Fig. 2. Ypt6p effectors. (A) Identification of proteins specifically bound to GST–Ypt6:GTPγS. Cytosol from a wild-type yeast strain was incubated with GST–Ypt6 affinity columns that had been pre-loaded with GDP or GTPγS and bound proteins eluted and analysed by SDS–PAGE followed by Coomassie staining as described in Materials and methods. Ypt6p effector proteins are numbered (1–4). Band 1 contains both Sgm1p and a second protein that binds in a nucleotide-independent manner. Band 3 corresponds to Vps53p and Mdr1p. (B) Western blot analysis of fractions eluted from GST–Ypt6:GTPγS or GST–Ypt6:GDP columns that had been incubated with cytosol from strains expressing Vps52p-PtA, Vps53p-PtA or Vps54p-PtA (strains SSY12, SSY13 and SSY14, respectively). Blots were probed with anti-PtA, anti-Pgk1p or anti-Imh1p antibodies. (C) As (B), but the columns contained GST–Ypt31p or GST–Ypt52p and were incubated with cytosol from a strain expressing Vps54p-PtA. The blot was probed with anti-PtA. (D) Left panel: binding of Sgm1p-PtA from cytosol to GST–Ypt6p as in (B). Right panel: the coiled-coil profile of Sgm1p as predicted using a 21 residue window and the program Coils (Lupas, 1996).