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Browsing by Author "Pollard, Thomas D"
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Item Purification of a Cortical Complex Containing Two Unconventional Actins from Acanthamoeba by Affinity Chromatography on Profilin-Agarose(1994-10) Machesky, Laura M; Atkinson, Simon J; Ampe, Christophe; Vandekerckhove, Joel; Pollard, Thomas DWe 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 WD40 motif of the G beta 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.Item Sequences, Structural Models, and Cellular Localization of the Actin-related Proteins Arp2 and Arp3 from Acanthamoeba(1995-10) Kelleher, Joseph F; Atkinson, Simon J; Pollard, Thomas DWe cloned and sequenced the two actin-related proteins (Arps) present in the profilin-binding complex of Acanthamoeba (Machesky, L. M., S. J. Atkinson, C. Ampe, J. Vandekerckhove, and T. D. Pollard. 1994, J. Cell Biol. 127:107-115). The sequence of Arp2 is more similar to other Arp2s than to actin, while the sequence of Arp3 is more similar to other Arp3s than to actin. Phylogenetic analysis of all known Arps demonstrates that most group into three major families, which are likely to be shared across all eukaryotic phyla. Together with conventional actins, the Arps form a larger family distinct from structurally related ATPases such as Hsp70's and sugar kinases. Atomic models of the Arps based on their sequences and the structure of actin provide some clues about function. Both Arps have atoms appropriately placed to bind ATP and divalent cation. Arp2, but not Arp3, has a conserved profilin-binding site. Neither Arp has the residues required to copolymerize with actin, but an Arp heterodimer present in the profilin-binding complex might serve as a pointed end nucleus for actin polymerization. Both Acanthamoeba Arps are soluble in cell homogenates, and both are concentrated in the cortex of Acanthamoeba. The cellular concentrations are 1.9 microM Arp2 and 5.1 microM Arp3, substoichiometric to actin (200 microM) but comparable to many actin-binding proteins.