A Complex of Pdi1p and the Mannosidase Htm1p Initiates Clearance of Unfolded Glycoproteins from the Endoplasmic Reticulum

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1 Article A Complex of Pdi1p and the Mannosidase Htm1p Initiates Clearance of Unfolded Glycoproteins from the Endoplasmic Reticulum Robert Gauss, 1,4 Kazue Kanehara, 2,4,5 Pedro Carvalho, 3 Davis T.W. Ng, 2, * and Markus Aebi 1, * 1 Institute of Microbiology, Department of Biology, Swiss Federal Institute of Technology Zurich, 8093 Zurich, Switzerland 2 Temasek Life Sciences Laboratory and Department of Biological Sciences, National University of Singapore, Singapore Department of Cell and Developmental Biology, Centre for Genomic Regulation (CRG), Barcelona, Spain 4 These authors contributed equally to this work 5 Present address: Max Planck Institute for Plant Breeding Research, Cologne, Germany *Correspondence: davis@tll.org.sg (D.T.W.N.), markus.aebi@micro.biol.ethz.ch (M.A.) DOI /j.molcel SUMMARY Endoplasmic reticulum (ER)-resident mannosidases generate asparagine-linked oligosaccharide signals that trigger ER-associated protein degradation (ERAD) of unfolded glycoproteins. In this study, we provide in vitro evidence that a complex of the yeast protein disulfide isomerase Pdi1p and the mannosidase Htm1p processes Man 8 GlcNAc 2 carbohydrates bound to unfolded proteins, yielding Man 7 GlcNAc 2. This glycan serves as a signal for HRD ligase-mediated glycoprotein disposal. We identified a point mutation in PDI1 that prevents complex formation of the oxidoreductase with Htm1p, diminishes mannosidase activity, and delays degradation of unfolded glycoproteins in vivo. Our results show that Pdi1p is engaged in both recognition and glycan signal processing of ERAD substrates and suggest that protein folding and breakdown are not separated but interconnected processes. We propose a stochastic model for how a given glycoprotein is partitioned into folding or degradation pathways and how the flux through these pathways is adjusted to stress conditions. INTRODUCTION Endoplasmic reticulum (ER) quality control (ERQC) monitors protein biogenesis and redirects unfolded and flawed polypeptides to the cytosol for destruction by the 26S proteasome (Buchberger et al., 2010; Määttänen et al., 2010; Stolz and Wolf, 2010). Asparagine (N)-linked oligosaccharides (NLOs) coupled to the majority of polypeptides synthesized in the ER are directly involved in this process (Aebi et al., 2010). Glycosylhydrolases process NLOs in a time- and protein conformationdependent manner and produce signals that reflect the current folding and maturation state of the attached polypeptide (Helenius and Aebi, 2004). Accordingly, molecular chaperones assist folding of nascent polypeptides presenting Glc 3 0 Man 9 GlcNAc 2 glycans, while specialized ubiquitin-protein ligases of the ERassociated protein degradation (ERAD) system clear glycoproteins displaying a bipartite signal unfolded domains and a Man 5 7 GlcNAc 2 sugar from the compartment (Hirsch et al., 2009). Thus, cleavage of more than one mannosyl residue by class 47-glycosylhydrolases (GH47s) extracts proteins from the folding process and directs them to disposal in the cytosol (Aebi et al., 2010; Helenius and Aebi, 2004). The yeast Saccharomyces cerevisiae expresses two ER-localized GH47-proteins with an a1,2-mannosidase homology (MH) domain: Mns1p (ER mannosidase 1) and Htm1p (homolog to ER mannosidase 1, also termed Mnl1p for mannosidase-like 1). In vitro, Mns1p removes the terminal mannosyl residue of the B branch of free Man 9 GlcNAc 2 oligosaccharides (Jelinek- Kelly et al., 1985; Jelinek-Kelly and Herscovics, 1988). In vivo, Mns1p processes the entire pool of protein-bound Man 9- GlcNAc 2 present in the ER (Jakob et al., 1998). In MNS1-deficient cells, however, breakdown of ERAD model proteins from the ER is impeded (Jakob et al., 1998). Although Mns1p has no peptide specificity, glycoproteins appear to be protected from degradation until Mns1p processes attached glycans. Deletion of the second GH47 gene, HTM1, also delays turnover of ERAD model substrates (Jakob et al., 2001; Nakatsukasa et al., 2001). While initially considered a lectin, recent genetic experiments provide evidence that Htm1p actively engages in removing the outmost mannosyl moiety from the C branch of the glycan (Clerc et al., 2009). The product Man 7 GlcNAc 2 oligosaccharide exposes an a1,6-linked mannosyl residue that is the substrate of Yos9p, the lectin subunit of the HRD ligase (Carvalho et al., 2006; Denic et al., 2006; Gauss et al., 2006; Quan et al., 2008). Mns1p activity is a precondition for Htm1p cleavage, suggesting that the two enzymes function in subsequent steps (Clerc et al., 2009). In addition to its N-terminal MH domain, Htm1p features a large C-terminal (CT) domain that is conserved only among fungal orthologs of Htm1p and does not display significant homology to any known functional domain (Sakoh-Nakatogawa et al., 2009). Truncating this domain disturbs the function of Htm1p and prevents association with its binding partner, the oxidoreductase Pdi1p (Clerc et al., 2009). Yeast Pdi1p is an essential folding catalyst that introduces disulfide bonds into its substrates and rearranges or reduces 782 Molecular Cell 42, , June 24, 2011 ª2011 Elsevier Inc.

2 pre-existing ones (Farquhar et al., 1991; Gilbert, 1997; Hatahet and Ruddock, 2009). Besides its oxidoreductase activity, Pdi1p acts as a molecular chaperone and also assists in the folding of proteins that do not contain disulfide bonds (Cai et al., 1994; Hatahet and Ruddock, 2009; Song and Wang, 1995; Wang and Tsou, 1993). Moreover, Pdi1p was assigned a function in retrotranslocation of proteins into the cytosol or unfolding of proteins prior to their export (Gillece et al., 1999; Lee et al., 2010; Tsai et al., 2001). Pdi1p also introduces a disulfide bond into the MH domain of Htm1p that is essential for its activity (Sakoh-Nakatogawa et al., 2009). In this study, we expressed the Pdi1p-Htm1p complex in insect cells and demonstrated that the purified complex processed Man 8 GlcNAc 2 glycans bound to unfolded glycoproteins, yielding Man 7 GlcNAc 2. Moreover, we showed that Pdi1p played a direct role in the clearance of unfolded glycoproteins from the ER. In summary, we propose that the Pdi1p-Htm1p complex is a functional unit in ERQC and put forward a model of how this complex initiates breakdown of unfolded proteins. RESULTS Figure 1. Purification of Mns1p and Pdi1p-Htm1p Complex and In Vitro Activity of Mns1p (A) Insect cells were infected with viruses carrying expression copies of GST, GST-Mns1p, GST-Htm1p, and HIS-Pdi1p. Cells were harvested and lysed and recombinantgst-fusionproteins were bound to glutathionesepharose-beads. Input (in), flowthrough (out), and bead fractions were separated by SDS-PAGE. Proteins were visualized by staining with Coomassie Brilliant Blue. Signals representing GST, GST-Mns1p, GST-Htm1p, and HIS-Pdi1p are indicated. (B) Denatured glycoproteins were extracted from 3 H-mannose-labeled YJU90 cells (Dmns1) and incubated with GST, or Mns1p. When indicated, EDTA was addedtoafinalconcentration of5 mm. Glycanswerereleasedfrompolypeptides using PNGase F, purified, and separated by HPLC. Elution of radiolabeled oligosaccharides was recorded by flow scintillation detection. Retention times of Man 8 GlcNAc 2 (M8) and Man 9 GlcNAc 2 (M9) were determined by comparison to an LLO standard profile. Displayed are representative HPLC elution profiles. (C) 3 H-mannose-labeled N-linked glycans from strain YJU90 were liberated from polypeptides by PNGase F treatment and purified. Oligosaccharides were incubated with GST or Mns1p, purified, and analyzed as described in (B). The Pdi1p-Htm1p Complex Converts Man 8 GlcNAc 2 to Man 7 GlcNAc 2 In Vitro To express and purify Htm1p, we took advantage of the insect cell-based baculovirus system. As a control protein, we included Mns1p in our analysis. The genes of interest were fused to N-terminal affinity tags and equipped with a C-terminal ERretention signal. GST-Mns1p was readily expressed and purified with GSH Sepharose (Figure 1A). In contrast to GST-Mns1p, only a small fraction of GST-Htm1p was soluble when expressed as a single protein (data not shown). Coexpression of its complex partner HIS-Pdi1p, however, increased the solubility of GST- Htm1p, and the Pdi1p-Htm1p complex was efficiently purified (data not shown and Figure 1A). Copurification of HIS-Pdi1p was dependent on GST-Htm1p because HIS-Pdi1p did not bind to the affinity matrix. The purified complex allowed us to study the activity of Htm1p in vitro. We metabolically labeled N-linked oligosaccharides with 3 H-mannose and prepared yeast whole cell extracts yielding unfolded, reduced, and alkylated polypeptides. We used defined mutant strains to obtain glycoproteins with defined oligosaccharide structures. Extracts were incubated with GSH beads that were loaded with proteins purified from insect cells. After removing the beads by centrifugation, N-linked glycans were liberated from the polypeptides by PNGase F and analyzed by HPLC to monitor glycan processing. To validate our experimental approach, we first analyzed the activity of recombinant Mns1p. Incubation of 3 H-mannose-labeled glycoproteins from Dmns1 cells that predominantly display Man 9 GlcNAc 2 NLOs with GST did not alter the NLO profile (Figure 1B). GST-Mns1p converted the whole pool of protein-bound Man 9 GlcNAc 2 sugars to Man 8 GlcNAc 2, unless Mns1p activity was suppressed by addition of EDTA (Figure 1B). In line with previous findings demonstrating that Mns1p does not require the presence of a polypeptide attached to its glycan substrate (Jakob et al., 1998; Jelinek-Kelly et al., 1985), GST-Mns1p was active on free oligosaccharides in our assay (Figure 1C). Molecular Cell 42, , June 24, 2011 ª2011 Elsevier Inc. 783

3 When we incubated glycoprotein extracts from wild-type cells that predominantly carry Man 8 GlcNAc 2 NLOs with recombinant Pdi1p-Htm1p, about 10% of Man 8 GlcNAc 2 was trimmed to Man 7 GlcNAc 2 (Figures 2A and 2D). Concomitantly, the Man 8- GlcNAc 2 population was decreased, indicating that it served as substrate for the trimming reaction. Application of twice the volume of Pdi1p-Htm1p-loaded beads did not increase the amount of Man 7 GlcNAc 2 produced in the assay, suggesting that the available substrate pool for the processing enzyme was depleted (data not shown). Addition of EDTA abolished the activity of Pdi1p-Htm1p, indicating that Htm1p required divalent cations to be functional (Figures 2A and 2D). Reduction and alkylation of cell lysates was required to achieve maximal conversion of Man 8 GlcNAc 2 NLOs by the mannosidase complex (Figure S1). Pdi1p-Htm1p was not active on Man 9 GlcNAc 2 NLOs (Figures 2C and 2E); only when Mns1p was included in the reaction did we observe an increased signal for Man 7 GlcNAc 2. Moreover, Pdi1-Htm1p was much less active on free oligosaccharides than Mns1p (Figures 2B and 2D). From these data we concluded that the Pdi1p-Htm1p complex is an active mannosidase that preferentially processes a fraction of protein-bound Man 8- GlcNAc 2 oligosaccharides. Importantly, our in vitro results accurately replicated in vivo experiments showing that Htm1p activity depends on preceding glycan trimming by adding Mns1p and that Htm1p only processes a portion of the total N-linked glycan pool (Clerc et al., 2009). It is possible that the peptide linked to the N-glycan determined whether the oligosaccharide was a substrate for the Pdi1p-Htm1p mannosidase. Isolation of a Viable PDI1 Point Mutation that Depends on an Intact UPR Pathway The in vitro experiments suggested that Pdi1p and Htm1p form a functional unit. We wanted to corroborate this hypothesis in invivo experiments. In a screen for mutant strains that depend on an intact unfolded protein response (UPR) for viability, we isolated a missense mutation in the PDI1 gene, named pdi1-1, that resulted in a leucine to proline exchange at position 313 of the protein (see Supplemental Experimental Procedures for details on the screening procedure). Located near the center of the b 0 domain of the enzyme (Figure 3A), the L313P mutation might interfere with substrate binding or crosstalk between the b 0 and a 0 domains and impair the functions of Pdi1p (Hatahet and Ruddock, 2009; Serve et al., 2010). Importantly, pdi1-1 cells were viable, suggesting that the essential functions of the oxidoreductase were maintained. Cells expressing Pdi1-1p grew indistinguishably from wild-type cells when incubated at 30 Cor37 C (Figure 3B). Only when we exposed the cells to the reducing agent DTT did we observe a mild growth defect (Figure 3B). Analyzing Pdi1-1p via immunoblotting, we detected a slight hypoglycosylation of the protein and a minor increase in protein level as compared to wild-type Pdi1p (Figure S2A). The stability of Pdi1-1p was the same as for the wild-type protein, and UPR was not induced in pdi1-1 cells (Figures S2B and S2C). The pdi1-1 Mutation Does Not Affect Oxidative Protein Folding but Impairs Disposal of Glycoproteins To substantiate these findings at a molecular level, we analyzed the biogenesis of two model proteins, CPY and Gas1p, in pulsechase experiments. While CPY/Prc1p is a soluble carboxypeptidase of the yeast vacuole, Gas1p is an extracellular glucanosyl transferase bound to the plasma membrane by a GPI anchor. The native structure of both proteins is stabilized by disulfide bonds. Maturation of both proteins can be easily monitored since posttranslational modifications change their electrophoretical mobility as they travel along the secretory pathway. Compared to wild-type cells, maturation of neither of the two proteins was delayed in cells expressing Pdi1-1p (Figures 3C and 3D), demonstrating that the mutation did not interfere with functions of Pdi1p in oxidative folding. Next, we analyzed the breakdown of CPY*, a mutant form of CPY that is not properly folded. Since fully glycosylated CPY* can use both the ERAD system and the vacuolar pathway for degradation, we used a single-glycan variant, CPY*abcD, that can only use the Htm1p-Yos9p-dependent pathway of ERAD (Kawaguchi et al., 2010). Compared to wild-type cells, disposal of CPY*abcD was delayed in pdi1-1 cells (Figure 3E). We observed the same effect on degradation of PrA* (data not shown). Importantly, as observed for Dhtm1, the pdi1-1 allele slightly increased the degradation of a glycan-independent ERAD substrate, ngprad (Kanehara et al., 2010) (Figure 3F). These findings demonstrated that the pdi1-1 mutation deteriorated efficient clearance of unfolded glycoproteins from the ER but did not generally impair Hrd1p-dependent protein degradation. Overexpression of Htm1p and Deletion of the ALG3 Gene Suppress the pdi1-1 Phenotype Since Pdi1p forms a complex with Htm1p, we asked whether elevated levels of Htm1p suppressed the degradation phenotype of pdi1-1. In wild-type cells, overexpression of HTM1 from a PRC1 promoter accelerated degradation of CPY*abcD, but slightly delayed breakdown of ngprad (Figures 4A and S3). Increased levels of HTM1 in cells carrying the pdi1-1 mutation restored breakdown of CPY*abcD to wild-type levels (Figures 4A and 4B). In cells lacking Alg3p, Man 5 GlcNAc 2 oligosaccharides are transferred onto proteins. These sugars expose an a1,6-linked mannosyl moiety and serve as signals for Yos9pmediated disposal. Eliminating the activity of Alg3p in cells lacking the HTM1 gene restores breakdown of CPY* and CPY*abcD (Clerc et al., 2009) (Figure 4C). Interestingly, the same effect was observed when ALG3 was deleted in cells expressing Pdi1-1p (Figure 4D). These results indicated that the pdi1-1 phenotype was suppressed by altering the structure of glycans linked to proteins and suggested that Pdi1p was engaged in manufacturing the Man 7 GlcNAc 2 glycan prior to Yos9p-mediated degradation. The pdi1-1 Mutation Disrupts Association of Pdi1p and Htm1p and Decreases Htm1p Stability Next, we asked whether the pdi1-1 mutation affected the ability of Pdi1p to associate with Htm1p and employed nondenaturing immunoprecipitation experiments. C-terminally tagged Htm1p (Htm1p myc ) was expressed from its chromosomal locus. Yeast cells were lysed and Htm1p myc and Pdi1p were precipitated with anti-myc and anti-pdi1p antibodies, respectively (Figures 5A and 5B). Htm1p myc was efficiently depleted from cell extracts, 784 Molecular Cell 42, , June 24, 2011 ª2011 Elsevier Inc.

4 Figure 2. The Pdi1p-Htm1p Complex Processes Man 8 GlcNAc 2 Glycans In Vitro (A) Denatured glycoproteins from 3 H-mannose-labeled wild-type strain YWO1 were incubated with GST or Pdi1-Htm1p complex. When indicated, EDTA was added. After incubation, oligosaccharides were purified and analyzed as described in Figure 1B. Displayed are representative HPLC elution profiles. M7, Man 7 GlcNAc 2 ; M8, Man 8 GlcNAc 2. (B) 3 H-labeled N-linked glycans from YWO1 were liberated from polypeptides using PNGase F and purified. Oligosaccharides were incubated with GST or Pdi1p-Htm1p, purified, and analyzed as described in Figure 1B. (C) Radiolabeled glycoproteins from YJU90 (Dmns1) were isolated as in Figure 1A and incubated with the indicated purified proteins. NLO profiles were recorded, and representative HPLC elution profiles are displayed. (D) Relative peak areas of M7 and M8 from three independent experiments, as shown in (A) and (B), were quantified. Error bars indicate the standard deviation of mean (SEM) values. P values were determined using paired Student s t test: *p < 0.05; **p < (E) Relative peak areas M7, M8, and M9 from four independent experiments, as shown in (C), were quantified relative to the sum of all peak areas. P values: *p < 0.01; **p < 0.001; ***p < See also Figure S1. while Pdi1p levels remained unchanged. In wild-type cells, Pdi1p efficiently coprecipitated with Htm1p myc, and vice versa. Contrarily, Pdi1-1p was almost absent from the Htm1p myc precipitate, and Htm1p was barely detectable when Pdi1-1p was pulled down, suggesting that the association of Htm1p and Pdi1-1p was abrogated because of the L313P mutation. Molecular Cell 42, , June 24, 2011 ª2011 Elsevier Inc. 785

5 Figure 3. The pdi1-1 Mutation Only Affects Breakdown of Glycoproteins, Not Oxidative Protein Folding (A) Location of the pdi1-1 (L313P) mutation in the crystal structure of Pdi1p. The four thioredoxinlike domains (a, b, b 0,a 0 ) and two active sites (CxxC) are indicated. (B) Serial 10-fold dilutions of logarithmically growing strains W303a [WT] and KKY415 [pdi1-1] were spotted on YPD and YPD containing 5 mm DTT and incubated at 30 Cor37 C for 2 days. (C) Proteins of logarithmically growing strains W303a and KKY415 were labeled with 35 S- cysteine/methionine for 5 min. Samples were taken at the indicated time after initiating a chase by addition of nonradioactive amino acids. CPY was immunoprecipitated from whole cell extracts and analyzed by SDS-PAGE. Representative phosphor scan images of the gels are shown. Signal intensities of precursor and matured forms were quantified, and the ratio was plotted against time. The data reflect three independent experiments with error bars indicating the SEM values. (D) As in (C), but with Gas1p immunoprecipitated. (E) As in (C), but with CPY*abcD HA was expressed in KKY854 (WT, pwx60) and KKY853 (pdi1-1, pwx60), and cells were labeled for 10 min. CPY*abcD HA was immunoprecipitated and analyzed. Representative phosphor scan images are shown. The intensities of CPY*abcD HA signals were quantified relative to the signal at the start of the chase. The data reflect three independent experiments with error bars indicating the SEM values. (F) As in (E), but with ngprad HA was expressed in KKY622 (WT, pkk261), KKY789 (pdi1-1, pkk261) and KKY624 (Dhrd1, pkk261). See also Figure S2. Moreover, our findings indicated that only a small fraction of Pdi1p associated with Htm1p. Although we depleted an HA- HDEL-tagged version of Pdi1p from cell extracts, Htm1p myc was not codepleted in this experiment (Figure S4). It is possible that the Pdi1p-Htm1p complex is not stable under the conditions used or the addition of the tag to Pdi1p affected complex formation. Alternatively, Htm1p might have other yet unknown binding partners. Importantly, we also observed decreased steady-state levels of Htm1p in pdi1-1 cells (Figure 5A, compare lanes 3 and 4) and we, therefore, monitored the stability of Htm1p in pulsechase experiments. In wild-type cells, the level of labeled Htm1p was reduced by 25% in cell extracts after a chase period of 2 hr (Figure 5C). When we introduced the pdi1-1 mutant, clearance of labeled Htm1p was increased (40% loss after 2 hr). Because Htm1p does not carry an ER-retention signal, abolishing its association to Pdi1p that carries an HDEL sequence might result in leakage of Htm1p into the Golgi. In fact, Htm1p displayed an altered electrophoretic mobility in pdi1-1 cells, arguing for Golgi modification of attached glycans and breakdown of the protein in the vacuole (Figures 5A and 5C). Alternatively, Htm1p can be removed from the ER by the ERAD system. When Htm1p was overexpressed in wild-type cells, the protein was stable (Figure 5D). However, when we introduced the pdi1-1 mutation, Htm1p was rapidly lost from cell extracts. When HRD1, the catalytic subunit of the HRD ligase, was deleted in addition, Htm1p was stabilized. These findings indicated that a significant fraction of Htm1p was removed from the ER by the HRD pathway when not associated with Pdi1p. In addition, the cellular concentration of Pdi1p exceeded that of Htm1p and, thus, Pdi1p was 786 Molecular Cell 42, , June 24, 2011 ª2011 Elsevier Inc.

6 able to bind overexpressed Htm1p and prevented its degradation. The observation that Htm1p is unstable when binding to Pdi1p is disrupted could also explain why we were not able to express Htm1p in insect cells without its complex partner. The pdi1-1 Mutation Diminishes Production of Man 7 GlcNAc 2 by Htm1p In Vivo Finally, we asked whether the pdi1-1 mutation also interfered with the mannosidase function of Htm1p. We analyzed NLO profiles of wild-type, pdi1-1, and Dhtm1 strains that harbored either a control plasmid or a 2m plasmid encoding for an expression copy of HTM1. We observed a reduced Man 7 GlcNAc 2 signal in pdi1-1 cells (Figures 6A and 6B). This effect was comparable to the situation observed in Dhtm1 cells and validated our assumption that Htm1p and Pdi1p act in the same pathway. Overexpression of HTM1 in wild-type and Dhtm1 cells increased the Man 7 GlcNAc 2 signal. The pdi1-1 mutation inhibited an increase in the Man 7 GlcNAc 2 population when HTM1 was overexpressed. We concluded that the function of Pdi1p-Htm1p is limiting in degradation of glycoproteins. A small, mass-actiondriven increase of mannosidase activity upon overexpression of HTM1 in pdi1-1 cells results in increased disposal of CPY*abcD (Figure 4B), but is not sufficient to yield elevated levels of Man 7 GlcNAc 2 in the NLO profiles. We evaluated the structure of N-linked glycans in cells deficient for HRD1. Unfolded polypeptides accumulate in these cells, and we expected an increase of the Man 7 GlcNAc 2 pool when compared to wild-type. However, overexpression of HTM1 in Dhrd1 cells did not result in the anticipated outcome (Figures 6C and 6D). The result suggested that, under the conditions employed, the majority of Man 7 GlcNAc 2 oligosaccharides were present on folded proteins that were not disposed of by the HRD pathway. More interestingly, these data also implied that the activity of the Pdi1p-Htm1p complex was decreased when ERAD was blocked, indicating that the flux through the degradation pathway was regulated by the activity of the Pdi1p-Htm1p complex. DISCUSSION Figure 4. Overexpression of HTM1 or Deletion of ALG3 Supresses the pdi1-1 Mutation (A) Proteins of strains KKY855 (wild-type [WT], pwx60, pdn251) and KKY856 (wild-type, pwx60, pkk225) were radioactively labeled for 10 min and decay kinetics of CPY*abcD HA were determined as described (Figures 3C and 3E). Representative phosphor scan images are shown. The quantified data reflect three independent experiments with error bars indicating the SEM values. (B) As in (A), but strains KKY857 (pdi1-1, pwx60, pdn251) and KKY858 (pdi1-1, pwx60, pkk225) were used. (C) As in (A), but strains KKY853 (wild-type [WT], pwx60), KKY899 (Dhtm1, pwx60), and KKY893 (Dhtm1, Dalg3, pwx60) were used. (D) As in (A), but strains KKY853 (wild-type [WT], pwx60), KKY854 (pdi1-1, pwx60), and KKY884 (pdi1-1, Dalg3, pwx60) were used. See also Figure S3. In this study, we provided in vitro and in vivo evidence that the yeast protein disulfide isomerase Pdi1p and the mannosidase Htm1p assembled into an operative complex initiating clearance of unfolded glycoproteins from the ER. Using recombinant proteins, we demonstrated that the Pdi1p-Htm1p complex acted as a peptide-dependent exomannosidase, generating N-linked Man 7 GlcNAc 2 oligosaccharides that can serve as signals for degradation by the HRD ligase. In contrast to Mns1p, which consumed the entire Man 9 GlcNAc 2 NLO pool, Pdi1p-Htm1p processed about 10% of protein-bound Man 8 GlcNAc 2 under the experimental conditions used (Figures 1 and 2). This outcome was consistent with earlier observations that overexpression of HTM1 in vivo results in about 15% of Man 8 GlcNAc 2 converted to Man 7 GlcNAc 2 (Clerc et al., 2009) and that not all glycans attached to a protein are used as a signal for ERAD. Only the very C-terminal glycan bound to CPY* and the N-terminal oligosaccharide of PrA*are mandatory for efficient clearance of the protein from the ER (Kostova and Wolf, 2005; Molecular Cell 42, , June 24, 2011 ª2011 Elsevier Inc. 787

7 Figure 5. The pdi1-1 Mutation Disrupts the Pdi1p-Htm1p Complex and Decreases Htm1p Stability (A) Whole cell extracts were prepared from strains W303a (wild-type; loaded in lanes 1, 5, 9), KKY415 (pdi1-1; lanes 2, 6, 10), YRG421 (wild-type, HTM1 myc ; lanes 3, 7, 11), and YRG422 (pdi1-1, HTM1 myc ; lanes 4, 8, 12), and Htm1p myc was immunoprecipitated. Input (in), flowthrough (ft), and precipitated (IP) fractions were analyzed by SDS-PAGE and western blotting (WB). Samples for detection of the precipitated protein were diluted 5-fold (WB anti-myc, lanes 9 12). The symbol + indicates the presence of Htm1p myc, wild-type PDI1, and pdi1-1. The symbol * indicates that the secondary antibody used in the WB also binds to the heavy chain of the antibody used for IP. (B) As in (A), but with Pdi1p immunoprecipated from lysates of YRG421 (lanes 1, 3, 5) and YRG422 (lanes 2, 4, 6). Samples for detection of the precipitated protein were diluted 5-fold (WB anti- Pdi1p, lanes 5 and 6). (C) Cells from strains W303a and KKY415 were radioactively labeled, and degradation kinetics of Htm1p was determined as described (Figures 3C and 3E). Htm1p was immunoprecipitated by anti- Htm1p antibodies. Representative phosphor scan images are shown. The quantified data reflect three independent experiments with error bars indicating the SEM values. (D) As in (C), but with Htm1p HA expressed in strains KKY866 (wild-type [WT], pkk207), KKY868 (pdi1-1, pkk207), and KKY917 (pdi1-1, Dhrd1, pkk207). See also Figure S4. Spear and Ng, 2005). Moreover, distinct regions within a protein appear to be essential for effective degradation (Spear and Ng, 2005; Vashist et al., 2001; Xie et al., 2009). Taken together, these findings imply that the mannosidase activity of the Pdi1p-Htm1p complex is restricted to glycans that are in a compatible spatial orientation to a distinct, presumably unfolded, polypeptide sequence bound by the complex. Indeed, the Pdi1p-Htm1p mannosidase did not efficiently process free Man 8 GlcNAc 2 oligosaccharides (Figure 2B). Pdi1p has a modular and dynamic domain structure, and the functions of its four thioredoxin domains (a, b, b 0, and a 0 ) are asymmetrical; while domains a and a 0 are involved in thiol exchange reactions, b and b 0 are catalytically inactive, and domain b 0 is considered the primary substrate-binding site (Hatahet and Ruddock, 2009; Klappa et al., 1998). However, b 0 is not only involved in client-protein capture; in addition, it also serves as the main binding interface for partner proteins of PDI1 or its homologs (Hatahet and Ruddock, 2009). The P domain of calnexin binds to a highly charged region of the b 0 domain of human ERp57, located opposite to the substrate binding surface (Ellgaard and Frickel, 2003; Kozlov et al., 2006; Russell et al., 2004). Conversely, mutations in the substrate binding site of human PDI b 0 domain do not inhibit its assembly into collagen prolyl 4-hydroxylase tetramers (Koivunen et al., 2005; Pirneskoski et al., 2004). Introducing a single point mutation into b 0 domain of Pdi1p disrupted its ability to bind Htm1p, and we speculate that the Pdi1p-Htm1p complex follows the same structural theme as described for ERp57- CNX and the collagen prolyl 4-hydroxylase tetramers. The CT domain of Htm1p might bind to the b 0 domain of Pdi1p; such an arrangement would recruit Htm1p to Pdi1p substrates and enable the mannosidase to process N-linked Man 8 GlcNAc 2 glycans. As compared to the system in S. cerevisiae, the ERQC network of mammalian cells is more complex. It is extended by the calnexin-calreticulin cycle, and there are multiple members of the GH47 family found in the ER: ER a1,2-mannosidase-i (ER- ManI); and three homologs of Htm1p, i.e., EDEM1, EDEM2, and EDEM3 (Aebi et al., 2010; Olivari and Molinari, 2007). Interestingly, EDEM1 forms a complex with BiP and ERdj5, a DnaJtype chaperone with six thioredoxin-like domains (Hagiwara et al., 2011; Ushioda et al., 2008). In vitro and in vivo experiments revealed that it can act as a reductase, cleaving disulfide bonds in unfolded model substrates, and can accelerate the turnover of disulfide bonds containing EDEM1 substrates (Hagiwara et al., 2011; Ushioda et al., 2008). In view of our results, we speculate 788 Molecular Cell 42, , June 24, 2011 ª2011 Elsevier Inc.

8 Figure 6. The pdi1-1 Mutation Abrogates Generation of Man 7 GlcNAc 2 NLOs In Vivo (A) Cells from strains W303a (wild-type [WT]), KKY415 (pdi1-1), and ESY679 (Dhtm1), carrying either an empty vector (YEp352; [e.v.]) or a 2m plasmid encoding for Htm1p (phtm1u-1; [HTM1]) were labeled with 3 H-mannose, and whole cell lysates were prepared. Glycans were released from polypeptides by PNGase F treatment, purified, and separated by HPLC. Elution profiles of radiolabeled oligosaccharides were recorded by flow scintillation detection. Retention times of Man 5 GlcNAc 2 (M5), Man 7 GlcNAc 2 (M7), and Man 8 GlcNAc 2 (M8) are indicated. Displayed are representative HPLC elution profiles. Note that the Man 5 GlcNAc 2 glycan is not a product of the Pdi1p-Htm1p mannosidase (data not shown); its origin and function will be addressed elsewhere. (B) Relative peak areas of M7 and M8 from three independent experiments, as shown in (A), were quantified. Error bars indicate the standard deviation of mean (SEM) values. P value was determined using paired Student s t test: *p < (C) As in (A), but with strains W303a and ESY279 (Dhrd1) carrying either an empty vector (YEp352; [e.v.]) or a 2m plasmid encoding for Htm1p (phtm1u-1; [HTM1]) used. (D) As in (B), but quantifying peak areas from (C). P value was determined using paired Student s t test: *p < that, comparably to Pdi1p in yeast, ERdj5 acts as a chaperone adaptor of EDEM1 to capture substrate proteins. Based on our results, we suggest a stochastic model of how the Pdip-Htm1p complex initiates breakdown of unfolded proteins and contributes to ER protein homeostasis (Figure 7). Only a fraction of the whole Pdi1p pool associates with Htm1p, and we speculate that unfolded polypeptides bind to free Pdi1p and Htm1p-bound Pdi1p with the same affinity. In addition Molecular Cell 42, , June 24, 2011 ª2011 Elsevier Inc. 789

9 displays features recognized by Pdi1p. The slower it folds i.e., the longer it exposes hydrophobic patches the higher the probability of encountering the Pdi1p-Htm1p complex that trims Man 8 GlcNAc 2 glycans. Mutant proteins like CPY* are trapped in an unfolded state and, thus, the majority of these proteins is removed by the ERAD pathway. Mns1p provides directionality and attenuates the system by producing the glycan detected by Pdi1p-Htm1p. Since free and Htm1p-bound Pdi1p compete for the same substrates, the relative levels of Pdi1p modulate the net output of the system. The higher the relative concentration of Pdi1p-Htm1p, the higher the fraction of proteins flagged with a Man 7 GlcNAc 2 glycan. It is noteworthy that in yeast, PDI1 but not HTM1 is a target of the UPR (Travers et al., 2000). ER stress condition will, therefore, trigger upregulation of the oxidoreductase, shift the ratio toward monomeric Pdi1p, and favor folding over degradation. Thus, turnover of secretory glycoproteins in the ER is regulated by adjusting the relative level of the Pdi1p-Htm1p mannosidase to ensure protein homeostasis. EXPERIMENTAL PROCEDURES Figure 7. A Stochastic Model for the Function of the Pdi1p-Htm1p Complex in ER Protein Homeostasis (1 9) A schematic illustration of the folding and glycan-processing pathways of a soluble glycoprotein in the ER is shown. After translocation into the lumen and covalent attachment of a Glc 3 Man 9 GlcNAc 2 oligosaccharide, initial trimming steps by glucosidase I and II generate a Man 9 GlcNAc 2 (M9) glycan on the unfolded polypeptide (1). Mns1p trims glycans of unfolded (2) or folded proteins (4). Pdi1p assists oxidative protein folding irrespective of the glycan status of the protein (3). Folded proteins leave the ER (5). Free Pdi1p and Htm1p-bound Pdi1p bind unfolded polypeptides with equal affinities (3) and (6), but only the Pdi1p-Htm1p can process the Man 8 GlcNAc 2 (M8) oligosaccharide to yield Man 7 GlcNAc 2 (M7) (7). The HRD ligase captures unfolded proteins flagged with M7 (8) and initiates clearance from the compartment (9). Materials and Reagents The monoclonal 9E10 anti-myc antibody was purchased from Sigma-Aldrich (M5546). The monoclonal HA-11 anti-ha antibody was obtained from Covance Research Products (Princeton, NJ). Polyclonal anti-htm1p antibodies were raised in rabbits against an Htm1p-glutathione S-transferase fusion protein purified from E. Coli BL21 cells, using the pgex-4t-3 expression system (GE Healthcare Life Sciences, Uppsala, Sweden). The polyclonal anti-gas1p was produced as described (Spear and Ng, 2003). Anti-Kar2p and anti- Sec61p antibodies were provided by Peter Walter (University of California San Francisco, San Francisco, CA). Anti-Pdi1p antibodies were kind gifts from Karin Römish (University of Cambridge, Cambridge, UK) and Jakob Winther (University of Copenhagen, Copenhagen, Denmark). Yeast Strains and Plasmids Standard yeast media and genetic techniques were used as described (Guthrie and Fink, 1991). Yeast strains used in this study are listed in Table S1. PCR-based methods were implemented to introduce C-terminal tags and deletions into the yeast genome (Janke et al., 2004; Knop et al., 1999). Plasmids and primers used in this study are listed in Tables S2 and S3. Plasmids were constructed using standard cloning protocols. See Supplemental Experimental Procedures for further details on the plasmids. Open reading frames of all constructs were confirmed by nucleotide sequencing. to oxidoreductase and chaperone functions, the Pdi1p-Htm1p complex possesses exomannosidase activity. If present at a compatible position relative to the Pdi1p-binding site, Htm1p trims the Man 8 GlcNAc 2 glycan to produce Man 7 GlcNAc 2. Once released from Pdi1p, the polypeptide can fold, reassociate with Pdi1p, or bind to the HRD ligase. Binding of a Man 7 GlcNAc 2 oligosaccharide to the lectin subunit Yos9p either increases the binding of polypeptides or triggers removal of the polypeptide from the compartment (Carvalho et al., 2006, 2010; Denic et al., 2006; Gauss et al., 2006; Quan et al., 2008). It is important to note that, within the framework of our hypothesis, Pdi1p does not distinguish between folding intermediates and polypeptides trapped in an unfolded conformation; Htm1p can process both specimens. The decisive variable determining the net output of the system is the time a given polypeptide Metabolic Labeling with 35 S and Immunoprecipitation Cell labeling and pulse-chase analysis were performed as described (Kanehara et al., 2010). Cells were grown to log phase and cells were harvested and resuspended in 0.9 ml synthetic complete media lacking methionine and cysteine. Cells were incubated at the appropriate temperature for 30 min. Then, cells were pulse-labeled in the presence of 82.5 mci 35 S-methionine/cysteine (1 mci/mmol) (EasyTag EXPRESS 35 S, Perkin Elmer) for 5 min (maturation assays) or 10 min (degradation assays). Nonradioactively labeled methionine/cysteine was added to a final concentration of 2 mm to initiate the chase. To terminate label incorporation at each time point, TCA was added to a final concentration of 10%, and cells were subsequently disrupted with the glass bead method. The homogenate was cleared by centrifugation at 18,000 g for 15 min. Pellets were resuspended in 120 ml of TCA suspension buffer (100 mm Tris-HCl, 3% SDS, and 3 mm DTT) and heated at 100 C for 15 min. Insoluble material was removed by centrifugation, and 40 ml of the lysate was added to 560 ml of IPS II (13.3 mm Tris-HCl, 150 mm NaCl, 1% Triton X-100, 0.02% NaN 3, 1 mm PMSF, and 1 ml protease inhibitor cocktail [Sigma-Aldrich]) and the appropriate antiserum. Samples were 790 Molecular Cell 42, , June 24, 2011 ª2011 Elsevier Inc.

10 incubated at 4 C for 2 hr and then centrifuged at 18,000 g for 15 min. The supernatant was recovered and protein A-Sepharose beads were added. Samples were incubated for 1 hr and washed three times with IPS I (0.2% SDS, 1% Triton X-100, 20 mm Tris-HCl [ph 7.4], 150 mm NaCl, and 0.02% NaN 3 ) and once with TBS (20 mm Tris-HCl [ph 7.4] and 150 mm NaCl). Immunoprecipitated proteins were eluted with SDS gel sample buffer and separated by SDS-PAGE. Gels were exposed to phosphor screens for hr and the exposed screens were scanned using a phosphorimager (Typhoon, GE Healthcare). Signals were quantified using ImageQuant TL software (GE Healthcare). All data plots reflect three independent experiments with SEM indicated with error bars. 3 H-Mannose Labeling and NLO Profiling To analyze NLO profiles of yeast cells, a modified version of the protocol described was implemented (Clerc et al., 2009; Jakob et al., 1998). Yeast cells were grown in appropriate medium at 30 C to early logarithmic phase corresponding to an OD 600 of An equivalent of 50 OD cells ( cells) was harvested and washed once with 25 ml of YP0.1D (1% yeast extract, 2% peptone, and 0.1% glucose). Cells were resuspended in 200 ml YP0.1D and incubated at 30 C with shaking (1000 rpm) for 5 min. Metabolic labeling was started by adding 100 ml YP0.1D containing 100 mci D-2-3 H-mannose (23.7 Ci/mmol, Perkin Elmer) and performed at 30 C for 20 min. To stop the label incorporation, 25 ml 100% TCA was added, and the suspension was incubated on ice for 5 min. After centrifugation at 20,000 g for 5 min, the precipitate was washed twice with ice-cold acetone and dried at 50 C for 10 min. The pellet was resuspended in 200 ml buffer S2 (50 mm sodium buffer [ph 7.5], 0.4% SDS, and 40 mm DTT) and vortexed with glass beads at 50 C for 60 min. The slurry was extracted twice with 250 ml S2 buffer and once with 200 ml S2 buffer. Supernatants were collected, cleared by centrifugation, and 500 ml of the lysate was transferred to a new tube. Fifty five ml of a 450 mm iodoacetamide was added, the mixture incubated at 37 C for 15 min, and 62 ml of NP-40 and 69 ml of 500 mm sodium buffer ph 7.5 were added. This mixture was used as substrate for the in vitro mannosidase assay. Finally, the sugars were cleaved from the polypeptides with 1 ml PNGase (NEB) at 37 C for 4 hr or 16 hr. Clean-up and HPLC analysis of oligosaccharides and data evaluation was performed as described (Clerc et al., 2009; Jakob et al., 1998). Coimmunoprecipitations Yeast cells were grown at 30 C in YPD to early logarithmic phase, corresponding to an OD 600 of 0.8 to 1.2. An equivalent of 50 OD cells ( cells) was harvested and washed once with ice-cold 1 mm PMSF in H 2 0. Cells were washed with 1 ml ice-cold IP33 (50 mm HEPES-NaOH [ph 7.2], 50 mm NaCl, 125 mm KOAc, 2 mm MgCl 2, 1 mm EDTA, 3% glycerol, 0.5% Nonidet P-40, and 1 mm PMSF), resuspended in 400 ml IP33, and disrupted with the glass beads method. The slurry was extracted twice with 750 ml IP33, and the supernatants were collected. Cellular debris was removed by high speed centrifugation (20,000 g at 4 C for 12 min). Htm1p myc and Pdi1p were precipitated from the supernatant by addition of 1 ml anti-myc (Sigma-Aldrich, M5546), or 1 ml polyclonal anti-pdi1p antibodies and 25 ml protein A-Sepharose beads (GE Healthcare, ) and incubation at 4 C for 3 hr. Beads were recovered by centrifugation and washed three times with 1 ml of IP33. Bound proteins were eluted by adding 50 ml of SDS sample buffer and incubation at 65 C for 15 min. When indicated, samples were diluted 1:5 in sample buffer. Proteins were separated by SDS-PAGE and immunoblotting, using the indicated antibodies. Protein Expression and Purification in Insect Cells Recombinant baculoviruses expressing GST-Mns1p, GST-Htm1p, HIS-Pdi1p, or GST were produced according to the manufacturer s instructions (Invitrogen). Following three rounds of amplification, the virus stocks were used for protein expression. For small scale analysis of protein expression, Spodoptera frugiperda (Sf9) cells were infected with 150 ml baculovirus solution and incubated at 27 C for 72 hr. Cells were harvested, washed once with ice-cold PBS, and lysed in 250 ml PBS-Tx (PBS + 1% Triton X-100) supplemented with 1 mm E-64 (Roche Applied Science) at 4 C for 20 min. Insoluble material was removed by centrifugation at 20,000 g for 5 min, and the supernatant was used for further experiments. For protein purification, cells were infected with 1.3 ml virus suspension. Cells were lysed in 1.5 ml PBS-Tx containing E-64, and recombinant proteins were bound to 100 ml GSH-Sepharose beads (50% suspension, GE Healthcare) at 4 C for 4 hr. Sepharose beads were washed three times with ice-cold PBS and stored as a 50% suspension in PBS at 4 C. In Vitro Mannosidase Assay Whole-cell lysates containing denatured, reduced, and alkylated glycoproteins with 3 H-mannose labeled NLOs were prepared, implementing the NLO extraction protocol described above. Prior to addition of PNGase F, 20 ml GSH-Sepharose beads loaded with purified Mns1p or 100 ml of GSH-beads loaded with Pdi1p-Htm1p was added and incubated for 16 hr. Beads were removed by centrifugation, and NLOs were cleaved by PNGase F. Glycans were isolated and analyzed as described above. SUPPLEMENTAL INFORMATION Supplemental Information includes four figures, three tables, Supplemental Experimental Procedures, and Supplemental References and can be found with this article online at doi: /j.molcel ACKNOWLEDGMENTS We thank Karelia Velez de Joerg, Jeremy Brodhead, Sandy Toh, Songyu Wang, and Jun Yang for excellent technical assistance; Anna Mohr and Christine Neupert for critical comments on experiments and the manuscript; Karin Römisch, Peter Walter, Jakob Winther, and Thomas Sommer for providing materials. The identification of the pdi1-1 allele was part of an expanded UPR synthetic lethal screen carried out by Woong Kim and isolation of the complementing PDI1 clone by Nurzian Ismail. Funding for this work was provided by funds from the Swiss National Science Foundation (grant for M.A.), the Swiss Federal Institute of Technology, and the Singapore Millennium Foundation. R.G. received an EMBO long-term fellowship. 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