-RTA to png1Δ cells. Pulse-chase experiments were done as described in Fig. 2 C. (C) Rad23^uba, which contains the double mutation L183A and L392A, binds Rpn1 and Png1 but not Ub. A two-hybrid assay was used to examine the interactions between Rad23 derivatives and the Rad23 binding partners Png1, Rpn1, and Ub. (D) Glycosylated RTA degradation is impaired in Rad23 mutants defective in Ub binding. Effect of Rad23 mutations on RTA degradation was determined by pulse-chase analysis.

Ufd2 and Png1 define two distinct Rad23-dependent pathways. (A) Ufd2 does not interact with Png1. This shows a two-hybrid analysis of interactions between Ufd2 and Png1 or Rad23. The bait and preys used are indicated. (B) Coimmunoprecipitation analysis of interactions between Ufd2 and Png1 or Rad23. Proteins were extracted from yeast cells expressing various epitope-tagged proteins as indicated and immunoprecipitated with beads coupled to various antibodies. Immunoprecipitates were separated on SDS-PAGE, transferred to a polyvinylidene difluoride membrane, and probed with antibodies. The identity of the bands is indicated on the left. The antibodies used for immunoprecipitation (IP) and Western blot (blot) are indicated to the right of the panels. (C) Degradation of RTA is not regulated by Ufd2. Flag-RTA was expressed in wild-type (BY4741) and ufd2Δ cells. The experiments were performed as in Fig. 2 B. (D) Deg1-Sec62 is glycosylated. Flag-tagged Deg1-Sec62 was recovered by immunoprecipitation with Flag beads and incubated with or without EndoH. Proteins were resolved by SDS-PAGE and immunoblotted with anti-Flag antibody. (E) Degradation of Deg1-Sec62 is Png1 independent. Flag-tagged Deg1-Sec62 was expressed in wild-type (W303-1A) and png1Δ cells. Pulse-chase experiments were performed as described in Material and methods. The asterisk denotes a protein cross-reacting with anti-Flag antibody. The amount of Deg1-Sec62 left is indicated at the bottom of each lane.