Signal Peptide-Binding Drug as a Selective Inhibitor of Co-Translational Protein Translocation
Figure 2
CADA specifically inhibits the biogenesis of human CD4.
(A, B) CADA inhibits the biosynthesis of CD4. CD4+.CHO cells were washed and kept in methionine and cysteine-free medium in the presence or absence of 16 µM CADA for 45 min before exposure to [35S]methionine/cysteine (Met/Cys) for 30 min. Pulsed-labelled cells were then washed, lysed, and analyzed directly (A) or incubated in normal medium for up to 4 h (chase) in the presence or absence of 16 µM CADA (B). At specified time points cell lysates were immunoprecipitated for CD4. The flow through fraction (FT) of the CD4-immunoprecipitated samples is also presented. Note that the weaker CD4 bands in the control samples at longer chase time points are the result of the high turnover of hCD4 in CHO cells. Molecular mass is in kDa. (C–F) CD4 negative and stably CD4-YFP transfected CHO cells were pretreated with CADA (5 µM) or DMSO for 1 h before starvation in Met/Cys free medium with CADA, DMSO, or 50 µg/ml CHX. Cells were pulsed for 30 min, washed, and incubated in fresh medium without serum for 90 min. After collection of supernatant proteins (Media) cells were first permeabilized with digitonin buffer to obtain the cytosolic cell fraction before lysis in NP-40 buffer to collect the membrane proteins. Membrane fractions were further incubated with Concanavalin A (ConA) agarose beads (Glycosylated). Molecular mass is in kDa. (D) Quantification of 35S incorporation in (C) by scintillation counting (n = 4). NS, not significant; *p<0.01. (F) ConA fraction from a repeat experiment with CHO.CD4+ cells treated with DMSO or CADA. Note that the expression of CD4-YFP (∼80 kDa) is clearly reduced by CADA-treatment (indicated by arrow).