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Calcineurin regulates the yeast synaptojanin Inp53/Sjl3 during membrane stress.

Guiney EL, Goldman AR, Elias JE, Cyert MS - Mol. Biol. Cell (2014)

Bottom Line: By activating Inp53, calcineurin repolarizes the actin cytoskeleton and maintains normal plasma membrane morphology in synaptojanin-limited cells.This response has physiological and molecular similarities to calcineurin-regulated activity-dependent bulk endocytosis in neurons, which retrieves a bolus of plasma membrane deposited by synaptic vesicle fusion.We propose that activation of Ca(2+)/calcineurin and PI(4,5)P2 signaling to regulate endocytosis is a fundamental and conserved response to excess membrane in eukaryotic cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Stanford University, Stanford, CA 94305.

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Molecular mechanism of Inp53 regulation by calcineurin. Wild-type cells (A) or cells expressing the indicated GFP fusion from endogenous loci (B–E) and expressing GST-Inp53, GST-Inp53 ARAQAA, or GST were treated with or without 1.25 M KCl for 10 min, and GST- tagged proteins were purified with glutathione-Sepharose. (C) Soluble (Input) and insoluble fractions (20,000 × g pellet). Copurification of the GFP fusion proteins with indicated GST-fusions was assessed by Western blotting; representative blots from more than three independent experiments are shown.
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Figure 9: Molecular mechanism of Inp53 regulation by calcineurin. Wild-type cells (A) or cells expressing the indicated GFP fusion from endogenous loci (B–E) and expressing GST-Inp53, GST-Inp53 ARAQAA, or GST were treated with or without 1.25 M KCl for 10 min, and GST- tagged proteins were purified with glutathione-Sepharose. (C) Soluble (Input) and insoluble fractions (20,000 × g pellet). Copurification of the GFP fusion proteins with indicated GST-fusions was assessed by Western blotting; representative blots from more than three independent experiments are shown.

Mentions: Therefore we tested whether any of the established or predicted protein–protein interactions for Inp53 were altered by hyperosmotic shock or its regulation by CN. In unstressed cells, Inp53 functions primarily to regulate phosphoinositide-dependent sorting of proteins at the TGN and interacts directly with clathrin via an LLDID motif in the Inp53-PRD (Ha et al., 2001, 2003; Daboussi et al., 2012). Equivalent amounts of clathrin heavy chain (Chc1) copurified with Inp53 and Inp53ARAQAA, but copurification of Chc1 with both proteins decreased after hyperosmotic shock (Figure 9A). Hyperphosphorylation of the Inp53-PRD may be responsible for reduced clathrin binding; we identified numerous phosphorylation sites in the Inp53-PRD that increased during osmotic stress, including S914, which immediately precedes LLDID915-919 (unpublished data). Loss of interaction with clathrin may indicate a repurposing of Inp53 from its normal role at the TGN to a function at actin patches and is consistent with reports of Inp53 translocation to actin patches during hyperosmotic shock (Ooms et al., 2000).


Calcineurin regulates the yeast synaptojanin Inp53/Sjl3 during membrane stress.

Guiney EL, Goldman AR, Elias JE, Cyert MS - Mol. Biol. Cell (2014)

Molecular mechanism of Inp53 regulation by calcineurin. Wild-type cells (A) or cells expressing the indicated GFP fusion from endogenous loci (B–E) and expressing GST-Inp53, GST-Inp53 ARAQAA, or GST were treated with or without 1.25 M KCl for 10 min, and GST- tagged proteins were purified with glutathione-Sepharose. (C) Soluble (Input) and insoluble fractions (20,000 × g pellet). Copurification of the GFP fusion proteins with indicated GST-fusions was assessed by Western blotting; representative blots from more than three independent experiments are shown.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4325846&req=5

Figure 9: Molecular mechanism of Inp53 regulation by calcineurin. Wild-type cells (A) or cells expressing the indicated GFP fusion from endogenous loci (B–E) and expressing GST-Inp53, GST-Inp53 ARAQAA, or GST were treated with or without 1.25 M KCl for 10 min, and GST- tagged proteins were purified with glutathione-Sepharose. (C) Soluble (Input) and insoluble fractions (20,000 × g pellet). Copurification of the GFP fusion proteins with indicated GST-fusions was assessed by Western blotting; representative blots from more than three independent experiments are shown.
Mentions: Therefore we tested whether any of the established or predicted protein–protein interactions for Inp53 were altered by hyperosmotic shock or its regulation by CN. In unstressed cells, Inp53 functions primarily to regulate phosphoinositide-dependent sorting of proteins at the TGN and interacts directly with clathrin via an LLDID motif in the Inp53-PRD (Ha et al., 2001, 2003; Daboussi et al., 2012). Equivalent amounts of clathrin heavy chain (Chc1) copurified with Inp53 and Inp53ARAQAA, but copurification of Chc1 with both proteins decreased after hyperosmotic shock (Figure 9A). Hyperphosphorylation of the Inp53-PRD may be responsible for reduced clathrin binding; we identified numerous phosphorylation sites in the Inp53-PRD that increased during osmotic stress, including S914, which immediately precedes LLDID915-919 (unpublished data). Loss of interaction with clathrin may indicate a repurposing of Inp53 from its normal role at the TGN to a function at actin patches and is consistent with reports of Inp53 translocation to actin patches during hyperosmotic shock (Ooms et al., 2000).

Bottom Line: By activating Inp53, calcineurin repolarizes the actin cytoskeleton and maintains normal plasma membrane morphology in synaptojanin-limited cells.This response has physiological and molecular similarities to calcineurin-regulated activity-dependent bulk endocytosis in neurons, which retrieves a bolus of plasma membrane deposited by synaptic vesicle fusion.We propose that activation of Ca(2+)/calcineurin and PI(4,5)P2 signaling to regulate endocytosis is a fundamental and conserved response to excess membrane in eukaryotic cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, Stanford University, Stanford, CA 94305.

Show MeSH
Related in: MedlinePlus