Assay for endoplasmic reticulum-associated degradation (ERAD) in mammalian cells

Assay for endoplasmic reticulum-associated degradation (ERAD) in mammalian cells

Cultured cells: approximately 1 × 10⁶ cells

A truncation mutant of α1-antytrypsin null Hong Kong (NHK) is a glycoprotein that is degraded by ERAD ⁴⁾. NHK degradation was enhanced by co-transfection with ER degradation enhancing α-mannosidase-like protein 3 (EDEM3) ⁵⁾, as observed in a pulse-chase experiment (Fig. 1). NHK degradation was inhibited following the addition of the proteasome inhibitor Lactacystin (Fig. 2), indicating that the proteasome was responsible for this degradation.

SEL1L, the human homolog of C. elegans sel-1, is an ER membrane protein that forms a stoichiometric complex with the ubiquitin ligase, HRD1. Knockdown of endogenous SEL1L by siRNA treatment inhibited NHK degradation (Fig. 3) ^{6) 7)}, suggesting that SEL1L is required for the degradation of NHK.

The degradation of ERAD substrates can be quantitatively assessed using these pulse-chase experiments. This method may be useful to identify components involved in ERAD and to assess their roles within the pathway. Although cycloheximide chase experiments are more convenient than pulse-chase experiments, it should be noted that the total amount of protein accumulated in cells is detected by this method, and that not just ERAD substrates but also the synthesis of most cellular proteins is inhibited. Thus, the addition of cycloheximide inhibits the degradation of some ERAD substrates, including NHK ⁸⁾.

Figure & Legends

Fig. 1. EDEM3 enhances the degradation of NHK.

HEK (human embryonic kidney) 293 cells were transfected with plasmids encoding NHK, together with an empty vector or EDEM3-HA using FuGENE 6 transfection reagent (Roche), and labeled with [³⁵S]-protein labeling mixture for 15 min. NHK and EDEM3 were immunoprecipitated with an anti- α1-antytrypsin antibody (lanes 1–6) or with an anti-HA-tag antibody (lanes 7–9). Immunoprecipitates were separated on a 10 % SDS-PAGE gel, which was then dried and exposed to an imaging plate. The radioactive signal was quantified using a Typhoon PhosphorImager (GE Healthcare Life Sciences) (right panel).

This figure was originally published in J. Biol. Chem. Hirao K. et al. “EDEM3, a soluble EDEM homolog, enhances glycoprotein endoplasmic reticulum-associated degradation and mannose trimming” 2006, 281: 9650–9658. © the American Society for Biochemistry and Molecular Biology.

Fig. 2. Lactacystin inhibits the degradation of NHK.

Cells were plated and transfected as described in the legend of Figure 1. Cells were treated with 20 μM lactacystin for 3 h prior to pulse-labeling (lanes 4–6, indicated as +) or were untreated (lanes 1–3, indicated as -).

This figure was originally published in the J. Biol. Chem. Hirao K. et al. “EDEM3, a soluble EDEM homolog, enhances glycoprotein endoplasmic reticulum-associated degradation and mannose trimming” 2006, 281: 9650–9658/ © the American Society for Biochemistry and Molecular Biology.

Fig. 3. SEL1L is required for the degradation of NHK.

HEK293 cells were treated with 30 nM of negative control Stealth™ siRNAs (Invitrogen/Life Technologies) (control-1 and -2, lanes 1–6) or specific siRNAs targeting SEL1L (SEL1L-2 and -3, lanes 7-12), and NHK degradation was analyzed as described in the legend of Fig. 1.

This figure was originally published in the J. Biol. Chem. Hosokawa N. et al. “Human XTP3-B Forms an Endoplasmic Reticulum Quality Control Scaffold with the HRD1-SEL1L Ubiquitin Ligase Complex and BiP” 2008, 283: 20914–20924. © the American Society for Biochemistry and Molecular Biology.