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Lysosomal sorting receptors are essential for secretory granule biogenesis in Tetrahymena.

Briguglio JS, Kumar S, Turkewitz AP - J. Cell Biol. (2013)

Bottom Line: We show that assembly of mucocysts depends on proteins classically associated with lysosome biogenesis.In addition, sortilins are required for delivery of a key protease involved in T. thermophila mucocyst maturation.Our results suggest potential similarities in the formation of regulated secretory organelles between even very distantly related eukaryotes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637.

ABSTRACT
Secretory granules, such as neuronal dense core vesicles, are specialized for storing cargo at high concentration and releasing it via regulated exocytosis in response to extracellular stimuli. Here, we used expression profiling to identify new components of the machinery for sorting proteins into mucocysts, secretory granule-like vesicles in the ciliate Tetrahymena thermophila. We show that assembly of mucocysts depends on proteins classically associated with lysosome biogenesis. In particular, the delivery of nonaggregated, but not aggregated, cargo proteins requires classical receptors of the sortilin/VPS10 family, which indicates that dual mechanisms are involved in sorting to this secretory compartment. In addition, sortilins are required for delivery of a key protease involved in T. thermophila mucocyst maturation. Our results suggest potential similarities in the formation of regulated secretory organelles between even very distantly related eukaryotes.

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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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fig3: 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.

Mentions: To ask whether the SOR4 gene product, Sor4p, played a role in sorting of mucocyst cargo proteins, we immunolocalized members of the Grt and Grl families (Grt1p and Grl3p, respectively) using previously characterized monoclonal antibodies. In wild-type cells, Grt1p localizes in a polarized fashion to the docked end of mucocysts (Bowman et al., 2005a; Fig. 3 A). Remarkably, we found that Δsor4 cells were completely defective in accumulation of Grt1p in mucocysts, as judged by indirect immunofluorescence (Fig. 3 B), and confirmed by Western blotting of whole cell lysates using a polyclonal antibody (Fig. 3 C and Fig. S3 A). The Δsor4 cells had no defect in Grt1p synthesis per se, as Grt1p was readily detected in cell culture medium. Because sortilins function as ligand-binding receptors, these results suggested that Sor4p acts as a sorting receptor for Grt1p. Consistent with this idea, we could immunoprecipitate Grt1p using an antibody against GFP in cells that were expressing Sor4p-GFP at the endogenous SOR4 locus (Fig. 3 D). Importantly, the GFP fusion protein is functional, as cells expressing Sor4p-GFP in lieu of the wild-type protein were fully exocytosis competent; i.e., did not manifest any SOR4 deficiency (Fig. S3 B). The robust interaction between Sor4p and Grt1p indicated by coprecipitation suggests that the interaction between these proteins is likely to be direct. Moreover, the SOR4-dependent missorting of Grt1p was specific, as disruption of the related T. thermophila paralogue, SOR2, did not produce any apparent defect in the accumulation of Grt1p (Fig. 3 B).


Lysosomal sorting receptors are essential for secretory granule biogenesis in Tetrahymena.

Briguglio JS, Kumar S, Turkewitz AP - J. Cell Biol. (2013)

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.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
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fig3: 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.
Mentions: To ask whether the SOR4 gene product, Sor4p, played a role in sorting of mucocyst cargo proteins, we immunolocalized members of the Grt and Grl families (Grt1p and Grl3p, respectively) using previously characterized monoclonal antibodies. In wild-type cells, Grt1p localizes in a polarized fashion to the docked end of mucocysts (Bowman et al., 2005a; Fig. 3 A). Remarkably, we found that Δsor4 cells were completely defective in accumulation of Grt1p in mucocysts, as judged by indirect immunofluorescence (Fig. 3 B), and confirmed by Western blotting of whole cell lysates using a polyclonal antibody (Fig. 3 C and Fig. S3 A). The Δsor4 cells had no defect in Grt1p synthesis per se, as Grt1p was readily detected in cell culture medium. Because sortilins function as ligand-binding receptors, these results suggested that Sor4p acts as a sorting receptor for Grt1p. Consistent with this idea, we could immunoprecipitate Grt1p using an antibody against GFP in cells that were expressing Sor4p-GFP at the endogenous SOR4 locus (Fig. 3 D). Importantly, the GFP fusion protein is functional, as cells expressing Sor4p-GFP in lieu of the wild-type protein were fully exocytosis competent; i.e., did not manifest any SOR4 deficiency (Fig. S3 B). The robust interaction between Sor4p and Grt1p indicated by coprecipitation suggests that the interaction between these proteins is likely to be direct. Moreover, the SOR4-dependent missorting of Grt1p was specific, as disruption of the related T. thermophila paralogue, SOR2, did not produce any apparent defect in the accumulation of Grt1p (Fig. 3 B).

Bottom Line: We show that assembly of mucocysts depends on proteins classically associated with lysosome biogenesis.In addition, sortilins are required for delivery of a key protease involved in T. thermophila mucocyst maturation.Our results suggest potential similarities in the formation of regulated secretory organelles between even very distantly related eukaryotes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637.

ABSTRACT
Secretory granules, such as neuronal dense core vesicles, are specialized for storing cargo at high concentration and releasing it via regulated exocytosis in response to extracellular stimuli. Here, we used expression profiling to identify new components of the machinery for sorting proteins into mucocysts, secretory granule-like vesicles in the ciliate Tetrahymena thermophila. We show that assembly of mucocysts depends on proteins classically associated with lysosome biogenesis. In particular, the delivery of nonaggregated, but not aggregated, cargo proteins requires classical receptors of the sortilin/VPS10 family, which indicates that dual mechanisms are involved in sorting to this secretory compartment. In addition, sortilins are required for delivery of a key protease involved in T. thermophila mucocyst maturation. Our results suggest potential similarities in the formation of regulated secretory organelles between even very distantly related eukaryotes.

Show MeSH
Related in: MedlinePlus