Fig. 1. Vps4p interacts with Vps20p and Vta1p. (A) A schematic showing the predicted domain structure of Vps20p, Vta1p and Vps4p, the fragments of Vps20p and Vta1p encoded by the two-hybrid library plasmids pB42AD-VPS20 (pAM349) and pB42AD-VTA1 (pAM398), and the various constructs used to map the domains that interact. Cross-hashed boxes represent domains predicted to have a high propensity (0.5) to form coiled-coil structure as assessed using the COILS algorithm (Lupas et al., 1991). Numbers refer to amino acid residues. Library plasmids are depicted using solid lines and constructs used for mapping using broken lines. (B) Yeast two-hybrid interactions of Vps4p with Vps20p and Vta1p. Yeast strain EGY48 carrying the reporter plasmid p8op-LacZ together with either pLexA vector alone or pLexA-VPS4 (pAM333), and either pB42AD vector alone, pB42AD-VPS20 or pB42AD-VTA1 were plated on SD complete medium lacking uracil, histidine and tryptophan to select for the p8op-LacZ, pLexA-based and pB42AD-based plasmids, respectively. After the strains had grown sufficiently they were replica plated to synthetic galactose/raffinose (SG) complete medium containing X-gal to test expression of the lacZ two-hybrid interaction reporter gene. The plates were photographed after 4 days at 30°C. Shown are patches representing four independent transformants for each plasmid combination (a-f). (C) Quantification of yeast two-hybrid interactions. Each of the strains in B was assayed for ß-galactosidase activity as a measure of the strength of two-hybrid interaction. Activities (in Miller units) represent the means obtained from assaying three independent transformants. Error bars, +/-s.e.m.

Return to article