FIG. 3.

Myriocin does not block secretory transport in trypanosomes. (A) Bloodstream-form trypanosomes constitutively expressing the soluble secretory reporter BiPN were cultured for 14 h in the absence (−) or presence (+) of 200 nM myriocin (myr.). Cells were pulse-radiolabeled (15 min) with [³⁵S]Met-Cys and then chased for the indicated times in the continued presence or absence of the inhibitor. The BiPN reporter was immunoprecipitated from cell and medium fractions and analyzed by SDS-PAGE and phosphorimaging. The mobilities of endogenous BiP and VSG, and of the BiPN reporter, are indicated on the left. All lanes contain 5 ×10⁶ cell equivalents. Note that the time points used in this assay fall in the linear range for BiPN transport in bloodstream cells (60). (B) Bloodstream cells were cultured for 14 h in the presence or absence of 200 nM myriocin. Cells were then pulse-radiolabeled (3 min) with [³⁵S]Met-Cys and chased in the continued presence (myr) or absence (control [cont]) of the inhibitor. At the indicated times, samples were washed and subjected to hypotonic lysis as described in Materials and Methods, and VSG polypeptides were immunoprecipitated from the released fractions. VSG was also immunoprecipitated from total-cell fractions at the beginning and end of the chase period (T₀ and T₃₀), and all samples were analyzed as for panel A. The recovery of released VSG as a fraction of total VSG (average of T₀ and T₃₀) is given for each lane. All lanes contain 2 × 10⁶ cell equivalents.