Substrate-Specific Requirements for UGT1-Dependent Release from Calnexin  Tatiana Soldà, Carmela Galli, Randal J. Kaufman, Maurizio Molinari  Molecular Cell  Volume 27, Issue 2, Pages 238-249 (July 2007) DOI: 10.1016/j.molcel.2007.05.032 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Consequences of UGT1 Deletion on Substrate Release from Calnexin (A) Structure of a protein-bound N-glycan. Triangles are glucose, circles are mannose, and squares are N-acetylglucosamine residues. GI and GII are ER α-glucosidase I and II, respectively. (B) ugt1−/− MEFs do not contain UGT1 protein, as determined by immunoprecipitation of labeled proteins with a UGT1-specific polyclonal antibody. (C) Labeled calnexin with associated cellular substrates has been immunoisolated from lysates of WT and ugt1−/− cells not exposed (lanes 1 and 3) or exposed (lanes 2 or 4) to 1 mM castanospermine. (D) Same as (C), but cells were chased for 3–54 min (without castanospermine) before lysis and immunoisolation of calnexin-substrate complexes. Release kinetics of proteins (U, unaffected; F, faster release upon UGT1 deletion; and S, slower release) has been quantified and plotted in (E). Position of molecular weight markers (200, 116, 66, 45, and 31 kDa) is shown with lines. (E) Quantification of release kinetics (values have been normalized for the content of labeled calnexin in each lane). Molecular Cell 2007 27, 238-249DOI: (10.1016/j.molcel.2007.05.032) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 UGT1 Deletion Does Not Affect Release from Calnexin and Maturation of VSV G Protein (A) Calnexin and the associated labeled cellular and viral substrates were immunoisolated from lysates of VSV-infected WT and ugt1−/− MEFs. Arrows show the G protein and endogenous proteins released with faster (F) or slower (S) kinetics from calnexin upon UGT1 deletion. (B) For plotting, the amount of labeled G, F, and S has been normalized for labeled calnexin content in each lane. (C) Native G protein has been immunoisolated with a B2-conformation-specific monoclonal antibody (I14; Doms et al., 1988). (D) Rate of G protein export from the WT and the ugt1−/− ER has been determined by assessing acquisition of EndoH-resistant N-glycans. Molecular Cell 2007 27, 238-249DOI: (10.1016/j.molcel.2007.05.032) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 UGT1 Deletion Accelerates BACE501 Release from Calnexin and Slightly Reduces Folding Efficiency (A) Calnexin and the associated labeled cellular substrates were immunoisolated from lysates of WT and ugt1−/− MEFs. Lines show calnexin, BACE501 (faster release upon UGT1 deletion), and cellular proteins released with slower kinetics. In lane B, BACE501 is shown as a mobility standard. (B) The same detergent extracts shown in (A) were first immunoprecipitated with a calnexin-specific antibody, then, upon dissociation of the calnexin-substrate complexes, they were subjected to a second round of immunoprecipitation with an antibody to BACE501. Lane B is as in (A). (C) More persistent association of BACE501 with BiP in ugt1−/− MEFs (left panel). No association with ERp57 could be detected (right panel). (D) BACE501 release from the ER monitored by increase in the polypeptide size upon complex glycosylation in the Golgi (I is immature; M is mature BACE501). (E) Export of BACE501 from the WT and the ugt1−/− ER monitored by assessing acquisition of EndoH-resistant N-glycans. In the untreated samples (lanes 1, 3, 5, and 7), I is immature and M is mature BACE501. In treated samples (lanes 2, 4, 6, and 8), S is the EndoH-sensitive and R the EndoH-resistant fraction of the labeled BACE501. Molecular Cell 2007 27, 238-249DOI: (10.1016/j.molcel.2007.05.032) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 UGT1 Deletion Inhibits Maturation of Influenza Virus HA (A) Labeled HA, a glycoprotein expressed in the ER, and NP, a protein expressed in the cell cytosol, are the only labeled proteins immunoisolated from the total cell extract (TCE) with the virus-specific polyclonal antibody used in this study. In reducing gels (Red), HA migrates as a single polypeptide band (HAR). In nonreducing gels (NRed), the HA folding intermediates are separated in three distinct bands shown as IT1, IT2, and NT. The schematics shows the native HA disulfides and the position of N-linked glycans. (B) Progression of the oxidative folding of HA during the chase analyzed in nonreducing gels. The folding intermediates are named IT1′, IT2′, and NT′ in ugt1−/− MEFs. DBC are disulfide-bonded complexes containing misfolded HA. (C) HA export from the ER monitored as in Figures 2D and 3E. (D) Same as (C), but for longer chase times. Molecular Cell 2007 27, 238-249DOI: (10.1016/j.molcel.2007.05.032) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 UGT1 Deletion Delays Influenza Virus HA Release from Calnexin and Elicits BiP Binding (A) HA association with calnexin (upper panels) and with BiP (lower panels) analyzed in reducing gels. (B) Analysis at longer chase times of HA release from calnexin (lanes 1–3) and hand off to BiP (lanes 4–6) in ugt1−/− MEFs. No association with ERp57 was detected (lanes 7–9). (C) JBαM only removes terminal mannose residues. (D) After a 2 min chase (lanes 1 and 2) and after a 20 min chase in the presence of bDNJ to prevent removal of the innermost glucose residues (lanes 5 and 6), influenza virus HA is glucosylated and JBαM treatment results in a small mobility shift. At longer chase time (20 min, lanes 3 and 4), HA has been extensively deglucosylated as shown by the exaggerated mobility shift upon JBαM treatment. (E) Same as (D) for HA synthesized in WT (lanes 1–4) and ugt1−/− MEFs (lanes 5–8). (F) Calnexin and the associated HA folding intermediates were separated in nonreducing gels. (G) At the end of a chase, cells were mock treated (lanes 1 [WT] and 4 [ugt1−/−]) or exposed for 5 min to 20 mM diamide (lanes 2 [WT] and 3 [ugt1−/−]) to exaggerate mobility shifts of different HA conformers. Molecular Cell 2007 27, 238-249DOI: (10.1016/j.molcel.2007.05.032) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 Protein-Specific Consequences of UGT1 Back Transfection (A) Labeled UGT1 and protein C-tagged UGT1 are immunoisolated from WT and from back-transfected cells, respectively (ugt1−/− + UGT1). (B) Same as Figure 3B in order to show that UGT1 back transfection delays BACE501 release from calnexin. (C) UGT1 back transfection accelerates HA release from calnexin. (D) The efficiency of folding for the recombinant HA is about 50% in WT MEFs as determined by acquisition of EndoH-resistant N-glycans (R is for resistant, S is for sensitive). Efficiency drops in cells lacking UGT1 (middle panel) and is partially recovered upon UGT1 back transfection (lower panel). Molecular Cell 2007 27, 238-249DOI: (10.1016/j.molcel.2007.05.032) Copyright © 2007 Elsevier Inc. Terms and Conditions