Cofilin recruits F-actin to SPCA1 and promotes Ca2+-mediated secretory cargo sorting.
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.
Affiliation: Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.Show MeSH
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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a single nor a double substitution to alanine (S608A/K634A) impaired TGN Ca2+ uptake and secretory cargo sorting (unpublished data).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.
Affiliation: Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.