FIG. 3.

Analysis of direct septin interactions and the role of GTP binding in septin complex formation in vitro. ³⁵S-labeled septins made individually by coupled in vitro transcription and translation were incubated together at 23°C for 1 h, and complexes were isolated by immunoprecipitation or binding to metal resin, as indicated. Septins in the unbound (UB) and bound (B) fractions were detected by autoradiography. The asterisk indicates a nonspecific band from the Cdc11p synthesis. “(6H)” indicates the presence of six N-terminal histidines. (A) Individual expression of Cdc3p, Cdc10p, Cdc11p, and Cdc12p and interactions among wild-type septins. For combinations of two septins, complexes were immunoprecipitated using anti-Cdc11p antibodies or, for the Cdc3p/Cdc12p pair, anti-Cdc12p antibodies. (B) Effects of P-loop mutations on the pair-wise interaction of full-length Cdc11p and Cdc12p. (C) Interaction of the N-terminal regions of Cdc11p and Cdc12p and the effects of mutations in the Cdc11p P-loop. Cdc11pN, residues 1 to 295; Cdc12pN, residues 1 to 311.