Figure 4.

ARF-GEF requirement for retrograde transport from the Golgi to the ER. Membranes of mutant and wild-type strains were prepared from cells grown at a permissive temperature. The membranes were added to stage III of the retrieval assay (for details see MATERIALS AND METHODS). After incubation at 20°C, the samples were complemented with coatomer and Arf1p. Retrieval reactions were incubated at 30°C. At the end of the reaction, samples were subjected to trypsin digestion, concanavalin A-Sepharose precipitation, and SDS-PAGE. The species migrating at the position of trimmed gpαF represents the reporter that has been transported back to the ER. Representative gels are shown. (A) Gea1p and Gea2p support retrograde transport. (B). Sec7p is not required for retrieval of reporter molecules back to the ER. Two different ts mutants were compared with wild-type membranes. In the control, the stage IV (retrieval from the Golgi to the ER) was performed at 4°C.