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 next question that we aimed to address was whether the expression of the actin and SPCA1 binding–deficient mutant has an effect on TGN Ca2+ uptake. We have shown previously that the knockdown of ADF/CFL-1 significantly impairs TGN Ca2+ uptake. This phenotype could be rescued by the expression of siRNA-resistant rCFL-1 wt (von Blume et al., 2011). To test the effect of CFL-S3E on TGN Ca2+ uptake, HeLa cells were treated with ADF/CFL-1 siRNAs and transfected with either Go-D1-cpv and a control plasmid or HA-rCFL-1-S3E, and processed for immunofluorescence microscopy (Fig. 8 A). The TGN Ca2+ uptake was significantly reduced in ADF/CFL-1–depleted cells expressing HA-rCFL-1-S3E compared with control cells (Fig. 8 B). These data further demonstrate that the binding between SPCA1, actin, and CFL-1 is crucial for Ca2+ pumping at the TGN.
Affiliation: Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.