The amphipathic alpha helices of the toxoplasma protein GRA2 mediate post-secretory membrane association

The Toxoplasma gondii protein GRA2 is secreted into the parasite-containing vacuole where it is rapidly and specifically targeted to a network of membranous tubules that connect with the vacuolar membrane. To examine the molecular basis of this association, we expressed an HA9 epitope-tagged form of GRA2 by stable transformation of Toxoplasma. GRA2-HA9 was correctly packaged inside the dense granules, secreted into the PV and targeted to the network, as shown by immunoelectron microscopy, immunofluorescence and cell fractionation. Expression of deletion mutants of GRA2-HA9 lacking either of two amphipathic alpha helices resulted in the production and secretion of soluble proteins which were unable to stably associate with the network. A mutant in which the amino acids of the first alpha helix were rearranged to a non-amphipathic pattern localized correctly to the network but failed to remained stably associated with the membrane. Collectively, these results demonstrate that targeting and membrane association occur by separate mechanisms and that the combination of both alpha helices is essential for stable localization of GRA2 to the network.