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: The results of the sorting assays strongly suggested that the overexpression of SPCA1-L2-C1 interferes with SPCA1 function by capturing the necessary interaction partners required for activation and, thus, sorting. To test this hypothesis, we performed Ca2+ measurements with a TGN-targeted Ca2+ FRET sensor (Lissandron et al., 2010). TGN Ca2+ uptake solely relies on SPCA1, as demonstrated before (Lissandron et al., 2010). HeLa cells were transfected with a control plasmid, SPCA1 siRNA, SPCA1-L1-HA, or SPCA1-L2-C1-HA in combination with the Go-D1-cpv sensor to measure the TGN Ca2+ uptake of the TGN, as described previously (von Blume et al., 2011). The coexpression of Go-D1-cpv and SPCA1-L1-HA or SPCA1-L2-C1-HA was confirmed by immunofluorescence microscopy (Fig. 6 A).
Affiliation: Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.