Figure 2.

GRA5 is a membrane-associated protein within the PV. (A) Infected cells were disrupted by passages through needles, and parasites and cell debris were removed by low-speed centrifugation. The LSS was further fractionated by high-speed centrifugation into a membrane pellet (HSP) and a soluble fraction (HSS), separated by SDS-PAGE, and probed with the anti-GRA1, -GRA2, and -GRA5 monoclonal antibodies as described in Figure 1. Whereas GRA1 was found almost exclusively in the soluble fraction, GRA2 was detected in both the soluble and pellet fractions. GRA5 was predominantly detected as a membrane-associated form in the HSP. (B) The membrane pellet obtained in A was resuspended in 50 mM Tris, pH 8.0, incubated with different denaturing agents, and submitted to a high-speed spin. Membrane pellets (P) and soluble fractions (S) were analyzed by SDS-PAGE and Western blotting using the anti-GRA5 mAb. GRA5 behaved as an integral membrane protein, remaining in the membrane pellet after high pH (CO3), high salt concentration (KCl), or urea treatment but was released by NP-40 and partitioned in the detergent phase after treatment with Triton X-114.