The signal sequence and C-terminal hydrophobic domain are required for localization of the sexual stage antigen Pgs28 to the surface of P-gallinaceumookinetes

The Pgs28 protein is a major surface antigen of the sexual stages of Plasmo dium gallinaceum - the zygotes and the ookinetes. The protein contains cons erved motifs, namely an N-terminal signal sequence, four epidermal growth f actor-like repeats and a C-terminal hydrophobic domain that serves as a sig nal for glycosylphosphatidylinositol (GPI) - anchor modification. In this s tudy, we define the protein motifs required for the surface localization of Pgs28 in ookinetes, using transient transfection combined with immunofluor escence and confocal microscopy. Pgs28 fused to the green fluorescent prote in (Pgs28-GFP) is expressed in zygotes, intermediate retort forms and ookin etes. Mutational analyses of Pgs28 coding regions reveal that deletions of the signal sequence and the C-terminal domain result in intracellular reten tion of the fusion protein. Therefore, the signal sequence and C-terminal d omain are required for cell surface localization. Additionally, the Pgs28-G FP fusion proteins are shed from the surface of live ookinetes, suggesting that Pgs28 may be involved in interactions with the cells of the mosquito m idgut or during motility. (C) 2000 Elsevier Science B.V. All rights reserve d.