Volume 19, Issue 6, Pages 765-775 (September 2005) Yos9 Protein Is Essential for Degradation of Misfolded Glycoproteins and May Function as Lectin in ERAD  Reka Szathmary, Regula Bielmann, Mihai Nita-Lazar, Patricie Burda, Claude A. Jakob  Molecular Cell  Volume 19, Issue 6, Pages 765-775 (September 2005) DOI: 10.1016/j.molcel.2005.08.015 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Amino Acid Alignment of OS-9 Family Member Proteins, Cation-Dependent, and Cation-Independent Mannose-6-Phosphate Receptor Proteins The amino acid sequence of the S. cerevisiae Yos9 was aligned with cation-dependent mannose-6-phosphate receptors (CD-MPRs), the domain3/9 (dom3, dom9) of the cation-independent mannose-6-phosphate receptor (CI-MPR/IGF-II), and with the amino acid sequence of other members of the OS-9 family. The sequence alignment was generated with the ClustalW algorithm and manually adjusted with Genedoc. Conserved residues are shaded (black, 80% similarity; dark gray, 50% similarity; and light gray, 40% similarity). Amino acid residues subjected to site-directed mutagenesis are indicated with an asterisk. Amino acid positions involved in carbohydrate binding in CD-MPR are underlined; the amino acid residues involved in Mn2+ coordination are boxed. The sequences shown, with their corresponding NCBI database accession numbers, are as follows: bovine (Bt) CD-MPR (P11456); mouse (Mm) CD-MPR (P24668); human (Hs) (NP002346) CD-MPR; bovine (Bt) IGF-II (P08169); mouse (Mm) IGF-II (P2468); human (Hs) IGF-II (P11717); mouse (Mm) OS-9 (NP808282); S. cerevisiae (Sc) Yos9 (NP010342); yeast S. pombe (Sp) OS-9 (CAB61460); caenorhabditis elegans (Ce) OS-9 (CAB60843); D. melanogaster (Dm) OS-9 (NP788078); human (Hs) OS-9 (Q13438); and human XTP3-B (NP056515). Molecular Cell 2005 19, 765-775DOI: (10.1016/j.molcel.2005.08.015) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 YOS9 Is Required for Degradation of the Mutant Proteins CPY*, PrA*, and Pdr5*, but Not for Sec61-2 Protein Isogenic cells were grown to midlog phase at 30°C. Equal cell numbers were harvested, incubated with 100 μg/ml cycloheximide, and aliquots removed at given time points. Crude protein extract was prepared and separated by SDS-PAGE. Proteins were visualized by Western analysis using specific antisera and ECL. Degradation rates of CPY* (A and D), PrA* (B), Pdr5* (C), and Sec61-2 (E) are shown. Hexokinase (HXK) protein levels served as loading control. Protein amounts from independent experiments were quantified and plotted against time, and the amount of protein of time point 0 min was set to 100%. Degradation rates, average ± SD. Molecular Cell 2005 19, 765-775DOI: (10.1016/j.molcel.2005.08.015) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Conserved Amino Acids of MRH Domain of Yos9p Are Required for Efficient CPY* Degradation (A) Degradation of CPY* was studied in Δyos9 cells expressing either epitope-tagged wild-type (wt) Yos9p, different mutant forms of Yos9-HA protein, or cells containing empty vector. Protein degradation was analyzed by cycloheximide chase (see Figure 2 for details). Crude protein extract was separated by SDS-PAGE, and CPY* was visualized by Western analysis. In cells where CPY* degradation occurred at rates comparable to wt cells, the expressed Yos9 protein complemented the Δyos9 deletion (marked with [+]). Lack of complementation is indicated by (−). (B) Expression levels and stability of wt and mutant epitope-tagged Yos9 proteins are shown at time points 0 and 60 min of the CHX chase. Molecular Cell 2005 19, 765-775DOI: (10.1016/j.molcel.2005.08.015) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 Steady-State CPY* Carried N-Glycans of Man8GlcNAc2 Structure N-glycans of glycoproteins of 2 × 1010 yeast cells were metabolically in vivo labeled with [3H] mannose. Cells were broken with glass beads, and protein A-fused CPY and CPY* proteins were specifically immunoprecipitated by using IgG Sepharose. N-glycans were enzymatically released by PNGaseF and analyzed by HPLC. Arrows indicate different oligosaccharides. Abbreviations: G, glucose and M, mannose. Molecular Cell 2005 19, 765-775DOI: (10.1016/j.molcel.2005.08.015) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Yos9p and CPY* Coprecipitate in an Oligosaccharide Structure-Specific Manner Yeast cells (wt, Δalg3 [A], or Δalg9, Δalg12, and Δmns1 [B]) expressing integrated Yos9-ProteinA-His7 containing CPY*-HA or empty vector were grown to midlog phase and harvested. Cells were broken, and protein was solublized by using 1% Triton X-100. Yos9 protein was specifically precipitated with IgG Sepharose beads. Immobilized protein was separated by SDS-PAGE. CPY* and Yos9 protein was detected by Western analysis using anti-HA antiserum and ECL. Oligosaccharide structures on glycoproteins found in respective cells are depicted as cartoons. Abbreviations: M5, Man5GlcNAc2; M6, Man6GlcNAc2; M7, Man7GlcNAc2; M8, Man8GlcNAc2; and M9, Man9GlcNAc2. Molecular Cell 2005 19, 765-775DOI: (10.1016/j.molcel.2005.08.015) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 Yos9 Specifically Binds to CPY*, but Not CPY, and HTM1 Is Required for Efficient Yos9p and CPY* Interaction (A) Yeast cells containing CPY were transformed with a plasmid encoding CPY*-HA, Yos9-ProteinA-His7, or empty vector. Native protein extract was prepared from midlog-phase cells, and Yos9-ProteinA-His7 was precipitated. CPY and CPY* were detected by using Western analysis anti-CPY serum and ECL. (B) Yeast cells proficient or deficient of the HTM1 locus expressing Yos9-ProteinA-His7 and CPY*-HA were used for pull-down experiments. The experiments were performed as described in Figure 4. (C) The CPY* protein amounts from four independent experiments were determined densitometrically. The CPY* protein amount in protein extracts prior to precipitation of wt cells was set to 100%; data presented as average ± SD. Molecular Cell 2005 19, 765-775DOI: (10.1016/j.molcel.2005.08.015) Copyright © 2005 Elsevier Inc. Terms and Conditions