FIGURE 1:

SubAB-mediated cleavage of BiP impairs WT rod opsin traffic. (A) SK-N-SH cells were transfected with WT rod opsin-GFP (WT-GFP) and were left untreated or treated with 1.5 μg/ml SubAB or SubA_A272B for 18 h before fixation, as indicated. Cnx antibody (Sigma-Aldrich) was used at 1:600. The cells were fixed with 4% PFA and permeabilized and were analyzed by confocal microscopy using the intrinsic GFP fluorescence and fluorescence from anti-rabbit Alexa 594 secondary antibodies. The scanning intensity levels between control and SubAB treated cells were kept the same. Scale bar, 10 μm. (B) SubAB reduces the amount of EndoH-resistant WT rod opsin. Immunoprecipitated (IP) material from SK-N-SH cells was transfected with WT rod opsin, untreated or treated with SubAB during depletion, labeling, and chase-time intervals, as indicated. Cells were pulsed for 20 min with [³⁵S]methionine/cysteine and were chased in unlabeled methionine media for up to 60 min. The 1D4 IPs were digested with 1500 U of EndoH overnight, as indicated. Digests were analyzed by SDS–PAGE and were visualized by autoradiography. Exposures have been adjusted to show similar levels at time 0. (C) SubAB results in a decrease in cell surface expression of WT rod opsin. “In-cell Western” analysis was performed on WT rod opsin–transfected SK-N-SH cells untreated or treated with SubAB for 16 h. Cells were fixed with 3% PFA and immunostained with 4D2 mAb to the extracellular N-terminus of rod opsin. The fluorescence on the plasma membrane (nonpermeabilized) was determined as a percentage of total fluorescence (permeabilized with 0.025% Triton); n = 3. The data were normalized to untreated WT rod opsin traffic to compensate for difference in expression level; *p < 0.05; p = 0.017.