A major agonist-regulated capping activity in Dictyostelium is due to the capping protein, cap32/34.
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 Ca(2+)-insensitive capping activity [1]. Further characterization of this capping activity, called aginactin, led to the isolation of an Hsc70 [2]. 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 Kd 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 Kd 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.
Eddy
R J
RJ
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Han
J
J
Sauterer
R A
RA
Condeelis
J S
JS
D000199:Actins / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis / Q000737:chemistry; D004023:Dictyostelium / Q000737:chemistry*; D005656:Fungal Proteins / Q000737:chemistry*; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D007700:Kinetics; D008840:Microfilament Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D008970:Molecular Weight; D011485:Protein Binding; D015800:Protozoan Proteins
D000199:Actins; D002352:Carrier Proteins; D005656:Fungal Proteins; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D008840:Microfilament Proteins; D015800:Protozoan Proteins; C072098:aginactin protein, Dictyostelium; C069272:cap32-34 protein, Dictyostelium; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate