FIG 6 

Motif 1 and 2 are essential and sufficient in localizing VSP to the cell membrane surface. VSP-175(VSPA6-S1) and non-VSP-344 were each divided into N domain, motif 1, and motif 2. Chimera proteins generated from exchanging the three domains between the two proteins were expressed in transfected Giardia trophozoites. (A) Motif 1 in non-VSP-344 was replaced by motif 1 from VSP-175(VSPA6-S1); (B) motif 2 in non-VSP-344 was replaced by motif 2 from VSP-175(VSPA6-S1); (C) both motif 1 and motif 2 in non-VSP-344 were replaced by the corresponding motifs from VSP-175(VSPA6-S1); (D) both motifs in VSP-175(VSPA6-S1) were replaced by the two corresponding motifs from non-VSP-344. Results from immunofluorescence assays showed that only the chimera between the N domain from non-VSP-344 and the two motifs from VSP-175(VSPA6-S1) localizes to the membrane surface (see panel C). This is also the only chimera that was primarily distributed to the organic phase in Western blot analysis. (E) The N domain of VSP-175(VSPA6-S1) was truncated to a 20-amino-acid stretch upstream from motif 1. The truncated mutant was expressed on the membrane surface. (F) The entire N domain of VSP-175(VSPA6-S1) was removed to leave only motif 1 and motif 2. The mutant protein was expressed on the cell membrane. Bars = 10 µm.