Figure 4.

Exchanging the arginine between Sec22p and Bos1p [sec22(Q)/bos1(R)] renders the mutant cells temperature sensitive and causes a block in anterograde traffic from ER to Golgi. (A) Cells with the ratio in the central layer of the ER-to-Golgi SNARE complex restored to 3Q:1R, but with the arginine moved to Qb, are viable but show a stronger growth defect than cells with a 4Q ratio. When incubated for 48 h on YEPD plates sec22(Q)/bos1(R) mutants (YCG38) failed to grow at 37°C and grow poorly even at medium or low temperatures. (B) Analysis of CPY processing in sec22(Q)/bos1(R) mutants. The cells were pulse labeled at 37°C (see legend to Figure 3). In these mutant cells, a block in an early step in transport of CPY was observed, resembling the transport defect seen in the sec22(Q) mutant. After a 30-min chase most of the labeled CPY accumulated in the ER form (p1CPY), and only a minor fraction was detected in the vacuolar mature form (mCPY).