Figure 2
RHBDL4 localizes to ER and rescues proTGFα from ERAD.
(a) RHBDL4-GFP co-localizes to the ER marker RFP-KDEL in HCT116 cells. Scale bar, 5 μM. (b) Fluorescent micrograph of HCT116 cells co-expressing RHBDL2-GFP and RHBDL4-RFP. Scale bar, 5 μM. (c) Immunofluorescence analysis of untransfected COS7 cells shows RHBDL4 co-localization with the ER protein BAP31. Scale bar, 5 μM. (d) Metabolic pulse label and chase experiment shows that the 37-kDa proTGFα species is generated with a lag phase of 30 minutes, whereas lower molecular weight degradation intermediates (white arrow and grey dot) appear directly after the pulse and chase away over time. Asterisk, unspecific band. (e) Treatment of Hek293T cells ectopically expressing proTGFα-FLAG with the proteasome inhibitor MG132 (mg; 5 μM) and epoxomicin (ep; 2 μM) leads to massive increase of the 37-kDa species (black arrow) and to various unglycosylated forms (circles) indicating that it is degraded to a high extent by the ERAD pathway. Whereas the 37-kDa form is secreted, mimicking the effect caused by RHBDL4 overexpression, the lower molecular weight species are only detected in the cell lysates (grey and open symbols).
