1. Volume 15, Issue 6, Pages 913-923 (September 2004)
A Polypeptide Binding Conformation of Calreticulin Is Induced by Heat Shock, Calcium Depletion, or by Deletion of the C-Terminal Acidic Region 
Syed Monem Rizvi, Laura Mancino, Vilasack Thammavongsa, Richard Louis Cantley, Malini Raghavan 
Molecular Cell 
Volume 15, Issue 6, Pages (September 2004)
DOI: /j.molcel

2. Figure 1
VMAPCFITCTLLL Binds to Calreticulin and Inhibits the Chaperone Activity of Calreticulin
(A and B) Indicated proteins (6 μM each) were incubated for 1 hr with (A) 60 μM VMAPCFITCTLLL at 37°C, 42°C, or 47°C or (B) with 120 μM VEICFITCPYKPTW (VEIC) (lanes 1–4) or VMAPCFITCTLLL (VMA) (lanes 5–8) at 42°C. Mixtures were separated by native-PAGE and proteins visualized by fluorimaging (top panel) or Coomassie staining (bottom panel).
(C) Thermal aggregation of HLA-B35 heavy chain. Left panel, HLA-B35 (12 μM) was incubated at 50°C for 1 hr with buffer, 1.5 μM calreticulin (CRT), 1.5 μM CRT μM VMA, or 1.5 μM CRT μM VEI, followed by centrifugation at 100,000 × g. Pellets (P) and supernatants (S) were resuspended in equal volumes. Percentages of HLA-B5 heavy chain in the P and S fractions were quantified following SDS-PAGE and Coomassie staining. Right panel, analyses as in the left panel but calreticulin samples (1 μM) were preincubated at 50°C in the presence of buffer or indicated peptides (80 μM), then HLA-B35 (8 μM) was added, and samples were heated an additional 1 hr at 50°C. Data are representative of several ([A], [B], and left panel in [C]) or two independent analyses ([C], right panel). All analyses were in 50 mM Tris, 150 mM NaCl, 1 mM CaCl2 (pH 7.5).
Molecular Cell  , DOI: ( /j.molcel )

3. Figure 2
Heat Shock Enhances Calreticulin Binding to VMAPCFITCTLLL and HLA-A2 Heavy Chains
(A) Calreticulin (6 μM) was preheated at 37°C (lane 1), 42°C (lane 2), or 47°C (lane 3) for 1 hr. VMAPCFITCTLLL (60 μM) was added, and incubation continued at 37°C for 1 hr. Samples were separated by native-PAGE and visualized by fluorimaging.
(B) Calreticulin (8.7 μM) and VMAPCFITCTLLL (87 μM) were incubated at 42°C for 1 hr. Unbound VMAPCFITCTLLL was removed with a Biospin 30 column. Unlabeled VMAPCTLLL (875 μM) was added to eluates, and mixtures were incubated at 4°C, 37°C, and 42°C. Total florescence signals (monomer + oligomer) were quantified at each time point following native-PAGE and fluorimaging analyses.
(C) Calreticulin (6 μM) and VMAPCFITCTLLL (60 μM) were incubated for the indicated time, and complex formation quantified as described in (B).
(D) Coomassie-stained SDS-PAGE gels. Lanes 1 and 5, direct protein loads of 3 μg HLA-A2 or 6 μg calreticulin. Lanes 2–4, anti-His immunoprecipitations. HS-CRT (heat-shocked calreticulin, prepared by heating calreticulin [6 μM] at 50°C for 1 hr) (lanes 2 and 4) or untreated calreticulin (CRT, 6 μM, lane 3), were incubated with HLA-A2 (6 μM, lanes 2 and 3) or buffer (lane 4) at 37°C for 1 hr, followed by immunoprecipitations. IgG heavy chain (IgG(HC)), A2 heavy chain (A2), and calreticulin (CRT) are indicated. Data are representative of several analyses (A, B, and D) or one analysis (C). All analyses were in 50 mM Tris, 150 mM NaCl, 1 mM CaCl2 (pH 7.5).
Molecular Cell  , DOI: ( /j.molcel )

4. Figure 3
Calcium Depletion Reduces the Critical Oligomerization Temperature of Calreticulin and Enhances Polypeptide Binding by Calreticulin
(A and B) Calreticulin samples (12 μM in 20 mM HEPES, 150 mM NaCl, pH 7.5) were incubated for 1 hr in the presence of 5 mM CaCl2 (A) or 10 mM EGTA (B) at the indicated temperatures. Protein was analyzed by native-PAGE and Sypro Orange staining. Percentage monomers were estimated as a fraction of total protein in each lane. Estimated sizes of the oligomeric species were determined as described in the Sigma Technical Bulletin number MKR-137.
(C) Samples obtained as described in (B), lane 3, were EGTA depleted using a Biospin 30 column. Equal amounts of eluate were incubated with 2 mM EGTA (lane 1) or 5 mM CaCl2 (lane 2) for 1 hr at 37°C and visualized by 10% native-PAGE and Coomassie staining.
(D) Calreticulin (12 μM) in 50 mM Tris, 150 mM NaCl (pH 7.5) was incubated with VMAPCFITCTLLL (120 μM) at 37°C for 1 hr in the presence of 2 mM EGTA or 1 mM CaCl2. Coomassie-stained and fluorescently imaged native gels are shown.
(E) Coomassie-stained SDS-PAGE gels. Lanes 1 and 8; direct protein loads of 3 μg HLA-A2 or 4 μg calreticulin. Lanes 2–7, anti-His immunoprecipitations. Indicated proteins (12 μM each) in 50 mM Tris, 150 mM NaCl containing 2 mM EGTA (lanes 2–4) or 1 mM CaCl2 (lanes 5–7) were incubated at 37°C for 3 hr (lanes 2–6), or 50°C for 1 hr (lane 7), followed by immunoprecipitations.
(F) Quantification of CRT/A2 intensity ratios from lanes 3, 5, and 7. Data are representative of at least two independent analyses each.
Molecular Cell  , DOI: ( /j.molcel )

5. Figure 4
The ΔC Mutant Oligomerizes and Binds Polypeptide Substrates at 37°C
(A and B) ΔC or full-length calreticulin were incubated with 200 μM VMAPCFITCTLLL at 37°C for 1 hr. Following native-PAGE, proteins were visualized by fluorimaging (A) or Coomassie staining (B).
(C) Binding analyses were undertaken as described in (A), but using 24 μM ΔC calreticulin and 240 μM VMAPCFITCTLLL (lane 1) or VEICFITCPYKPTW (lane 2). Only fluorescent panels are shown.
(D) Thermal aggregation of HLA-A2 in the presence of buffer, ΔC, or full-length calreticulin (10 μM each protein), analyzed as described in Figure 1C for HLA-B35.
(E) Coomassie-stained SDS-PAGE gels. Lanes 1, 2, and 8 respectively, direct protein loads of 3 μg calreticulin, 2 μg ΔC calreticulin (asterisk indicates a degradation product), or 2 μg HLA-A2. Lanes 3–7, anti-His immunoprecipitations. The indicated protein(s) (6 μM each) were incubated at 37°C for 1 hr, followed by immunoprecipitation. IgG (LC) denotes IgG light chain.
(F) Thermal denaturation of ΔC calreticulin (12 μM in 20 mM HEPES, 150 mM NaCl, and 1 mM CaCl2, pH 7.5) was monitored by a circular dichroism assay at 280 nm as described (Li et al., 2001). Data are representative of several independent analyses (A and B), three independent analysis (C, D, and F), or 2 independent analyses (E). All analyses (except [F]) were in 50 mM Tris, 150 mM NaCl, 1 mM CaCl2 pH 7.5.
Molecular Cell  , DOI: ( /j.molcel )

6. Figure 5
Specific Structural Forms of Calreticulin Promote Folding and Assembly of a Nonglycosylated Substrate
(A–C) 1 μl HLA-E heavy chain and 1 μl light chain (β2-microglobulin [β2m]) in 6 M guanidine hydrochloride were diluted with rapid stirring into 100 μl refolding buffer alone or buffer containing indicated concentrations of calreticulin (CRT), heat-shocked calreticulin (HS-CRT), ΔC calreticulin (ΔC CRT), or IgG. HLA-E and β2m were at 2 μM after dilution. Proteins were centrifuged at 100,000 × g. Pellets were resuspended in 100 μl SDS-PAGE buffer.
(A) Equal volumes of supernatant (S) and pellet (P) fractions were analyzed by SDS-PAGE and Coomassie staining, and HLA-E heavy chains were quantified. Data is the average of six independent experiments for all samples except IgG and ΔC, which are the average of four and three independent analyses respectively.
(B) A representative Coomassie-stained gel of HLA-E refolding in the presence of buffer or the indicated concentrations of proteins (lanes 1–8). In lanes 9–12, CRT or HS-CRT in refolding buffer were centrifuged, and the S and P fractions were included as controls.
(C) 80 μl supernatants from the refolding analyses in (B) were incubated overnight at 4°C with 50 μg β2m and 100 μM VMAPCFITCTLLL. Samples were separated on a Superose 6 column, and fraction fluorescence was measured using a PTI fluorimeter. HLA-E heterodimers elute in fractions 31–34 (peak at 32 and 33), and calreticulin monomers elute in fractions 28–31 (peak at 29 and 30), as detected by absorbance measurements of the corresponding protein standards (data not shown).
(D–G) HLA-A2 heterodimers were dialyzed into 6 M guanidine hydrochloride and concentrated to 100 μM. 1 μl protein was diluted with rapid stirring into 50 μl buffer alone (lanes 1 and 2) or buffer containing the indicated proteins at 4 μM (lanes 3–8). Samples were centrifuged, and S and P fractions obtained.
(D) Equal volumes of S and P fractions from each refolding were visualized by SDS-PAGE and Coomassie staining.
(E) Quantifications of HLA-A2 heavy chain in S and P fractions, averaged over three independent experiments.
(F) Native-PAGE and fluorimaging analyses (lanes 1–4), 30 μl of the indicated S fractions, incubated overnight at 4°C with β2m and LLDCFITCPTAAV (20 μM each); lane 5, direct incubation of purified HLA-A2 heterodimers (2 μM) with LLDCFITCPTAAV (20 μM) overnight at 4°C (0.2 μg were loaded); and lanes 6–8, incubation of CRT, HS-CRT, and IgG (2 μM each) with LLDCFITCPTAAV (20 μM) overnight at 4°C.
(G) Quantification of fluorescence signals from lanes 1–4 in (F), averaged over three (buffer, HS-CRT, and IgG) or two (CRT) independent experiments each.
Molecular Cell  , DOI: ( /j.molcel )

7. Figure 6
Disulfide-Linked Dimers of Calreticulin Are Observable In Vitro and in HeLa Cells
(A and B) Heat-shocked calreticulin (12 μM) was incubated at room temperature with DTT or SDS as indicated for 1 hr. Samples were separated by native-PAGE and visualized by Sypro Orange staining.
(B) Untreated and NEM-modified calreticulin (12 μM) were incubated at 50°C for 1 hr and oligomerization analyzed by native-PAGE.
(C and E) Immunoblotting analyses with anti-calreticulin antisera, following 8% SDS-PAGE under reducing or nonreducing conditions (C), or native-PAGE (E). 25 μg of lysates from HeLa cells that were maintained at 37°C (lanes 1 and 5), heat shocked for 2 hr at 42°C (lanes 2 and 6) or 45°C (lanes 3 and 7), or incubated at 37°C in the presence of 5 μM thapsigargin (thap) for 21 hr (lanes 4 and 8) were loaded. “*” denotes the position of unidentified higher molecular weight calreticulin species induced upon stress conditions. “CRT-g” denotes a glycosylated calreticulin monomer species.
(D) 25 μg of HeLa cell lysates obtained as described in (C) were digested with 250 U (New England Biolabs) PNGase F (lanes 1 and 4) or left undigested (lanes 2, 3, 5, and 6) and analyzed by immunblotting. Data are representative of several independent analyses (A, B, C, and E) or a single analysis (D).
Molecular Cell  , DOI: ( /j.molcel )

8. Figure 7
Calreticulin-MHC Class I Interactions in HeLa Cells as a Function of the Appearance of Calreticulin Oligomerization Intermediates
(A–D) Proteins in lysates from HeLa cells that were maintained at 37°C, heat shocked at 45°C for 2 hr, treated with tunicamycin (10 μg/ml) for 48 hr, 5 μM thapsigargin for 24 hr, or tunicamycin and thapsigargin for 48 and 24 hr respectively (both) were immunoprecipitated with anti-calreticulin antisera as described in Experimental Procedures. Immunoprecipitated samples (IP) or 5 μg lysates as indicated were separated by 10% SDS-PAGE, and proteins transferred to PVDF membrane.
(A and D) Immunoblotting analyses to detect MHC class I heavy chains (class I HC) or deglycosylated heavy chains (class I HC-dg) using HC-10 antibody.
(B) Heavy chains band intensities were quantified from (A). First panel, lane 1/lane 2; second panel, lane 3/lane 4.
(C and F) Immunoblotting analyses with anti-calreticulin antisera.
(E) Quantitation of heavy chain (first and third panels) and deglycosylated heavy chain (second and fourth panels) bands from (D). First panel, lane 2/lane 1; second panel, lane 4/lane 3; third panel, lane 6/lane 5; fourth panel, lane 8/lane 7. The immunoblot quantifications are not linear with exposure time and therefore do not linearly correlate with the extent of binding under each condition. Thus, we cannot present error bars that are averaged across experiments. However, the results shown in (B) and (E) are representative of at least three independent analyses each.
Molecular Cell  , DOI: ( /j.molcel )