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 elucidate how F-actin binds to the SPCA1–CFL-1 complex, we established a reconstitution assay to visualize the process. To set up the assay, we attached His-Sumo, His-Sumo-CFL-1-S3E, and His-Sumo-CFL-1 to Ni-NTA beads. As a negative control, we incubated Ni-NTA beads with BSA. To visualize the proteins, beads were incubated with an anti-His antibody and an Alexa Fluor 488–labeled secondary antibody (Fig. 2 A), and analyzed by fluorescence microscopy. The His-tagged proteins were visible on the beads in equal amounts, whereas the BSA-incubated Ni-NTA beads did not show a fluorescent signal (Fig. 2 A). To test if actin can be recruited to CFL-1 under these conditions, we incubated the beads with fluorescently labeled F-actin and monitored them by fluorescence microscopy. F-actin strongly associated to His-Sumo-CFL-1 beads but not to beads alone, His-Sumo, or His-Sumo-CFL-1-S3E (Fig. 2 B).
Affiliation: Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.