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Cofilin recruits F-actin to SPCA1 and promotes Ca2+-mediated secretory cargo sorting.

Kienzle C, Basnet N, Crevenna AH, Beck G, Habermann B, Mizuno N, von Blume J - J. Cell Biol. (2014)

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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.

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CFL-1 directly interacts with a small domain of SPCA1 in vitro. (A, top) Schematic representation of the His-Sumo-SPCA1 expression constructs: SPCA1-N-term (aa 1–70), SPCA1-L1 (aa 124–252), and SPCA1-L2 (aa 329–680) comprise the A-, N-, and P-domain of SPCA1, which are the major cytosolic domains responsible for Ca2+ pumping activity. (A, bottom) To perform His pull-downs, E. coli purified His-Sumo-SPCA1 fusion proteins were immobilized on Ni-NTA agarose beads and incubated with equal amounts of HeLa cell lysates. Protein–bead complexes were analyzed by Western blotting using antibodies recognizing His tag, CFL-1, and β-actin. (B, top) To restrict the binding site of actin and CFL-1 to SPCA1-L2 to a smaller region, full-length L2 (aa 329–680) was subdivided into two fragments. The first part is referred to as SPCA1-L2-N1 (aa 329–548), the second part as SPCA1-L2-C1 (aa 549–680). (B, bottom) His pull-downs were performed and analyzed with recombinant His-Sumo-SPCA1-L2 full length, -SPCA1-L2-N1, and -SPCA1-L2-C1 as described in A. (C) His-Sumo-CFL-1 was expressed in E. coli, purified, and the His-tag was subsequently cleaved. CFL-1 was incubated with a CFL-1 antibody and immobilized on agarose beads. Then, His-Sumo tag, His-Sumo-SPCA1-L1, or His-Sumo-SPCA1-L2-C1 were added. Proteins were eluted and separated by SDS-PAGE and stained with Coomassie blue. (D) CFL-1 and mutant CFL-1-S3E constructs were expressed, purified, and cut from His-Sumo tag. Recombinant proteins were incubated with purified His-Sumo-SPCA1-L2-C1 attached to Ni-NTA beads. Proteins were eluted and analyzed by SDS-PAGE and Coomassie blue staining.
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fig1: CFL-1 directly interacts with a small domain of SPCA1 in vitro. (A, top) Schematic representation of the His-Sumo-SPCA1 expression constructs: SPCA1-N-term (aa 1–70), SPCA1-L1 (aa 124–252), and SPCA1-L2 (aa 329–680) comprise the A-, N-, and P-domain of SPCA1, which are the major cytosolic domains responsible for Ca2+ pumping activity. (A, bottom) To perform His pull-downs, E. coli purified His-Sumo-SPCA1 fusion proteins were immobilized on Ni-NTA agarose beads and incubated with equal amounts of HeLa cell lysates. Protein–bead complexes were analyzed by Western blotting using antibodies recognizing His tag, CFL-1, and β-actin. (B, top) To restrict the binding site of actin and CFL-1 to SPCA1-L2 to a smaller region, full-length L2 (aa 329–680) was subdivided into two fragments. The first part is referred to as SPCA1-L2-N1 (aa 329–548), the second part as SPCA1-L2-C1 (aa 549–680). (B, bottom) His pull-downs were performed and analyzed with recombinant His-Sumo-SPCA1-L2 full length, -SPCA1-L2-N1, and -SPCA1-L2-C1 as described in A. (C) His-Sumo-CFL-1 was expressed in E. coli, purified, and the His-tag was subsequently cleaved. CFL-1 was incubated with a CFL-1 antibody and immobilized on agarose beads. Then, His-Sumo tag, His-Sumo-SPCA1-L1, or His-Sumo-SPCA1-L2-C1 were added. Proteins were eluted and separated by SDS-PAGE and stained with Coomassie blue. (D) CFL-1 and mutant CFL-1-S3E constructs were expressed, purified, and cut from His-Sumo tag. Recombinant proteins were incubated with purified His-Sumo-SPCA1-L2-C1 attached to Ni-NTA beads. Proteins were eluted and analyzed by SDS-PAGE and Coomassie blue staining.

Mentions: Thereafter, we incubated the major three cytosolic domains known to be required to pump Ca2+ (SPCA1-N-term, A-domain; SPCA1-L1-, A-domain; and SPCA-L2, N- and P-domains; Fig. 1 A) coupled to nickel nitrilotriacetic acid (Ni-NTA) agarose beads with equal amounts of HeLa cell lysates. The beads were then washed, and bound proteins were eluted. Analysis of the samples by Western blotting with antibodies against CFL-1 and β-actin showed that CFL-1 and actin specifically interacted only with SPCA1-L2 (Fig. 1 A).


Cofilin recruits F-actin to SPCA1 and promotes Ca2+-mediated secretory cargo sorting.

Kienzle C, Basnet N, Crevenna AH, Beck G, Habermann B, Mizuno N, von Blume J - J. Cell Biol. (2014)

CFL-1 directly interacts with a small domain of SPCA1 in vitro. (A, top) Schematic representation of the His-Sumo-SPCA1 expression constructs: SPCA1-N-term (aa 1–70), SPCA1-L1 (aa 124–252), and SPCA1-L2 (aa 329–680) comprise the A-, N-, and P-domain of SPCA1, which are the major cytosolic domains responsible for Ca2+ pumping activity. (A, bottom) To perform His pull-downs, E. coli purified His-Sumo-SPCA1 fusion proteins were immobilized on Ni-NTA agarose beads and incubated with equal amounts of HeLa cell lysates. Protein–bead complexes were analyzed by Western blotting using antibodies recognizing His tag, CFL-1, and β-actin. (B, top) To restrict the binding site of actin and CFL-1 to SPCA1-L2 to a smaller region, full-length L2 (aa 329–680) was subdivided into two fragments. The first part is referred to as SPCA1-L2-N1 (aa 329–548), the second part as SPCA1-L2-C1 (aa 549–680). (B, bottom) His pull-downs were performed and analyzed with recombinant His-Sumo-SPCA1-L2 full length, -SPCA1-L2-N1, and -SPCA1-L2-C1 as described in A. (C) His-Sumo-CFL-1 was expressed in E. coli, purified, and the His-tag was subsequently cleaved. CFL-1 was incubated with a CFL-1 antibody and immobilized on agarose beads. Then, His-Sumo tag, His-Sumo-SPCA1-L1, or His-Sumo-SPCA1-L2-C1 were added. Proteins were eluted and separated by SDS-PAGE and stained with Coomassie blue. (D) CFL-1 and mutant CFL-1-S3E constructs were expressed, purified, and cut from His-Sumo tag. Recombinant proteins were incubated with purified His-Sumo-SPCA1-L2-C1 attached to Ni-NTA beads. Proteins were eluted and analyzed by SDS-PAGE and Coomassie blue staining.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4151145&req=5

fig1: CFL-1 directly interacts with a small domain of SPCA1 in vitro. (A, top) Schematic representation of the His-Sumo-SPCA1 expression constructs: SPCA1-N-term (aa 1–70), SPCA1-L1 (aa 124–252), and SPCA1-L2 (aa 329–680) comprise the A-, N-, and P-domain of SPCA1, which are the major cytosolic domains responsible for Ca2+ pumping activity. (A, bottom) To perform His pull-downs, E. coli purified His-Sumo-SPCA1 fusion proteins were immobilized on Ni-NTA agarose beads and incubated with equal amounts of HeLa cell lysates. Protein–bead complexes were analyzed by Western blotting using antibodies recognizing His tag, CFL-1, and β-actin. (B, top) To restrict the binding site of actin and CFL-1 to SPCA1-L2 to a smaller region, full-length L2 (aa 329–680) was subdivided into two fragments. The first part is referred to as SPCA1-L2-N1 (aa 329–548), the second part as SPCA1-L2-C1 (aa 549–680). (B, bottom) His pull-downs were performed and analyzed with recombinant His-Sumo-SPCA1-L2 full length, -SPCA1-L2-N1, and -SPCA1-L2-C1 as described in A. (C) His-Sumo-CFL-1 was expressed in E. coli, purified, and the His-tag was subsequently cleaved. CFL-1 was incubated with a CFL-1 antibody and immobilized on agarose beads. Then, His-Sumo tag, His-Sumo-SPCA1-L1, or His-Sumo-SPCA1-L2-C1 were added. Proteins were eluted and separated by SDS-PAGE and stained with Coomassie blue. (D) CFL-1 and mutant CFL-1-S3E constructs were expressed, purified, and cut from His-Sumo tag. Recombinant proteins were incubated with purified His-Sumo-SPCA1-L2-C1 attached to Ni-NTA beads. Proteins were eluted and analyzed by SDS-PAGE and Coomassie blue staining.
Mentions: Thereafter, we incubated the major three cytosolic domains known to be required to pump Ca2+ (SPCA1-N-term, A-domain; SPCA1-L1-, A-domain; and SPCA-L2, N- and P-domains; Fig. 1 A) coupled to nickel nitrilotriacetic acid (Ni-NTA) agarose beads with equal amounts of HeLa cell lysates. The beads were then washed, and bound proteins were eluted. Analysis of the samples by Western blotting with antibodies against CFL-1 and β-actin showed that CFL-1 and actin specifically interacted only with SPCA1-L2 (Fig. 1 A).

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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Max Planck Institute of Biochemistry, 82152 Martinsried, Germany.

Show MeSH