Fig. 4.

CADA diverts the nonglycosylated preprotein for proteasomal degradation.A, autoradiogram of cell lysates from nontransfected (NT) and huCD4-V5 transfected CHO-K1 cells pulsed for 30 min with [³⁵S]methionine/cysteine. Pulsed-labeled cells were then chased for 45 min in the presence or the absence of CADA (10 μM) and/or MG132 (200 nM). Open arrowhead represents the translocated (and glycosylated) huCD4 mature protein fraction, and solid arrowhead represents the nonglycosylated huCD4 preprotein. Graph on the right shows the quantification of the presented radioblot. For each sample, the fraction of preprotein and mature protein is calculated and given as percentage of total amount of protein. B, Western blot images of cell lysates from nontransfected (NT) and huCD4-V5, SORT-V5, and PTK7-V5 transfected HEK293T cells treated for 24 h with different CADA concentrations and 200 nM MG132. Protein bands were visualized using an antibody against the V5 tag, whereas anticlathrin (huCD4) or anti-β-actin (SORT and PTK7) antibody was used for the cell loading controls. Do note that the combination of MG132 with 10 or 2 μM CADA strongly reduces the level of mature protein, presumably by blocking the residual translocation of substrate that is normally seen with CADA. C, same as in (B) but for 10 μM CADA and/or 200 nM MG132. Lysates were treated with endo H prior to SDS-PAGE and Western blotting to remove N-linked glycosylations. Protein bands were visualized through V5. Do note that, because of the stronger denaturation conditions during the endo H treatment, V5 detection of the endo H treated samples was more efficient, giving thicker protein bands (although the same sample loading was used). PTK7 is a highly glycosylated protein (ten potential N-glycosylations) that is partially resistant to endo H treatment, resulting in only a minor fraction that is deglycosylated by endo H (solid arrowhead). CADA, cyclotriazadisulfonamide; Endo H, endoglycosidase H; HEK293T, human embryonic kidney 293T cells; huCD4, human CD4; PTK7, inactive tyrosine-protein kinase 7; SORT, sortilin.