Purification of a cortical complex containing two unconventional actins from Acanthamoeba by affinity chromatography on profilin-agarose.

Abstract

Abstract. We identified four polypeptides of 47, 44, 40, and 35 kD that bind to profilin-Sepharose and elute with high salt. When purified by conventional chromatography using an antibody to the 47-kD polypeptide, these four polypeptides copurified as a stoichiometric complex together with three additional polypeptides of 19, 18, and 13 kD that varied in their proportions to the other polypeptides. Partial protein sequences showed that the 47-kD polypeptide is a homologue of S. pombe act2 and the 44-kD polypeptide is a homologue of S. cerevisiae ACT2, both unconventional actins. The 40-kD polypeptide contains a sequence similar to the WIM0 motif of the Ga subunit of a trimeric G-protein from Dictyostelium discoideum. From partial sequences, the 35-, 19-, and 18-kD polypeptides appear to be novel proteins. On gel filtration the complex of purified polypeptides cochromatograph with a Stokes' radius of 4.8 nm, a value consistent with a globular particle of 220 kD containing one copy of each polypeptide. Cell extracts also contain components of the complex that do not bind the profilin column. Affinity purified antibodies localize 47-and 18/19-kD polypeptides in the cortex and filopodia of Acanthamoeba. Antibodies to the 47-kD unconventional actin cross-react on immunoblots with polypeptides of similar size in Dictyostelium, rabbit muscle, and conventional preparations of rabbit muscle actin but do not react with actin.

Signaling mechanisms that regulate actin‐based motility processes in the nervous system

“…The Arp 2/3 complex is ubiquitous to eukaryotic cells, and was first identified in Acanthamoeba by affinity chromatography on profilin Sepharose (Machesky et al 1994). The complex consists of 7 proteins: Arp 2, Arp 3, p41Arc, p34Arc, p21Arc, p20Arc, and p16Arc.…”

Profilin is required for the normal timing of actin polymerization in response to thermal stress

“…Recent evidence suggests that this property is important for profilin's interactions with the Cappuccino gene product in Drosophila [7], and for its involvement in the motility of various pathogenic bacteria and viruses [8][9][10][11]. Less well characterized profilin interactions include the copurification of profilin with a seven-protein complex from Acanthamoeba [12], and our recent two-hybrid results that indicate a *Corresponding author. Fax: (1) (512) 471-9651.…”

Spatial and temporal regulation of actin polymerization for cytoskeleton formation through Arp2/3 complex and WASP/WAVE proteins

“…An abundant protein complex, actin-related protein (Arp) 2/3 complex, which functions as a nucleation core has been identified [Machesky et al, 1994]. The nucleation core activity of the Arp2/3 complex is tightly regulated by members of Wiskott-Aldrich syndrome family protein (WASP, N-WASP, and WAVE proteins; WAVE is also called SCAR), which enable rapid polymerization of actin for promotion of changes in the actin cytoskeleton [Machesky and Insall, 1998;Machesky et al, 1999;Rohatgi et al, 1999].…”