Figure 3.

Exchanging the arginine between Sec22p and Bet1p [sec22(Q)/bet1(R)] does not affect growth or ER-to-Golgi transport. (A) Cells expressing wild-type BET1 and sec22(Q) as the only source of SEC22 (4Q ratio, strain Y15177 +pUA4-sec22R157Q) show reduced growth at 35°C and failed to grow at 37°C when incubated for 48 h (see Figure 1). Growth was normal in cells where the 3Q:1R ratio was restored by introducing an arginine residue at the `0' layer position of Bet1p (second row, strain YCG8). (B) Processing of CPY, analyzed by SDS-PAGE and autofluorography. CPY was immunoprecipitated from cell extracts after a 15-min preincubation and pulse-chase labeling at 37°C (see Materials and Methods). In sec22(Q)/bet1(R) mutant cells, like in wild-type cells, all of the labeled CPY was transported to the vacuole. After a chase period of 30 min, only the mature form of CPY (mCPY) was detected, whereas the ER form (p1CPY) accumulated in sec22(Q) mutant cells.