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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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Inp53 is a calcineurin substrate. (A) Domain structure of yeast synaptojanins and creation of Inp53ARAQAA allele. IP5P, inositol-polyphosphate 5-phosphatase domain; PRD, proline-rich domain; SAC, SAC1-like domain. Yellow bar, PxIxIT-CN–docking site; below, alignment with consensus. Gray bars, Inp53 peptides identified by mass spectrometry (amino acids [aa] 358–373, 523–537, 818–830, 984–1001). (B) Copurification of Cna2 with Inp53. JRY11 cells expressing GST-Inp53, GST- Inp53ARAQAA, or GST alone were treated with 1.25 M KCl for 10 min where indicated and GST proteins purified with glutathione–Sepharose. Copurification of Cna2 with indicated GST-fusions was assessed by Western blotting. (C) JRY11 cells expressing GST-Inp53 or GST-Inp53(PRD)aa.887-1107 were pretreated with 1 μg/ml FK506 or vehicle (90% EtOH/10% Tween-20) for 30 min, then treated with 1.25 M KCl for 10 min where indicated and purified as in B. Phosphorylated protein (top) and total GST-tagged protein (bottom) were assessed by Western blotting. Representative blots from more than three independent experiments are shown.
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Figure 4: Inp53 is a calcineurin substrate. (A) Domain structure of yeast synaptojanins and creation of Inp53ARAQAA allele. IP5P, inositol-polyphosphate 5-phosphatase domain; PRD, proline-rich domain; SAC, SAC1-like domain. Yellow bar, PxIxIT-CN–docking site; below, alignment with consensus. Gray bars, Inp53 peptides identified by mass spectrometry (amino acids [aa] 358–373, 523–537, 818–830, 984–1001). (B) Copurification of Cna2 with Inp53. JRY11 cells expressing GST-Inp53, GST- Inp53ARAQAA, or GST alone were treated with 1.25 M KCl for 10 min where indicated and GST proteins purified with glutathione–Sepharose. Copurification of Cna2 with indicated GST-fusions was assessed by Western blotting. (C) JRY11 cells expressing GST-Inp53 or GST-Inp53(PRD)aa.887-1107 were pretreated with 1 μg/ml FK506 or vehicle (90% EtOH/10% Tween-20) for 30 min, then treated with 1.25 M KCl for 10 min where indicated and purified as in B. Phosphorylated protein (top) and total GST-tagged protein (bottom) were assessed by Western blotting. Representative blots from more than three independent experiments are shown.

Mentions: Because the altered subcellular distribution of CN likely reflected a change in protein–protein interactions, we sought to identify proteins whose association with CN increased during hyperosmotic stress. Mass spectrometry was used to identify proteins that copurified with endogenously expressed, epitope-tagged CN (Cna2-TEV-ZZ) isolated from either unstressed cells or cells exposed to 1.25 M KCl, as compared with purifications from untagged cells, which controlled for nonspecific background. These analyses suggested a possible interaction between CN and Inp53/Sjl3, one of three yeast synaptojanins (Figure 4A; Srinivasan et al., 1997; Stolz et al., 1998), which was enhanced during hyperosmotic stress. In unstressed cells, Inp53 contributes to protein sorting at the TGN but localizes to actin patches and stimulates repolarization of the actin cytoskeleton during osmotic stress (Ooms et al., 2000). Thus we hypothesized that CN might promote actin polarization during hyperosmotic stress by regulating Inp53.


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

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

Inp53 is a calcineurin substrate. (A) Domain structure of yeast synaptojanins and creation of Inp53ARAQAA allele. IP5P, inositol-polyphosphate 5-phosphatase domain; PRD, proline-rich domain; SAC, SAC1-like domain. Yellow bar, PxIxIT-CN–docking site; below, alignment with consensus. Gray bars, Inp53 peptides identified by mass spectrometry (amino acids [aa] 358–373, 523–537, 818–830, 984–1001). (B) Copurification of Cna2 with Inp53. JRY11 cells expressing GST-Inp53, GST- Inp53ARAQAA, or GST alone were treated with 1.25 M KCl for 10 min where indicated and GST proteins purified with glutathione–Sepharose. Copurification of Cna2 with indicated GST-fusions was assessed by Western blotting. (C) JRY11 cells expressing GST-Inp53 or GST-Inp53(PRD)aa.887-1107 were pretreated with 1 μg/ml FK506 or vehicle (90% EtOH/10% Tween-20) for 30 min, then treated with 1.25 M KCl for 10 min where indicated and purified as in B. Phosphorylated protein (top) and total GST-tagged protein (bottom) were assessed by Western blotting. Representative blots from more than three independent experiments are shown.
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Figure 4: Inp53 is a calcineurin substrate. (A) Domain structure of yeast synaptojanins and creation of Inp53ARAQAA allele. IP5P, inositol-polyphosphate 5-phosphatase domain; PRD, proline-rich domain; SAC, SAC1-like domain. Yellow bar, PxIxIT-CN–docking site; below, alignment with consensus. Gray bars, Inp53 peptides identified by mass spectrometry (amino acids [aa] 358–373, 523–537, 818–830, 984–1001). (B) Copurification of Cna2 with Inp53. JRY11 cells expressing GST-Inp53, GST- Inp53ARAQAA, or GST alone were treated with 1.25 M KCl for 10 min where indicated and GST proteins purified with glutathione–Sepharose. Copurification of Cna2 with indicated GST-fusions was assessed by Western blotting. (C) JRY11 cells expressing GST-Inp53 or GST-Inp53(PRD)aa.887-1107 were pretreated with 1 μg/ml FK506 or vehicle (90% EtOH/10% Tween-20) for 30 min, then treated with 1.25 M KCl for 10 min where indicated and purified as in B. Phosphorylated protein (top) and total GST-tagged protein (bottom) were assessed by Western blotting. Representative blots from more than three independent experiments are shown.
Mentions: Because the altered subcellular distribution of CN likely reflected a change in protein–protein interactions, we sought to identify proteins whose association with CN increased during hyperosmotic stress. Mass spectrometry was used to identify proteins that copurified with endogenously expressed, epitope-tagged CN (Cna2-TEV-ZZ) isolated from either unstressed cells or cells exposed to 1.25 M KCl, as compared with purifications from untagged cells, which controlled for nonspecific background. These analyses suggested a possible interaction between CN and Inp53/Sjl3, one of three yeast synaptojanins (Figure 4A; Srinivasan et al., 1997; Stolz et al., 1998), which was enhanced during hyperosmotic stress. In unstressed cells, Inp53 contributes to protein sorting at the TGN but localizes to actin patches and stimulates repolarization of the actin cytoskeleton during osmotic stress (Ooms et al., 2000). Thus we hypothesized that CN might promote actin polarization during hyperosmotic stress by regulating Inp53.

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