PURIFICATION OF A CORTICAL COMPLEX CONTAINING 2 UNCONVENTIONAL ACTINSFROM ACANTHAMOEBA BY AFFINITY-CHROMATOGRAPHY ON PROFILIN-AGAROSE

We identified four polypeptides of 47, 44, 40, and 35 kD that bind to profilin-Sepharose and elute with high salt. When purified by conventi onal 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 the ir proportions to the other polypeptides. Partial protein sequences sh owed that the 47-kD polypeptide is a homologue of S. pombe act2 and th e 44-kD polypeptide is a homologue of S. cerevisiae ACT2, both unconve ntional actins. The 40-kD polypeptide contains a sequence similar to t he WD40 motif of the G(beta) subunit of a trimeric G-protein from Dict yostelium discoideum. From partial sequences, the 35-, 19-, and 18-kD polypeptides appear to be novel proteins. On gel filtration the comple x 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 o f the complex that do not bind the profilin column. Affinity purified antibodies localize 47- and 18/19-kD polypeptides in the cortex and fi lopodia of Acanthamoeba. Antibodies to the 47-kD unconventional actin cross-react on immunoblots with polypeptides of similar size in Dictyo stelium, rabbit muscle, and conventional preparations of rabbit muscle actin but do not react with actin.