Figure 3.

The ost2-1D and ost2-2D genes complement the yeast RS-72. (A) Complementation of the yeast mutant RS-72 in which the essential gene encoding the H⁺-ATPase PMA1 was placed under the strict control of a galactose (GAL)-inducible promoter. The ost2 genes with the mutations P₆₈S (ost2-1D) and G₈₆₇S (in ost2-2D), but not the wild-type AHA1, are able to complement the growth defect of RS-72. (B) Testing intramolecular interaction between the C-terminal regulatory domain and the remaining protein fragment of AHA1 using the yeast two-hybrid test based on split ubiquitin. Both the N-terminal moiety (aa 1–842) and the C-terminal regulatory domain (aa 838–949) were expressed as Cub-PLV and NubHA fusion protein, respectively. Interaction between the two parts of AHA1 causes cleavage release of the synthetic PLV transcription factor from Cub. PLV is then transported to the nucleus to activate the LacZ gene whose reporter expression is detectable as a blue color in the presence of X-gal. The expression levels of the different protein domains were compared by immunoblotting using either anti-HA or anti-VP16.