FIG 4.

Monomeric GFP, but not Cth4p-GFP, is released upon mucocyst exocytosis. Fifty-milliliter cultures of wild-type or cth4p-GFP expressing cells (from the endogenous locus) were first grown overnight to ∼3 × 10⁵/ml at 30°C in SPP and then incubated for an additional 24 h at 22°C with gentle shaking. Two 15-ml samples were removed from each culture and pelleted and resuspended in 1.5 ml of medium, and one half were treated with dibucaine for 20 s. The cells were then diluted to 15 ml and centrifuged to generate cell pellet, flocculent, and supernatant fractions as in Fig. 5. After aspiration of the superatants, tubes were sliced with a razor below the flocculent-cell interface, and the flocculent was discarded. Cells were resuspended in 10 mM Tris (pH 7.4), repelleted, and then lysed with 10%TCA. Lysates (5 × 10⁴ cell equivalents) were resolved by 4 to 20% SDS-PAGE, transferred to PVDF, and Western blotted with anti-GFP (A) or anti-Grl1p (B) antibody. (Left blot) No release of cth4p-GFP from cells is seen, since the band intensities are equivalent in untreated and dibucaine-treated cells. In contast, a substantial fraction of free GFP is released by dibucaine stimulation. (Right blot) Grl1p is released from wild-type or cth4p-GFP cells during dibucaine stimulation.