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: Next, we wanted to know the functional consequence of SPCA1-L2-C1 overexpression for TGN protein sorting in these cells. In recent years we have established robust assays to determine CFL-1– and SPCA1-dependent TGN sorting (von Blume et al., 2009, 2011, 2012). First, HeLa cells stably expressing signal sequence HRP (ss-HRP) were transduced with a control plasmid, SPCA1-L1-HA, or SPCA1-L2-C1-HA. To test the expression of the HA-tagged proteins, lysates were analyzed by Western blotting with an HA antibody. The cells expressed SPCA1-L1-HA and SPCA1-L2-C1-HA at similar levels (Fig. 5 A). The HRP assay was performed by incubating the cells with serum-free medium, and the secretion of HRP was measured by chemiluminescence as described previously (Bard et al., 2006).
Affiliation: Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.