(A) Schematic illustration of the hamster FICD protein. The transmembrane region (TM), the two tetratricopeptide repeats (TPR) as well as the FIC-domain (purple) with its core sub-domain (dark purple) and the catalytic loop sequence are shown. Numbers represent amino acid positions. The amino acid sequence surrounding the mutations introduced into the CHO-K1 FICD-/- clone (#49) by CRISPR-Cas9-mediated genome editing are noted. Both alleles result in premature termination of translation deleting the active site (*). (B) Immunoblots of endogenous BiP from wildtype (wt) or FICD-/- CHO-K1 cell lysates from which ATP was either depleted by incubation with hexokinase and glucose (-ATP, top panel) or to which ATP (1 mM) had been added (+ATP, bottom panel), resolved by native-PAGE. Where indicated the cells were exposed to cycloheximide (CHX, 100 µg/ml) or thapsigargin (Tg, 0.5 µM) for 3 hr before lysis. The major species visible on the native gels are numbered by order of descending mobility (I-III) and the monomeric ‘B’ form induced by CHX treatment and the ‘A’ form, prominent in ATP-replete lysates of untreated cells, are marked. Immunoblots of the same samples resolved by SDS-PAGE report on total BiP loaded and on eIF2α as a loading control. The ATP-supplemented lysates (3 µg/µl protein) were in addition exposed to SubA (30 ng/µl) for 10 min at room temperature prior to separation by SDS-PAGE and immunodetection of BiP. The intact protein and the substrate binding domain (SBD), which are detected by the antibodies against a C-terminal epitope of BiP, are indicated. The asterisk marks a band of unknown identity. Note that neither CHX-dependent conversion of endogenous BiP into the monomeric ‘B’ form nor the CHX-mediated resistance of BiP towards proteolytic cleavage by SubA, were observed in FICD-/- cells. (C) Immunoblot of endogenous BiP from wildtype and FICD-/- CHO-K1 cell lysates resolved on an isoelectric focusing (IEF) gel. Where indicated the cells have been exposed to CHX (100 µg/ml) for 3 hr before lysis. Note that the more acidic (‘B’) form of BiP associated with CHX treatment was absent in FICD-/- cells. (D) IEF immunoblot of endogenous BiP from CHO-K1 FICD-/- cells transfected with plasmids encoding wildtype GST-FICD, the constitutively active GST-FICDE234G or the inactive GST-FICDE234G-H363A mutant. Mock transfected cells were analyzed as a control. The cells were treated with CHX (100 µg/ml) for 3 hr before lysis. A pulldown with GSH-Sepharose beads was performed with the same lysates to analyze expression levels of the plasmid-encoded GST-FICD fusion proteins. Note that formation of the acidic (‘B’) form of BiP was restored by expression of catalytically active GST-FICDE234G protein (despite its comparatively low expression level) but neither by expression of the catalytically inactive GST-FICDE234G-H363A mutant nor the regulated wildtype enzyme.