Figure 5.

Secretion defect in snc1-M43A yeast. (A) Wild-type (SP1), SNC1 snc2Δ (NY2264), and snc1-M43A snc2Δ (NY2265) yeast were pulse labeled for 15 min at 37°C with [³⁵S]methionine. Cells were removed from the media by centrifugation and secreted proteins were concentrated by TCA precipitation. ³⁵S-labeled proteins in the media (top) and cell pellet (bottom) were separated on polyacrylamide gels. (B) ³⁵S incorporated in the 190-kDa band identified with an arrow in A was quantified using a phosphor imager. The amount of ³⁵S-p190 secreted was compared with the amount of total ³⁵S-labeled proteins in the lysate. (C) Invertase secretion. Wild-type (SP1), SNC1 snc2Δ (NY2264), snc1-M43A snc2Δ (NY2265), snc1 Δ SNC2 (NY2204), and snc1Δ snc2-M42A (NY2205) yeast were shifted to 0.1% glucose media for 20 min at 30°C to derepress invertase synthesis. External invertase was measured from intact cells and internal invertase was measured after preparing spheroplasts.