A major agonist-regulated capping activity in Dictyostelium is due to the capping protein, cap32/34

Robert J. Eddy, Han Jinghua, Roger A. Sauterer, John S. Condeelis

Research output: Contribution to journalArticle

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Abstract

Stimulation of starved Dictyostelium amoebae with the chemoattractant cAMP produces a rapid increase in actin nucleation activity at 5 seconds which is cotemporal with an increase in actin assembly and a decrease in Ca2+-insensitive capping activity. Further characterization of this capping activity, called aginactin, led to the isolation of an Hsc70. Here, we demonstrate that purified aginactin contains both Hsc70 and the heterodimeric barbed-end capping protein, cap32/34. Immunoprecipitation of cap32/34 from purified aginactin removes all capping activity while immunoprecipitation of Hsc70 does not, indicating that the capping activity of aginactin is an intrinsic property of cap32/34. Gel filtration and immunoprecipitation assays fail to demonstrate the existence of a stable, high affinity complex between Hsc70 and cap32/34 in either lysate supernatants or aginactin pools but indicate the presence of a transient, ATP-sensitive interaction in cell lysates. Reconstitution experiments with purified Hsc70 and cap32/34 demonstrate that Hsc70 neither stimulates nor inhibits the capping activity of native cap32/34. Furthermore, we measured a K(d) of approx. 0.8 nM for the binding of cap32/34 to barbed ends of actin filaments in the absence or presence of Hsc70, in agreement with K(d) values measured for purified capping protein from other sources. We conclude, therefore, that cap32/34 is responsible for the capping activity called aginactin and that Hsc70 is not a regulatory cofactor for cap32/34 in Dictyostelium but may function as a chaperone during assembly of the cap32/34 heterodimer.

Original languageEnglish (US)
Pages (from-to)247-259
Number of pages13
JournalBiochimica et Biophysica Acta - Molecular Cell Research
Volume1314
Issue number3
DOIs
Publication statusPublished - Dec 12 1996

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Keywords

  • actin
  • aginactin
  • cell motility
  • chemotaxis
  • cytoskeleton

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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