Figure 7.

Lumenal portion of human Gpi8p is sufficient for its functions. (A) Schematic representation of wild-type and carboxyl-terminal deletion mutants of human Gpi8p. 310del, 321del, and 332del refer to deletion mutants of GPI8 encoding amino acids 1–310, 1–321, and 1–332, respectively. Segments are patterned in the same way as in Figure 5A. (B) Wild-type and deletion mutants of human GPI8 were transiently transfected into class K cells. Surface expression of CD59 was measured by flow cytometry 2 d later. Solid and dotted lines indicate staining with anti-CD59 antibody and that with secondary reagent alone, respectively. (C) Association of HA-tagged Gaa1p and FLAG-tagged deletion mutants of Gpi8p were analyzed by coprecipitation. Plasmids for HA- or FLAG-tagged proteins were transiently transfected into CHO cells in the indicated combinations. The precipitates from NP-40 lysates with anti-HA antibody were subjected to Western blotting with anti-HA (lanes 1–6) or anti-FLAG (lanes 7–12) antibodies. The remaining supernatants were then subjected to precipitation and blotted with anti-FLAG antibody (lanes 13–18). An asterisk indicates nonspecific bands of unknown origin that overlap with a band of 310del Gpi8p (lane 15). Molecular markers in kilodaltons are indicated on the left.