Figure 3.
Figure 3. Sortilin 4 is required for the sorting of Grt1p, a member of the Granule tip family of mucocyst cargo proteins. (A) Grt1p localizes to a subdomain of mucocysts, whereas Grl3p is found through the mucocyst core. Grl3p and Grt1p in wild-type cells were simultaneously visualized using mAbs (5E9 and 4D11, respectively) directly conjugated to two different fluorophores. Visualization along the long mucocyst axis (demonstrated best in a cross section of the cell, illustrated by the red plane in the diagram shown at the top for reference) demonstrates that Grt1p is concentrated at the pole where docking occurs (right). Bar, 1 µm. (B) Grt1p is mistargeted in Δsor4 cells. Immunolocalization of Grt1p in wild-type cells (top left) shows that Grt1p accumulates in the expected array of docked mucocysts at the surface (illustrated by the red plane in the diagram at the top), and the same pattern is seen in Δsor2 cells (top right). Grt1p was visualized using mAb 4D11. In contrast, there is only background staining of Grt1p in Δsor4 cells, comparable to the signal in Δgrt1/Δgrt2 cells that lack the mAb target entirely (bottom). Note that docked mucocysts are still present in the Δsor4 cells (Fig. 5, A and B). Images of Δsor4 and Δgrt1/Δgrt2 cells were auto-adjusted to show the cell outlines. Bars, 5 µm. (C) Grt1p is absent from Δsor4 cells. Western blotting of whole cell lysates, using polyclonal anti-Grt1p antiserum, confirms the defect in Grt1p accumulation in Δsor4 relative to wild type. An uncropped version of this Western blot is shown in Fig. S3 A. (D) Biochemical interaction between Sor4p and Grt1p: coprecipitation of Sor4p and Grt1p. Sor4p was immunoprecipitated using anti-GFP antiserum from lysates of cells that express Sor4p-GFP from the endogenous SOR4 locus and that were actively synthesizing new mucocysts. Immunoprecipitated samples were analyzed by Western blotting with anti-GFP antiserum (left), confirming that full-length Sor4p-GFP is expressed, and with anti-Grt1p antiserum (right) to show coprecipitation of Grt1p. (E) Sor4p-GFP localizes to mobile cytoplasmic vesicles but not to docked mucocysts. A frame from Video 1 is shown, in which Sor4p-GFP was tracked in immobilized live cells. Sor4p-GFP is present in mobile cytoplasmic puncta and not present in docked mucocysts. The gray line traces the approximate outline of the cell. Bar, 5 µm.

Sortilin 4 is required for the sorting of Grt1p, a member of the Granule tip family of mucocyst cargo proteins. (A) Grt1p localizes to a subdomain of mucocysts, whereas Grl3p is found through the mucocyst core. Grl3p and Grt1p in wild-type cells were simultaneously visualized using mAbs (5E9 and 4D11, respectively) directly conjugated to two different fluorophores. Visualization along the long mucocyst axis (demonstrated best in a cross section of the cell, illustrated by the red plane in the diagram shown at the top for reference) demonstrates that Grt1p is concentrated at the pole where docking occurs (right). Bar, 1 µm. (B) Grt1p is mistargeted in Δsor4 cells. Immunolocalization of Grt1p in wild-type cells (top left) shows that Grt1p accumulates in the expected array of docked mucocysts at the surface (illustrated by the red plane in the diagram at the top), and the same pattern is seen in Δsor2 cells (top right). Grt1p was visualized using mAb 4D11. In contrast, there is only background staining of Grt1p in Δsor4 cells, comparable to the signal in Δgrt1/Δgrt2 cells that lack the mAb target entirely (bottom). Note that docked mucocysts are still present in the Δsor4 cells (Fig. 5, A and B). Images of Δsor4 and Δgrt1/Δgrt2 cells were auto-adjusted to show the cell outlines. Bars, 5 µm. (C) Grt1p is absent from Δsor4 cells. Western blotting of whole cell lysates, using polyclonal anti-Grt1p antiserum, confirms the defect in Grt1p accumulation in Δsor4 relative to wild type. An uncropped version of this Western blot is shown in Fig. S3 A. (D) Biochemical interaction between Sor4p and Grt1p: coprecipitation of Sor4p and Grt1p. Sor4p was immunoprecipitated using anti-GFP antiserum from lysates of cells that express Sor4p-GFP from the endogenous SOR4 locus and that were actively synthesizing new mucocysts. Immunoprecipitated samples were analyzed by Western blotting with anti-GFP antiserum (left), confirming that full-length Sor4p-GFP is expressed, and with anti-Grt1p antiserum (right) to show coprecipitation of Grt1p. (E) Sor4p-GFP localizes to mobile cytoplasmic vesicles but not to docked mucocysts. A frame from Video 1 is shown, in which Sor4p-GFP was tracked in immobilized live cells. Sor4p-GFP is present in mobile cytoplasmic puncta and not present in docked mucocysts. The gray line traces the approximate outline of the cell. Bar, 5 µm.

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