Fig. 1. Ypt1p is required for retrograde transport from the Golgi to the ER. (A) Addition of GDI reduced the retrieval efficiency of [³⁵S]gpF-HDEL to the ER. A round-trip assay was performed with wild-type semi-intact cells. GDI was added to the round-trip assay after the fusion of COPII vesicles with the ER. The amount of trimmed [³⁵S]gpF-HDEL in lane 2 was reduced to 63% compared with that in the buffer control (100%). In the buffer control 1.14% and after addition of GDI 0.73% of the [³⁵S]gpF-HDEL incorporated into COPII vesicles at the donor ER reached the acceptor ER and was trimmed. (B) YPT mutants are not defective in retrograde transport at the permissive temperature. Semi-intact cells from different ts-mutants and wild type were used as acceptor membranes in a round-trip assay. The last step, the retrieval from the Golgi to the ER, was performed at the permissive temperature, 23°C. All semi-intact cells gave a comparable signal of trimmed [³⁵S]gpF-HDEL. (C) Deletion of YPT1 results in a reduction of retrograde transport from the Golgi to the ER. In parallel to the assay in B an assay was performed, in which the temperature was raised in the last step to 30°C. This temperature should represent at least a semi-restrictive temperature for most ts-mutants. The signal did not alter significantly for most mutants when compared with wild type, but the signal of retrieved and trimmed [³⁵S]gpF-HDEL was strongly diminished in ypt1/SLY1-20 acceptor membranes. (D) SLY1-20 expression does not contribute to the defect in retrograde transport. A retrieval assay was performed as described above. In ypt1/SLY1-20 membranes the signal in the retrograde transport assay was drastically reduced at 30°C, but the transport in WT/SLY1-20 was as efficient as in WT. (E) ypt1-3 is not defective in retrograde transport in vitro. A retrieval assay was performed to compare two different YPT1 mutants side by side. ypt1/SLY1-20 membranes were unable to allow retrograde transport of [³⁵S]gpF-HDEL at the restrictive temperature, ypt1-3 membranes behaved like wild-type semi-intact cells. `Bckgrd' is an assay using wild-type membranes, but cytosol was omitted in the last incubation step (transport from the Golgi to the ER). The retrieval efficiency was determined as percentage of trimmed [³⁵S]gpF-HDEL of the reporter that was incorporated into COPII-coated vesicles at the ER. At 20°C: WT 2.9 %, ypt1/SLY1-20 2.34 %, ypt1-3 3.15 %; at 30°C: WT 2.92 %, ypt1/SLY1-20 1.28%, ypt1-3 3.05%.