Figure 3. Cysteine mutants are enzymically active but unable to access VSG on hypotonic lysis.

Cell lines modified to contain a single copy of the wild type (CCGAC) or cysteine mutant GPI-PLC all modified with a C-terminal eYFP tag were assayed for hydrolysis of the VSG GPI-anchor. (A) Expression level of GPI-PLC-eYFP wild type and mutant transgenes detected by Western blots probed with anti-GPI-PLC or anti-DHH1 as a loading control. 2×10⁶ cell equivalents were loaded in each track. (B) Western blot lysates of cells expressing GPI-PLC wild type and mutants before and after 20 min incubation in 0.5% triton X-100. The GPI-PLC activity was detected by probing with anti-CRD and anti-BiP was used as a loading control. Two clones are shown for each transgene. (C) One clone expressing each transgene was lysed hypotonically and incubated for 20 minutes at 37°C. Pellet (P) and supernatant (S) fractions were separated at 0 and 20 minutes and analysed by Coomassie staining gels. The white arrows indicate the VSG released. 2×10⁶ cell equivalents were loaded in each track.