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Cofilin recruits F-actin to SPCA1 and promotes Ca2+-mediated secretory cargo sorting.

Kienzle C, Basnet N, Crevenna AH, Beck G, Habermann B, Mizuno N, von Blume J - J. Cell Biol. (2014)

Bottom Line: How these proteins interact and activate the pump to facilitate cargo sorting, however, is not known.A 132-amino acid portion of the SPCA1 phosphorylation domain (P-domain) interacted with actin in a CFL-1-dependent manner.Altogether, our findings reveal the mechanism of CFL-1-dependent recruitment of actin to SPCA1 and the significance of this interaction for Ca(2+) influx and secretory cargo sorting.

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Affiliation: Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.

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Screen for CFL-1 binding deficient SPCA1 mutants. (A) Schematic representation of wt and SPCA1 mutants (mut1, mut2, mut3, mut4, and ΔL2-C1) used for screening. (B) HeLa cells stably expressing ss-HRP and siRNA-resistant SPCA1-wt or mutants were transfected with control or SPCA1 siRNA, and cell lysates were analyzed by Western blotting with HA, SPCA1, and β-actin antibodies. (C) Medium and cells were analyzed for HRP activity. Error bars indicate the mean SD of HRP activity in the medium normalized by HRP activity in cell lysates collected from three independent experiments. (D) Structural model of human SPCA1 (top). The CFL-1 binding domain (SPCA1-L2-C1) is displayed in purple. The four residues required for CFL1 binding are indicated as red spheres (bottom).
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fig9: Screen for CFL-1 binding deficient SPCA1 mutants. (A) Schematic representation of wt and SPCA1 mutants (mut1, mut2, mut3, mut4, and ΔL2-C1) used for screening. (B) HeLa cells stably expressing ss-HRP and siRNA-resistant SPCA1-wt or mutants were transfected with control or SPCA1 siRNA, and cell lysates were analyzed by Western blotting with HA, SPCA1, and β-actin antibodies. (C) Medium and cells were analyzed for HRP activity. Error bars indicate the mean SD of HRP activity in the medium normalized by HRP activity in cell lysates collected from three independent experiments. (D) Structural model of human SPCA1 (top). The CFL-1 binding domain (SPCA1-L2-C1) is displayed in purple. The four residues required for CFL1 binding are indicated as red spheres (bottom).

Mentions: To finally prove that the observed phenotype is directly caused by the loss of CFL-1 binding to SPCA1, we aimed at identifying the crucial amino acids in SPCA1 required for CFL-1 binding. It was reported that CFL-1 binds to and activates the related rat Na+/K+-ATPase (ATP2A2) in a domain that corresponds to L2-C1 in SPCA1 (Lee et al., 2001; Kim et al., 2002). Based on these studies, two residues are required for the functional interaction: D672, which according to the alignment of SPCA1 with ATP2A2 corresponds to S608 in human SPCA1; and R700, which is equivalent to K634 (Fig. S3). However, neither a single nor a double substitution to alanine (S608A/K634A) impaired TGN Ca2+ uptake and secretory cargo sorting (unpublished data). We further reasoned that, potentially, a charged patch rather than a single amino acid could be responsible for CFL-1 binding. We therefore generated a structural model of SPCA1 to analyze the surface accessibility of charged residues in the CFL-1 binding domain (L2-C1). Based on the model structure, we decided to add Q605A to the S608A/K634A mutations (mut1) because this residue is strongly charged and lies in close proximity to S608. In addition, three additional patches were selected for mutagenesis (Fig. 9 A; mut2, R623A/K627A/K630A/K634A; mut3, K589A/K613A; mut4, Q605A/Q606A/Q609A/K634A). Moreover, we generated a L2-C1 deletion mutant (Fig. 9 A). First, stable HeLa cell lines expressing siRNA-resistant SPCA1-wt, -ΔL2-C1, or mutants (-mut1, -mut2, -mut3, -mut4) were generated, and the localization of SPCA1-HA was observed by immunofluorescence microscopy. All the point mutants localized correctly to the TGN, whereas the deletion mutant (ΔL2-C1) accumulated in the ER (Fig. S4). This mutant was therefore not further analyzed.


Cofilin recruits F-actin to SPCA1 and promotes Ca2+-mediated secretory cargo sorting.

Kienzle C, Basnet N, Crevenna AH, Beck G, Habermann B, Mizuno N, von Blume J - J. Cell Biol. (2014)

Screen for CFL-1 binding deficient SPCA1 mutants. (A) Schematic representation of wt and SPCA1 mutants (mut1, mut2, mut3, mut4, and ΔL2-C1) used for screening. (B) HeLa cells stably expressing ss-HRP and siRNA-resistant SPCA1-wt or mutants were transfected with control or SPCA1 siRNA, and cell lysates were analyzed by Western blotting with HA, SPCA1, and β-actin antibodies. (C) Medium and cells were analyzed for HRP activity. Error bars indicate the mean SD of HRP activity in the medium normalized by HRP activity in cell lysates collected from three independent experiments. (D) Structural model of human SPCA1 (top). The CFL-1 binding domain (SPCA1-L2-C1) is displayed in purple. The four residues required for CFL1 binding are indicated as red spheres (bottom).
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Related In: Results  -  Collection

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fig9: Screen for CFL-1 binding deficient SPCA1 mutants. (A) Schematic representation of wt and SPCA1 mutants (mut1, mut2, mut3, mut4, and ΔL2-C1) used for screening. (B) HeLa cells stably expressing ss-HRP and siRNA-resistant SPCA1-wt or mutants were transfected with control or SPCA1 siRNA, and cell lysates were analyzed by Western blotting with HA, SPCA1, and β-actin antibodies. (C) Medium and cells were analyzed for HRP activity. Error bars indicate the mean SD of HRP activity in the medium normalized by HRP activity in cell lysates collected from three independent experiments. (D) Structural model of human SPCA1 (top). The CFL-1 binding domain (SPCA1-L2-C1) is displayed in purple. The four residues required for CFL1 binding are indicated as red spheres (bottom).
Mentions: To finally prove that the observed phenotype is directly caused by the loss of CFL-1 binding to SPCA1, we aimed at identifying the crucial amino acids in SPCA1 required for CFL-1 binding. It was reported that CFL-1 binds to and activates the related rat Na+/K+-ATPase (ATP2A2) in a domain that corresponds to L2-C1 in SPCA1 (Lee et al., 2001; Kim et al., 2002). Based on these studies, two residues are required for the functional interaction: D672, which according to the alignment of SPCA1 with ATP2A2 corresponds to S608 in human SPCA1; and R700, which is equivalent to K634 (Fig. S3). However, neither a single nor a double substitution to alanine (S608A/K634A) impaired TGN Ca2+ uptake and secretory cargo sorting (unpublished data). We further reasoned that, potentially, a charged patch rather than a single amino acid could be responsible for CFL-1 binding. We therefore generated a structural model of SPCA1 to analyze the surface accessibility of charged residues in the CFL-1 binding domain (L2-C1). Based on the model structure, we decided to add Q605A to the S608A/K634A mutations (mut1) because this residue is strongly charged and lies in close proximity to S608. In addition, three additional patches were selected for mutagenesis (Fig. 9 A; mut2, R623A/K627A/K630A/K634A; mut3, K589A/K613A; mut4, Q605A/Q606A/Q609A/K634A). Moreover, we generated a L2-C1 deletion mutant (Fig. 9 A). First, stable HeLa cell lines expressing siRNA-resistant SPCA1-wt, -ΔL2-C1, or mutants (-mut1, -mut2, -mut3, -mut4) were generated, and the localization of SPCA1-HA was observed by immunofluorescence microscopy. All the point mutants localized correctly to the TGN, whereas the deletion mutant (ΔL2-C1) accumulated in the ER (Fig. S4). This mutant was therefore not further analyzed.

Bottom Line: How these proteins interact and activate the pump to facilitate cargo sorting, however, is not known.A 132-amino acid portion of the SPCA1 phosphorylation domain (P-domain) interacted with actin in a CFL-1-dependent manner.Altogether, our findings reveal the mechanism of CFL-1-dependent recruitment of actin to SPCA1 and the significance of this interaction for Ca(2+) influx and secretory cargo sorting.

View Article: PubMed Central - HTML - PubMed

Affiliation: Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.

Show MeSH