Supplemental Experimental Procedures

The sequence for S. cerevisiae Sec2 (residues 17-167) was cloned into the plasmid pET15b (Novagen). The construct includes an N-terminal hexahistidine tag. S. cerevisiae Sec4 (residues 18-187) was cloned into a modified version of the pGEX4T-3 plasmid (Amersham Pharmacia), where a sequence coding for hexahistidine was inserted between the glutathione S-transferase sequence and the SEC4 gene. Both tags can be removed by thrombin cleavage. Selenomethionine substituted Sec2p and Sec4p were each overexpressed in Escherichia coli BL21(DE3) cells at 20˚C according to established protocols (Doublie, 1997). The proteins were each purified by Ni-NTA chromatography (Qiagen) in the presence of 20 mM β-Me. Proteins were eluted from the Ni-NTA resin by cleavage with thrombin. The proteins were further purified by gel filtration on a Superdex-200 column (Amersham Pharmacia) in buffer containing 10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5 mM MgCl2, 10 mM DTT, and 5 % (v/v) glycerol. Sec2p/Sec4p Complex Formation and Purification To reconstitute the Sec2p/Sec4p complex, purified Sec2p and Sec4p were mixed togethe