CARMIL is a bona fide capping protein interactant.
CARMIL, also known as Acan 125, is a multidomain protein that was originally identified on the basis of its interaction with the Src homology 3 (SH3) domain of type I myosins from Acanthamoeba. In a subsequent study of CARMIL from Dictyostelium, pull-down assays indicated that the protein also bound capping protein and the Arp2/3 complex. Here we present biochemical evidence that Acanthamoeba CARMIL interacts tightly with capping protein. In biochemical preparations, CARMIL copurified extensively with two polypeptides that were shown by microsequencing to be the alpha- and beta-subunits of Acanthamoeba capping protein. The complex between CARMIL and capping protein, which is readily demonstratable by chemical cross-linking, can be completely dissociated by size exclusion chromatography at pH 5.4. Analytical ultracentrifugation, surface plasmon resonance and SH3 domain pull-down assays indicate that the dissociation constant of capping protein for CARMIL is approximately 0.4 microm or lower. Using CARMIL fusion proteins, the binding site for capping protein was shown to reside within the carboxyl-terminal, approximately 200 residue, proline-rich domain of CARMIL. Finally, chemical cross-linking, analytical ultracentrifugation, and rotary shadowed electron microscopy revealed that CARMIL is asymmetric and that it exists in a monomer <--> dimer equilibrium with an association constant of 1.0 x 10(6) m(-1). Together, these results indicate that CARMIL self-associates and interacts with capping protein with affinities that, given the cellular concentrations of the proteins ( approximately 1 and 2 microm for capping protein and CARMIL, respectively), indicate that both activities should be physiologically relevant.
Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Kirsten
K
Remmert
Thomas E
TE
Olszewski
M Blair
MB
Bowers
Mariana
M
Dimitrova
Ann
A
Ginsburg
John A
JA
Hammer
D000048:Acanthamoeba / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification; D008840:Microfilament Proteins / Q000032:analysis / Q000737:chemistry; D015800:Protozoan Proteins / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification
C096372:Acan125 protein, Acanthamoeba; D002352:Carrier Proteins; D008840:Microfilament Proteins; D015800:Protozoan Proteins