GRASP is required for Acb1 secretion. (A) Wild-type and grh1Δ yeast were incubated in starvation buffer for 0, 1, 4, and 16 h, and the supernatant was tested in the bioassay. (B) Wild-type and bug1Δ yeast were starved for 4 h and the supernatant analyzed in the bioassay. (C) A wild-type strain expressing a GFP-tagged version of Grh1 was starved for 4 h and visualized by fluorescence microscopy. Starvation does not change the punctate pattern of Grh1 localization. Bar, 5 µm. (D) Cells were treated as in A, and the cell lysates were analyzed to monitor the respective protein levels. Deletion of Grh1 does not affect the intracellular levels of 3-phosphoglycerate kinase or Acb1. The data show an average of three experiments.