Binding of Divalent Cation and Nucleotide to G-actin in the Presence of Profilin

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147

Profilin, an essential G-actin-binding protein, has two opposite regulatory functions in actin filament assembly. It facilitates assembly at the barbed ends by lowering the critical concentration (Pantaloni, D., and Carlier, M.-F. (1993) Cell 75, 1007-1014); in contrast it contributes to the pool of unassembled actin when barbed ends are capped. We proposed that the first of these functions required an input of energy. How profilin uses the ATP hydrolysis that accompanies actin polymerization and whether the acceleration of nucleotide exchange on G-actin by profilin participates in its function in filament assembly are the issues addressed here. We show that 1) profilin increases the treadmilling rate of actin filaments in the presence of Mg 2؉ ions; 2) when filaments are assembled from CaATP-actin, which polymerizes in a quasireversible fashion, profilin does not promote assembly at the barbed ends and has only a Gactin-sequestering function; 3) plant profilins do not accelerate nucleotide exchange on G-actin, yet they promote assembly at the barbed end. The enhancement of nucleotide exchange by profilin is therefore not involved in its promotion of actin assembly, and the productive growth of filaments from profilin-actin complex requires the coupling of ATP hydrolysis to profilin-actin assembly, a condition fulfilled by Mg-actin, and not by Ca-actin.Living cells undergo changes in shape and motile behavior by spatially and temporally controlled rearrangements of the actin cytoskeleton. In the physiological ionic conditions, F-actin is assembled at steady state in the cell medium. Changes in the F-actin/G-actin ratio, which occur in response to stimuli, are made possible by shifts in steady state, i.e. changes in the critical concentration for filament assembly. These changes are elicited by capping proteins and profilin (1, 2) and amplified by G-actin-binding proteins (3). A high level of capping of barbed ends maintains the high critical concentration of pointed ends in the cytoplasm. A steep energetic gradient is therefore created between the cell medium and the loci where uncapped barbed ends are nucleated at the plasma membrane. We understand that in this way capping of barbed ends in the cytoplasm is required for a more efficient local actin assembly. In support of this view, recent evidence indeed indicates that the level of motility in fibroblasts (4) and Dictyostelium (5) correlates with the level of barbed end capping. Similarly, the actinbased propulsive movement of Listeria results from the local creation and maintenance of new uncapped barbed ends at the bacterium surface, while filaments are capped in the bulk cytoplasm (6).Profilin has unique properties among G-actin-binding proteins. Under physiological ionic conditions (Mg-actin, 0.1 M KCl), it binds G-actin tightly (K ϭ 10 7 M Ϫ1 for vertebrate profilin I) and participates in the establishment of the pool of unassembled actin when barbed ends are capped. In contrast, when barbed ends are uncapped, the participation of profilinactin complex in...

Section: Profilin Does Not Promote Actin Assembly At the Barbed End Wsupporting

confidence: 79%

Section: The Ability Of Profilin To Increase the Rate Of Nucleotide Dmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Role of Nucleotide Exchange and Hydrolysis in the Function of Profilin in Actin Assembly

Perelroizen¹,

Didry²,

Christensen³

et al. 1996

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141

147

“…3B). Remarkably, despite extensive differences (Table 3), fission yeast and bovine profilins have the same affinity for rabbit muscle actin (K d ϭ 0.1-0.2 M) in fluorescence quenching and nucleotide exchange assays (1,37,38 Residues in the interface with actin that differ between human and S. pombe profilins appear to provide Hs profilin with more interactions with actin than S. pombe profilin (Table 3), so other interactions must compensate. We note three examples of possible compensation.…”

Section: Human Profilin-i Does Not Complement the Fission Yeastmentioning

confidence: 99%

Incompatibility with Formin Cdc12p Prevents Human Profilin from Substituting for Fission Yeast Profilin

Ezezika

Younger

et al. 2009

The small protein profilin not only helps to maintain a cytoplasmic pool of actin monomers ready to elongate actin filament barbed ends (2), but it also binds to type II poly-L-proline helices (3, 4). The actin (5) and poly-L-proline (6 -8) binding sites are on opposite sides of the profilin molecule, so profilin can link actin to proline-rich targets. Viability of fission yeast depends independently on profilin binding to both actin and poly-L-proline, although cells survive Ͼ10-fold reductions in affinity for either ligand (1).Fission yeast Schizosaccharomyces pombe depend on formin Cdc12p (9, 10) and profilin (11) to assemble actin filaments for the cytokinetic contractile ring. Formins are multidomain proteins that nucleate and assemble unbranched actin filaments (12). Formin FH2 domains form homodimers that can associate processively with the barbed ends of growing actin filaments (13,14). FH2 dimers slow the elongation of barbed ends (15). Most formin proteins have an FH1 domain linked to the FH2 domain. Binding profilin-actin to multiple polyproline sites in an FH1 domain concentrates actin near the barbed end of an actin filament associated with a formin FH2 homodimer. Actin transfers very rapidly from the FH1 domains onto the filament end (16) allowing profilin to stimulate elongation of the filament (15, 17).We tested the ability of human (Homo sapiens, Hs) 7 profilin-I to complement the temperature-sensitive cdc3-124 mutation (11) in the single fission yeast profilin gene with the aim of using yeast to characterize human profilin mutations. The failure of expression of Hs profilin-I to complement the cdc3-124 mutation prompted us to compare human and fission yeast profilins more carefully. We report here a surprising incompatibility of Hs profilin-I with fission yeast formin Cdc12p, a crystal structure of fission yeast profilin, which allowed a detailed comparison with Hs profilin, and mutations that revealed how overexpression of Hs profilin-I compromises the viability of wild-type fission yeast. EXPERIMENTAL PROCEDURESStrains, Media, and Chemicals-Kathleen Gould of Vanderbilt University provided strains of S. pombe: KYG491, a haploid temperature-sensitive strain with a point mutation in the * This work was supported, in whole or in part, by National Institutes of Health Grants GM-026338 and GM-066311 (to T. D. P.) and GM-079265 (to D. R. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S5. The atomic coordinates and structure factors (codes 3D9Y and 3DAV)

“…ATP hydrolysis is not necessary for polymerization, since ADP-G actin will polymerize (10). Several potential roles for ATP hydrolysis have been suggested, including regulating the structure of the actin filament (1-4, 31), regulating interactions with actin binding proteins (7,8,32,33), and regulating the dynamics of monomer and filament interaction (6,34), effects which are not necessarily mutually exclusive.…”

Section: Fig 3 Concentration and Ph Dependence Of The Inhibitory Efmentioning

confidence: 99%

Binding of Phosphate, Aluminum Fluoride, or Beryllium Fluoride to F-actin Inhibits Severing by Gelsolin

Allen

Laham

Way

et al. 1996

Actin exhibits ATPase activity of unknown function that increases when monomers polymerize into filaments. Differences in the kinetics of ATP hydrolysis and the release of the hydrolysis products ADP and inorganic phosphate suggest that phosphate-rich domains exist in newly polymerized filaments. We examined whether the enrichment of phosphate on filamentous ADP-actin might modulate the severing activity of gelsolin, a protein previously shown to bind differently to ATP and ADP actin monomers. Binding of phosphate, or the phosphate analogs aluminum fluoride and beryllium fluoride, to actin filaments reduces their susceptibility to severing by gelsolin. The concentration and pH dependence of inhibition suggest that HPO 4 2؊ binding to actin filaments generates this resistant state. We also provide evidence for two different binding sites for beryllium fluoride on actin. Actin has been postulated to contain two P i binding sites. Our data suggest that they are sequentially occupied following ATP hydrolysis by HPO 4 2؊ which is subsequently titrated to H 2 PO 4 ؊ . We speculate that beryllium fluoride and aluminum fluoride bind to the HPO 4 2؊ binding site. The cellular consequences of this model of phosphate release are discussed.