FIG 3 .

C-terminal CRGKA is not required for VSP expression on the Giardia trophozoite membrane surface. The three C-terminal myc-tagged VSPs and their three mutants, which have the C-terminal CRGKA replaced with CRRET, QSSKE, and WPGSL, were each expressed in transfected Giardia trophozoites: VSP-175(VSPA6-S1)-myc (A), VSP-175′(VSPA6-S1′)-myc (B), VSP-88(VSP9B10A)-myc (C), VSP-88′(VSP9B10A′)-myc (D), VSP-98.1(VSP1267)-myc (E), and VSP-98.1′(VSP1267′)-myc (F). Three other C-terminal myc-tagged proteins, previously designated VSPs, 333, 344, and 313, were also expressed in transfected Giardia trophozoites. Their respective C-terminal CRRET, QSSKE, and WPGSL were each replaced with CRGKA, designated 333′, 344′, and 313′, and expressed: 333-myc (G), 333′-myc (H), 344-myc (I), 344′-myc (J), 313-myc (K), and 313′-myc (L). Extracts from the 12 transfectants were analyzed by a Western blot stained with anti-myc-Ab. All 3 previously verified VSPs and their C-terminal pentapeptide mutants were present primarily in the organic phase, whereas protein 333 and its mutant were also largely in the organic phase, but 313 and 344 and their mutants were largely in the aqueous phase. Immunofluorescence assays of the 12 transfectants in both nonpermeabilized and permeabilized forms showed that all 3 VSPs and their mutants were expressed on the cell membrane surface, whereas proteins 333, 313, and 344 and their mutants were all confined in the cytoplasm. C-terminal CRGKA thus does not dictate membrane expression of proteins. Bars = 10 µm.