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Fig. 1. The Sec7 domain of Gea2p interacts with Drs2p. (A) Yeast two-hybrid strain Y190 carrying the C-terminal region of Drs2p (amino acids 1230-1355) in a two-hybrid prey vector and either the Gea2p Sec7 domain, full-length Gea1p or the two-hybrid bait vector alone were grown on selective medium lacking leucine and tryptophan (-L-T) that selects for each plasmid (right), or on medium containing X-gal (5-bromo-4-chloro-3-indolyl-ß-D-galactoside) (left). (B) Drs2-HA was expressed in E. coli, and cell lysates loaded onto an anti-HA antibody affinity column. The Sec7 domain of Gea2p was transcribed and translated in vitro in the presence of [³⁵S]methionine. The radiolabeled protein was passed over the Drs2-HA column, and a control column loaded with lysates from cells not expressing Drs2-HA. (C,D) The Gea2 Sec7 domain tagged with HA was expressed in E. coli and bound to an anti-HA antibody affinity column. The full-length Drs2p C terminus, amino acids 1230-1355 (C) and two halves, amino acids 1230-1305 (D) and amino acids 1306-1355 (not shown) were radiolabeled in vitro and passed over Gea2 Sec7 domain and control columns. The bound fraction was run on SDS-PAGE gels, which were exposed to film. Coomassie Blue-stained gels of the E. coli cell lysates used to prepare each column are also shown.

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