Grl protein sorting to mucocysts, though not subsequent proteolytic processing, is independent of SOR4. (A and B) Grl3p localizes to mature mucocysts in the absence of Sor4p. Localization of Grl3p, one of a family of proteins that assembles to form the mucocyst core, in wild-type, Δsor2, and Δsor4 cells. Grl3p was visualized by indirect immunofluorescence (IF) using mAb 5E9. At the cell surface (A; illustrated by the red plane in the diagram shown in the top right and in the differential interference contrast micrographs to the left), Grl3p can be seen localized to mucocysts in both wild-type (top) and Δsor4 (bottom) cells. When seen in cross section (B), the mucocysts of Δsor4 cells appear roughly spherical, in contrast to the elongated wild-type mucocysts (compare the bottom insets of the IF panel). Δsor2 mucocysts show an intermediate morphology. The cross-section image of the wild-type control is shown again in Fig. S4 C. Insets are enlarged from the indicated regions. Bars: (main panels) 5 µm; (inset) 0.5 µm. (C) Ultrastructure of Δsor4 mucocysts. Electron micrographs of docked mucocysts (labeled with asterisks) in wild-type (top) and Δsor2 and Δsor4 cells (bottom). The mucocyst cores in Δsor2 and Δsor4 cells do not contain the visible lattice that is characteristic of wild-type mucocyst cores. The same wild-type control is shown again in Fig. S4 D. Bars, 200 nM. (D) Grl proprotein processing is defective in Δsor4 cells. Whole cell lysates of wild-type and Δsor4 cells were separated by SDS-PAGE and Western blotted with an antibody against Grl1p, which undergoes proteolytic processing during mucocyst maturation. In wild-type cells, Grl1p accumulates primarily in its fully processed form. In Δsor4 cells, most Grl1p remains as an incompletely processed precursor. The unprocessed and processed forms of Grl1p are indicated by arrows.