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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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Cth3p, an aspartyl protease, is targeted to mucocysts in a Sor4p-dependent manner. Cth3p-CFP was inducibly expressed with 0.75 µg/ml CdCl2 for 2 h in wild-type and Δsor4 cells. Cth3p-CFP was localized in fixed, permeabilized cells using a polyclonal anti-GFP antibody, and endogenous Grl3p was immunolocalized using mAb 5E9. (A) Cth3p-CFP expressed in wild-type cells colocalizes with Grl3p in mucocysts at the cell periphery (top). In contrast, Cth3p-CFP expressed in Δsor4 cells shows reduced colocalization with Grl3p (middle). Bars, 5 µm. (B) Cth3p-CFP shows reduced colocalization with Grl3p in Δsor4 versus wild-type cells. Colocalization was quantified in 15 wild-type and Δsor4 cells, using the Manders’ correlation coefficient M2, and then a mean M2 value for each population was determined from the sample. Reduced colocalization was observed whether measuring all puncta (M2 values for wild type: mean = 0.615, SEM = 0.036; for Δsor4: M = 0.337, SEM = 0.049; P < 0.01 as determined by one-tailed t test) or the subset near the cell periphery, which is enriched in docked mucocysts (wild type: M = 0.731, SEM = 0.036; Δsor4: M = 0.373, SEM = 0.054; P < 0.01; right graph). Details of the image analysis are provided in Materials and methods, and a range of representative images is shown in Fig. S5.
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fig6: Cth3p, an aspartyl protease, is targeted to mucocysts in a Sor4p-dependent manner. Cth3p-CFP was inducibly expressed with 0.75 µg/ml CdCl2 for 2 h in wild-type and Δsor4 cells. Cth3p-CFP was localized in fixed, permeabilized cells using a polyclonal anti-GFP antibody, and endogenous Grl3p was immunolocalized using mAb 5E9. (A) Cth3p-CFP expressed in wild-type cells colocalizes with Grl3p in mucocysts at the cell periphery (top). In contrast, Cth3p-CFP expressed in Δsor4 cells shows reduced colocalization with Grl3p (middle). Bars, 5 µm. (B) Cth3p-CFP shows reduced colocalization with Grl3p in Δsor4 versus wild-type cells. Colocalization was quantified in 15 wild-type and Δsor4 cells, using the Manders’ correlation coefficient M2, and then a mean M2 value for each population was determined from the sample. Reduced colocalization was observed whether measuring all puncta (M2 values for wild type: mean = 0.615, SEM = 0.036; for Δsor4: M = 0.337, SEM = 0.049; P < 0.01 as determined by one-tailed t test) or the subset near the cell periphery, which is enriched in docked mucocysts (wild type: M = 0.731, SEM = 0.036; Δsor4: M = 0.373, SEM = 0.054; P < 0.01; right graph). Details of the image analysis are provided in Materials and methods, and a range of representative images is shown in Fig. S5.

Mentions: The simplest hypothesis was that Sor4p was required to deliver the proteases that process pro-Grl proteins. Those proteases have been studied indirectly but had not yet been identified in any ciliate. However, strong candidates for these enzymes emerged from the same expression screening approach, described earlier, which led us to focus on sortilins. Four of the putative processing enzymes are cathepsins, named CTH1–4, and disruption of the CTH3 gene in particular resulted in a near-complete failure to process Grl proproteins or synthesize mucocysts (unpublished data). To validate the inference that Cth3p functioned directly in mucocyst maturation, we transiently expressed the protein as a CFP fusion. Consistent with a role in pro-Grl processing, Cth3p-CFP showed significant localization to mucocysts (Fig. 6). This is likely due to specific targeting signals, as neither an unrelated cathepsin that has been studied in T. thermophila nor GFP attached to a signal sequence accumulate in mucocysts (Haddad et al., 2002; Jacobs et al., 2006). Importantly, the targeting of Cth3p-CFP to mucocysts, as measured by the colocalization of Cth3p-CFP with Grl3p, was reduced in Δsor4 cells (Fig. 6 and Fig. S5). These results support the hypothesis that Δsor4 cells are deficient in mucocyst delivery of both Grt family proteins and also one or more processing enzymes needed for Grl proprotein processing, with the latter defect sufficient to explain the aberrant mucocyst morphology shown in Fig. 5 C. Sor2p may also be involved in delivery to mucocysts of proteins required for pro-Grl processing, as Δsor2 cells showed comparable, though less severe, defects in Grl-based core formation (Fig. 5, A–C).


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

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

Cth3p, an aspartyl protease, is targeted to mucocysts in a Sor4p-dependent manner. Cth3p-CFP was inducibly expressed with 0.75 µg/ml CdCl2 for 2 h in wild-type and Δsor4 cells. Cth3p-CFP was localized in fixed, permeabilized cells using a polyclonal anti-GFP antibody, and endogenous Grl3p was immunolocalized using mAb 5E9. (A) Cth3p-CFP expressed in wild-type cells colocalizes with Grl3p in mucocysts at the cell periphery (top). In contrast, Cth3p-CFP expressed in Δsor4 cells shows reduced colocalization with Grl3p (middle). Bars, 5 µm. (B) Cth3p-CFP shows reduced colocalization with Grl3p in Δsor4 versus wild-type cells. Colocalization was quantified in 15 wild-type and Δsor4 cells, using the Manders’ correlation coefficient M2, and then a mean M2 value for each population was determined from the sample. Reduced colocalization was observed whether measuring all puncta (M2 values for wild type: mean = 0.615, SEM = 0.036; for Δsor4: M = 0.337, SEM = 0.049; P < 0.01 as determined by one-tailed t test) or the subset near the cell periphery, which is enriched in docked mucocysts (wild type: M = 0.731, SEM = 0.036; Δsor4: M = 0.373, SEM = 0.054; P < 0.01; right graph). Details of the image analysis are provided in Materials and methods, and a range of representative images is shown in Fig. S5.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3824020&req=5

fig6: Cth3p, an aspartyl protease, is targeted to mucocysts in a Sor4p-dependent manner. Cth3p-CFP was inducibly expressed with 0.75 µg/ml CdCl2 for 2 h in wild-type and Δsor4 cells. Cth3p-CFP was localized in fixed, permeabilized cells using a polyclonal anti-GFP antibody, and endogenous Grl3p was immunolocalized using mAb 5E9. (A) Cth3p-CFP expressed in wild-type cells colocalizes with Grl3p in mucocysts at the cell periphery (top). In contrast, Cth3p-CFP expressed in Δsor4 cells shows reduced colocalization with Grl3p (middle). Bars, 5 µm. (B) Cth3p-CFP shows reduced colocalization with Grl3p in Δsor4 versus wild-type cells. Colocalization was quantified in 15 wild-type and Δsor4 cells, using the Manders’ correlation coefficient M2, and then a mean M2 value for each population was determined from the sample. Reduced colocalization was observed whether measuring all puncta (M2 values for wild type: mean = 0.615, SEM = 0.036; for Δsor4: M = 0.337, SEM = 0.049; P < 0.01 as determined by one-tailed t test) or the subset near the cell periphery, which is enriched in docked mucocysts (wild type: M = 0.731, SEM = 0.036; Δsor4: M = 0.373, SEM = 0.054; P < 0.01; right graph). Details of the image analysis are provided in Materials and methods, and a range of representative images is shown in Fig. S5.
Mentions: The simplest hypothesis was that Sor4p was required to deliver the proteases that process pro-Grl proteins. Those proteases have been studied indirectly but had not yet been identified in any ciliate. However, strong candidates for these enzymes emerged from the same expression screening approach, described earlier, which led us to focus on sortilins. Four of the putative processing enzymes are cathepsins, named CTH1–4, and disruption of the CTH3 gene in particular resulted in a near-complete failure to process Grl proproteins or synthesize mucocysts (unpublished data). To validate the inference that Cth3p functioned directly in mucocyst maturation, we transiently expressed the protein as a CFP fusion. Consistent with a role in pro-Grl processing, Cth3p-CFP showed significant localization to mucocysts (Fig. 6). This is likely due to specific targeting signals, as neither an unrelated cathepsin that has been studied in T. thermophila nor GFP attached to a signal sequence accumulate in mucocysts (Haddad et al., 2002; Jacobs et al., 2006). Importantly, the targeting of Cth3p-CFP to mucocysts, as measured by the colocalization of Cth3p-CFP with Grl3p, was reduced in Δsor4 cells (Fig. 6 and Fig. S5). These results support the hypothesis that Δsor4 cells are deficient in mucocyst delivery of both Grt family proteins and also one or more processing enzymes needed for Grl proprotein processing, with the latter defect sufficient to explain the aberrant mucocyst morphology shown in Fig. 5 C. Sor2p may also be involved in delivery to mucocysts of proteins required for pro-Grl processing, as Δsor2 cells showed comparable, though less severe, defects in Grl-based core formation (Fig. 5, A–C).

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