Figure 7.

The glideosome complex is assembled in two stages and the presence of GAP50 correlates with membrane association. (A) Parallel cultures of Toxoplasma-infected HFF cells were metabolically labeled for 15 min at 37°C with [³⁵S]-labeled methionine and cysteine, and were either placed on ice (pulse) or incubated for an additional 4 h at 37°C after addition of unlabeled amino acids (chase). Parasites were isolated, lysed in TX100 lysis buffer, and subjected to immunoprecipitation with TgGAP45 antiserum. (B) Parallel cultures of Toxoplasma-infected HFF cells were pulse labeled and chased as described above. Parasites were homogenized by sonication and an aliquot was fractionated into soluble (SN) and particulate (P) fractions by centrifugation. These were solubilized in TX100 lysis buffer along with the nonfractionated sample and subjected to immunoprecipitation with the TgGAP45 antiserum. We routinely observed that TgGAP45 in the pulsed sample migrated as a doublet, irrespective of whether it was solubilized in TX100 or SDS.