METHOD DETAILS

Plasmids expressing ^FTALLC, ^FTJTO, or untagged ALLC in the pCMV1 or pDEST40 vector were described previously (Cooley et al., 2014). ^FTBiP and ERdj3 overexpression plasmids were used as described previously (Genereux et al., 2015). The GRP94 overexpression plasmid was prepared using GRP94.pDONR223 (Addgene; Cat #82130), which was recombined into pDEST40 using Gateway cloning according to the manufacturers protocol. Primary antibodies were acquired from commercial sources and used at the indicated dilutions in Antibody Buffer (50 mM Tris [pH 7.5], 150 mM NaCl supplemented with 5% BSA and 0.1% NaN₃). Mouse monoclonal antibodies were used to detect KDEL (1:1000, Enzo Life Sciences), M2 anti-FLAG (1:500, Sigma Aldrich), Tubulin [B-5-1-2] (1:4000,Sigma), BiP/GRP-28 (1:500, Santa Cruz Biotechnology), β-actin (1:10000, Sigma Aldrich). Polyclonal rabbit antibodies were used to detect GRP94 (1:1000, GeneTex), HYOU1 (1:1000, GeneTex), ERdj3 (DNAJB11) (1:1000, ProteinTech), PDIA4 (1:1000, ProteinTech), PDIA6 (1:1000, GenTex), PDIA3 (ERp57) (1:1000, CellSignaling).

In general, a 10 cm tissue culture plate of HEK293^DAX cells was transfected with the appropriate LC expression plasmids and a fully confluent plate (approximately 10⁷ cells) was used per condition. Cell harvest, cross-linking, lysis and co-immunoprecipitation were carried out as described in the Supplemental Materials and Methods. Proteins were eluted from anti-M1 FLAG agarose beads (Sigma) twice in 75μL elution buffer (10mM Tris [pH 7.5], 2% SDS, 1mM EDTA) by heating to 95°C for 5 min. Eluted fractions were combined and proteins were precipitated in methanol/chloroform, washed twice in methanol, and then air dried. For SILAC experiments, protein pellets were resuspended in 50μL 8M urea, 50mM Tris pH 8.0, reduced with 10mM TCEP (ThermoFisher) for 30 min at room temperature, and alkylated with 12 mM iodoacetamide (Sigma) for 30min in the dark. Samples were then diluted four-fold in 50mM Tris to lower the urea concentration. For TMT experiments, the protein pellets were resuspended in 3 – 5μL 1% RapiGest SF Surfactant (Waters) followed by addition of HEPES buffer (pH 8.0, 50 mM) to a volume of 50μL. Samples were reduced with 5mM TCEP for 30min at room temperature and alkylated with 10mM iodoacetamide for 30min in the dark. Trypsin (0.5μg, Sequencing grade, Promega) was then added to the SILAC or TMT samples and incubated for 16 hours at 37°C while shaking. After digestion, SILAC peptides samples were acidified with formic acid (5% final concentration) and directly proceeded to LC-MS analysis. TMT samples were first reacted with NHS-modified TMT sixplex reagents (ThermoFisher) in 40% v/v acetonitrile and incubated for 60 min at room temperature. Reactions were then quenched by addition of 0.4% (w/v) ammonium bicarbonate. The digested and labeled samples for a given sixplex experiment were pooled and acidified with formic acid (5% final concentration). Samples were concentrated on a SpeedVac and rediluted in buffer A (94.9% water, 5% acetonitrile, 0.1 formic acid, v/v/v). Cleaved Rapigest SF and debris was removed by centrifugation for 30min at 18,000× g.

MuDPIT microcolumns were prepared as described (Fonslow et al., 2012), peptide samples were directly loaded onto the columns using a high-pressure chamber (Shotgun Proteomics Inc), and the columns were washed for 30min with buffer A. LC-MS/MS analysis was performed using a Q-Exactive mass spectrometer equipped with an EASY nLC 1000 (Thermo Fisher). MuDPIT experiments were performed by 10 pL sequential injections of 0, 20, 50, 80, 100% buffer C (500 mM ammonium acetate in buffer A) and a final step of 90% buffer C / 10% buffer B (19.9% water, 80% acetonitrile, 0.1% formic acid, v/v/v) and each step followed by a gradient from buffer A to buffer B on a 18 cm fused silica microcapillary column (ID 100μm) ending in a laser-pulled tip filled with Aqua C18, 3μm, 100Å resin (Phenomenex). Electrospray ionization (ESI) was performed directly from the analytical column by applying a voltage of 2.5 kV with an inlet capillary temperature of 275Ό. Data-dependent acquisition of MS/MS spectra was performed with the following settings: eluted peptides were scanned from 400 to 1800 m/z with a resolution of 70,000 and the mass spectrometer in a data dependent acquisition mode. The top ten peaks for each full scan were fragmented by HCD using normalized collision energy of 30%, 2.0 m/z isolation window, 120 ms max integration time, a resolution of 7500, scanned from 100 to 1800 m/z, and dynamic exclusion set to 60s. Peptide identification and SILAC- or TMT-based protein quantification was performed using the Integrated Proteomics Pipeline Suite IP2 (Integrated Proteomics Applications, Inc.) and modules ProLuCID, DTASelect and Census (Park et al., 2014; Tabb et al., 2002; Xu et al., 2015). MS2 spectra were extracted from Thermo XCalibur .raw file format using RawConverter (He et al., 2015). Spectra were searched using ProLuCID against a Uniprot human proteome database (release date 05/2014). The database was curated to remove redundant protein and splice-isoforms, and the sequences for the variable domains of ^FTALLC and ^FTJTO and the shared constant domain were added. Searches were carried out using a decoy database of reversed peptide sequences and the following search parameters: 50 ppm peptide precursor tolerance, 0.6 Da fragment mass tolerance, 6 amino acid minimum peptide length, trypsin cleavage (max. 2 missed cleavage events), static Cys modification of 57.0215 (carbamidomethylation), and static N-terminal and Lys modification of 229.1629 (TMT-sixplex). ProLuCID search results were filtered using DTASelect using combined XCorr and DeltaCN scores to minimize the peptide false discovery rate at 1% and minimum of 2 peptides per protein ID. TMT reporter ion intensities were extracted in Census using a mass tolerance of 0.05 Da and summed for individual peptides belonging to the same protein. SILAC data was processed similarly, except static modification from the TMT-sixplex were omitted, and heavy [¹⁵N, ¹³C]-Lys and Arg modifications were included in the ProLuCID search.

Transfected HEK293^DAX were plated 150,000 cells/well in 2 identical 48-well plates (Genessee Scientific) containing 500 μL of media. Media was removed and wells were washed two times with 250 μL media containing 50 μg/mL cycloheximide (CHX). One plate was washed two times with 1× PBS and cell lysates prepared in RIPA buffer. This sample was used to monitor lysate levels of LC at t=0 h. The second plate was incubated 250 μL media with CHX for 4 h and conditioned media was collected. This sample was used to monitor secreted LC levels at 4 h. Free LC concentrations were determined by ELISA in 96-well plates (Immulon 4HBX, Thermo Fisher), as previously described (Cooley et al., 2014; Plate et al., 2016). Briefly, wells were coated overnight at 37 °C with sheep polyclonal free λ LC antibody (Bethyl Laboratories, A80-127A) at a 1:500 dilution in 50 mM sodium carbonate (pH 9.6). In between all incubation steps, the plates were rinsed extensively with Tris-buffered saline containing 0.05% Tween-20 (TBST). Plates were blocked with 5% non-fat dry milk in TBST for 1 hr at 37°C. Media analytes were diluted between 5 – 200-fold in 5% non-fat dry milk in TBST and 100 μL of each sample was added to individual wells. Light chain standards ranging from 3 – 300 ng/mL were prepared from purified human Bence Jones λ light chain (Bethyl Laboratories, P80-127). Plates were incubated at 37 °C for 1.5 h with shaking. Finally, HRP-conjugated goat anti-human λ light chain antibody (Bethyl Laboratories, A80-116P) was added at a 1: 5,000 dilution in 5% non-fat dry milk in TBST, followed by a 1.5 h incubation of the plates at 37 °C. The detection was carried out with 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS, 0.18 mg/mL) and 0.03% hydrogen peroxide in 100 mM sodium citrate pH 4.0. Detection solution (100 μL) was added to each well and the plates were incubated at room temperature. The absorbance was recorded at 405 nm and the values for the LC standards were fitted to a 4-parameter logistic function. LC concentrations were averaged from at least 3 independent replicates under each treatment and then normalized to vehicle conditions. Fraction secreted was then calculated using the equation: fraction secreted = [LC] in media at t=4 h / [LC] lysate at t = 0 h.

Cell lysates were prepared as previously described in RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1 % SDS, 1% Triton X-100, 0.5% deoxycholate and protease inhibitor cocktail (Roche). Total protein concentration in cellular lysates was normalized using the Bio-Rad protein assay. Lysates were then denatured with 1X Laemmli buffer + 100 mM DTT and boiled before being separated by SDS-PAGE. Samples were transferred onto nitrocellulose membranes (Bio-Rad) for immunoblotting and blocked with 5% milk in Tris-buffered saline, 0.5 % Tween-20 (TBST) following incubation overnight at 4°C with primary antibodies. Membranes were washed in TBST, incubated with IR-Dye conjugated secondary antibodies and analyzed using Odyssey Infrared Imaging System (LI-COR Biosciences). Quantification was carried out with LI-COR Image Studio software. For immunoprecipitations, cells were washed with PBS and then treated with the indicated concentration Dithiobis(succinimidiyl propionate) (DSP) for 30 min at room temperature. The crosslinking reaction was quenched by addition of 100 mM Tris pH 7.5 for 15 min, then lysates were prepared in RIPA buffer. Total protein concentration in cellular lysates was normalized using Bio-Rad protein assay. Cell lysates were then subjected to preclearing with Sepharose 4B beads (Sigma) at 4 °C for 1 h with agitation. The precleared lysates were then subjected to immunoprecipitation with a M1 anti-Flag agarose resin (Sigma) at 4 °C overnight. After four washes in RIPA buffer, proteins were eluted by boiling in 6× Laemmli buffer and 100 mM DTT. Blots from IPs and inputs were probed with the primary antibodies. Membranes were then treated as described above.

[³⁵S] metabolic labeling experiments were performed as previously described (Cooley et al., 2014; Shoulders et al., 2013). Briefly, transfected HEK293^DAX were plated on poly-D-lysine coated 6-well plates and metabolically labeled in DMEM-Cys/-Met (Corning CellGro, Mediatech Inc., Manassas, VA) supplemented with glutamine, penicillin/streptomycin, dialyzed fetal bovine serum, and EasyTag EXPRESS [³⁵S] Protein Labeling Mix (Perkin Elmer) for 30 min. Cells were washed twice with complete media and incubated in pre-warmed DMEM for the indicated times. Media or lysates were harvested at the indicated times. Lysates were prepared in RIPA buffer (50mM Tris [pH 7.5], 150mM NaCl, 1% Triton X100, 0.5% sodium deoxycholate, 0.1% SDS) containing proteases inhibitors cocktail (Roche). FLAG-tagged LC variants were immunopurified using M1 anti-FLAG agarose beads (Sigma Aldrich) and washed four times with RIPA buffer. The immunoisolates were then eluted by boiling in 6X Laemmli buffer and separated on 12% SDS-PAGE. Gels were stained with Coomassie Blue, dried, exposed to phosphorimager plates (GE Healthcare, Pittsburgh, PA) and imaged by autoradiography using a Typhoon Trio Imager (GE Healthcare). Band intensities were quantified by densitometry in ImageQuant. Fraction secreted was calculated using the equation: fraction secreted = [extracellular [³⁵S]-LC signal at t / (extracellular [³⁵S]-LC signal at t=0 + intracellular [³⁵S]-LC signal at t=0)]. Fraction remaining was calculated using the equation: [(extracellular [³⁵S]-LC signal at t + intracellular [³⁵S]-LC signal at t) / (extracellular [³⁵S]-LC signal at t=0 + intracellular [³⁵S]-LC signal at t=0)].