Abstract
Fission yeast Schizosaccharomyces pombe is an important genetic model organism for studying the mechanisms of endocytosis and cytokinesis. However, most work on the biochemical properties of fission yeast actin-binding proteins has been done with skeletal muscle actin for matters of convenience. When simulations of mathematical models of the mechanism of endocytosis were compared with events in live cells, some of the reactions appeared to be much faster than observed in biochemical experiments with muscle actin. Here, we used gelsolin affinity chromatography to purify actin from fission yeast. S. pombe actin shares many properties with skeletal muscle actin but has higher intrinsic nucleotide exchange rate, faster trimer nucleus formation, faster phosphate dissociation rate from polymerized actin, and faster nucleation of actin filaments with Arp2/3 complex. These properties close the gap between the biochemistry and predictions made by mathematical models of endocytosis in S. pombe cells.
Highlights
Fission yeast Schizosaccharomyces pombe is a genetic model organism used to study actin dynamics during endocytosis and cytokinesis [2,3,4]
We found that a modification of their method yields highly purified, active fission yeast actin
Purification of S. pombe Actin—After homogenization of fission yeast, a substantial fraction of the actin pelleted with cell debris as expected because ϳ13% of cellular actin is polymerized and associated with endocytic actin patches [2]
Summary
Purification of C-terminal Half of Mouse Gelsolin—We purified the C-terminal half of His6-tagged mouse gelsolin (G4-6) from Rosetta (DE3) pLysS-competent Escherichia coli cells (Novagen) [11] with the following modifications. After the beads settled and the supernatant was removed, actin was eluted by suspending the beads in 100 ml of elution buffer (10 mM Tris, 5 mM EGTA, 1 mM MgCl2, 1 mM ATP, 7 mM -mercaptoethanol, 1 mM NaN3, pH 8.0) for 1 min and pelleting the beads. The supernatant was discarded, and the surface of actin filament pellet was washed with 5 ml of G-buffer (2 mM Tris, 0.1 mM CaCl2, 0.2 mM ATP, 0.5 mM DTT, 1 mM NaN3, pH 8.0). Release of Phosphate during Actin Polymerization—ATP was removed from actin monomers in a Cortex 0.22-m spin column with 600 l of Sephadex G-25 resin (Sigma-Aldrich G2550) previously washed twice with nucleotide-free G-buffer (1 mM NaN3, 0.1 mM CaCl2, and 2 mM Tris, pH 8.0).
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