Hrd1 interaction proteomics
Endoplasmic reticulum (ER)-associated degradation (ERAD) mediates the degradation of misfolded and unoligomerized proteins in the early secretory pathway. ERAD substrates are detected and delivered to membrane-embedded dislocation complexes. Following transfer into the cytosol, substrates are rapidly ubiquitinated and targeted to the 26S proteasome for degradation. Hrd1 is a highly conserved, ER-resident E3 ubiquitin-protein ligase that functions in ERAD. In this study, we employed stable isotope labeling with amino acids in cell culture (SILAC) to quantitatively assess the impact of altered lipid homeostasis on the composition of S-tagged Hrd1 complexes affinity purified from HEK293 cells. Although lipid disequilibrium impaired ERAD substrate delivery to Hrd1, the overall composition of the Hrd1 complex was unaffected.
Sample Processing Protocol
Parental HEK293 cells or HEK293 cells expressing S-tagged Hrd1 were grown in DMEM lacking L-Arginine and L-Lysine supplemented with 10% dialyzed FBS (Gibco) that was supplemented with the appropriate SILAC amino acids: light, L-Arginine (Arg0) and L-Lysine (Lys0); medium, 13C6-L-arginine (Arg6) and 4,4,5,5-D4-L-lysine (Lys4); heavy, 13C615N4-L-arginine (Arg10) and 13C615N2-L-Lysine (Lys8). Cells were cultured for at least seven cell doublings to allow for complete incorporation of the stable isotope-labeled amino acids (Cambridge Isotope Laboratories, Inc). Parental HEK293 control cells were light SILAC labeled and S-tagged Hrd1 cells were either medium or heavy labeled. 16 hr prior to harvest, the S-tagged Hrd1 cells were incubated with either vehicle (medium SILAC labeled) or 1 µg/mL triacsin C (heavy SILAC labeled). Following several washes in PBS, cells were lysed in IP buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% digitonin, and protease inhibitor tablets (Thermo Fisher Scientific)), and 3 mg of protein lysate loaded onto 75 µL S-protein agarose beads (EMD Millipore). Lysates were rotated at 4ºC for 2 hr, and washed three times with IP buffer containing 0.1% digitonin and twice with 50 mM ammonium bicarbonate. Beads were resuspended in 75 µL 0.2% RapiGest SF (Waters) in 50 mM ammonium bicarbonate for 15 min at 65ºC, followed by incubation with 2.5 µg trypsin (Thermo Fisher Scientific) overnight at 37ºC. The affinity purification for each condition was performed separately to prevent exchange of interaction partners during the incubations. Following the proteolysis step, equal volumes of digested peptides were combined and acidified with HCl to pH 2.0. Rapigest SF precipitate was removed by centrifugation at 20,000xg for 30 min and the peptide solution concentrated to 40 µL using a SpeedVac. Digested peptides were analyzed by LC-MS/MS on a Thermo Scientific Q Exactive Orbitrap Mass spectrometer in conjunction Proxeon Easy-nLC II HPLC (Thermo Fisher Scientific) and Proxeon nanospray source at the University of California, Davis Proteomics Core Facility. The digested peptides were loaded onto a 100 micron x 25 mm Magic C18 100Å 5U reverse phase trap where they were desalted online before being separated using a 75 micron x 150 mm Magic C18 200Å 3U reverse phase column. Peptides were eluted using a 180 min gradient with a flow rate of 300 nl/min.
Data Processing Protocol
An MS survey scan was obtained for the m/z range 300-1600, MS/MS spectra were acquired using a top 15 method, where the top 15 ions in the MS spectra were subjected to HCD (High Energy Collisional Dissociation). An isolation mass window of 1.6 m/z was for the precursor ion selection, and normalized collision energy of 27% was used for fragmentation. A five second duration was used for the dynamic exclusion. The acquired MS/MS spectra were searched against a full UniProt database of human protein sequences and SILAC ratios determined using MaxQuant.
To M, Peterson CW, Roberts MA, Counihan JL, Wu TT, Forster MS, Nomura DK, Olzmann JA. Lipid disequilibrium disrupts ER proteostasis by impairing ERAD substrate glycan trimming and dislocation. Mol Biol Cell. 2016 Nov 23. pii: mbc.E16-07-0483 PubMed: 27881664