Figure 3. Catalytically inactive YOD1 C160S impairs the dislocation of truncated ribophorin.

(A) 293T cells were co-transfected with RI₃₃₂ and empty vector, YOD1 WT or YOD1 C160S. 24 hours after transfection, cells were pulse-labeled with ³⁵S for 10 min, chased for the indicated time points, lysed in 1% SDS, and the lysates were subjected to immunoprecipitation with anti-ribophorin antibodies. The eluates were resolved by 12% SDS-PAGE and visualized by autoradiography (upper panel). Unbound material was immunoprecipitated with anti-FLAG antibodies to verify equal expression of the YOD1 constructs (lower panel).

(B) Densiometric quantification of RI₃₃₂ levels. Plotted are the mean values from three independent experiments. Error bars depict the standard deviation.

(C) 293T cells were co-transfected with a cytosolic variant of RI₃₃₂ lacking its N-terminal signal sequence (ΔSS RI₃₃₂) and with either empty vector (pcDNA), YOD1 WT or catalytically inactive YOD1 C160S, and processed as in (A).

(D) ΔSS RI₃₃₂ stability was quantified as in (B).

(E) Cells co-transfected with YOD1 C160S and RI₃₃₂ were metabolically labeled as in (A). Cell extracts were prepared in hypotonic buffer by homogenization in absence of detergent. The homogenate was incubated on ice in presence and absence of 100 μg/ml proteinase K. 0.5% NP40 was added as indicated. Proteinase K was inactivated after 15 min by inclusion of PMSF. The resulting material was solubilized with 1% SDS, immunoprecipitated with anti-ribophorin antibodies and processed as in (A).