Figure 6.

Inhibition by Ypt1p-D124N of the in vitro transport reaction is relieved by Ypt1p-GEF, HAP peak A. The indicated amounts of exchange activity from HAP Pool A (1 unit is defined as 1 fmol of GDP released from 0.1 μM Ypt1p per minute per milligram of GEF) were mixed with 1.1 μg of Ypt1p-D124N protein (2 μl) in a total volume of 38 μl of Buffer 88 and were incubated on ice for 20 min. This amount of Ypt1p-D124N protein inhibits the ER-to-Golgi transport reaction 50% under these conditions. The S1 cell fraction (2 μl, ∼100 μg) was added to each reaction, and then incubations were continued on ice for 20 more minutes before the reactions were initiated by the addition of 10 μl of permeabilized yeast cells preloaded with ³⁵S-α-factor. The reactions were incubated at 20°C for 90 min. The percent rescue of inhibition of transport was calculated by setting 0% inhibition equal to the amount of transport in the uninhibited control reaction and by setting 100% inhibition equal to the amount of ER-to-Golgi transport in the fully inhibited reaction (without HAP Pool A). This figure represents the average of two independent experiments with each sample measured as duplicates, and the error bars represent the SD of all four measurements for each point.