A change in actin conformation associated with filament instability after Pi release - PubMed (original) (raw)

A change in actin conformation associated with filament instability after Pi release

L D Belmont et al. Proc Natl Acad Sci U S A. 1999.

Abstract

The ability of actin to both polymerize into filaments and to depolymerize permits the rapid rearrangements of actin structures that are essential for actin's function in most cellular processes. Filament polarity and dynamic properties are conferred by the hydrolysis of ATP on actin filaments. Release of inorganic phosphate (Pi) from filaments after ATP hydrolysis promotes depolymerization. We identify a yeast actin mutation, Val-159 to Asn, which uncouples Pi release from the conformational change that results in filament destabilization. Three-dimensional reconstructions of electron micrographs reveal a conformational difference between ADP-Pi filaments and ADP filaments and show that ADP V159N filaments resemble ADP-Pi wild-type filaments. Crystal structures of mammalian beta-actin in which the nucleotide binding cleft is in the "open" and "closed" states can be used to model actin filaments in the ADP and ADP-Pi conformations, respectively. We propose that these two conformations of G-actin may be related to two functional states of F-actin.

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Figures

Figure 1

Figure 1

Polymerization and depolymerization of V159N actin were measured by light scattering at 400 nm. (A) Polymerization of 5 μM wild-type or V159N actin was initiated by the addition of 0.1 M KCl, 2 mM MgCl2, and 0.5 mM ATP. (B) Depolymerization was measured by diluting 5 μM F-actin to 0.5 μM in the polymerization buffer. The half time of depolymerization for wild-type actin is 114 s (n = 4, SD = 18), and for V159N actin is 355 s (n = 5, SD = 155). (C) Cofilin-accelerated depolymerization was observed by diluting 5 μM F-actin to 0.5 μM in polymerization buffer containing 0.5 μM yeast cofilin. (D) The critical concentration was measured by pelleting filamentous actin from steady-state reactions containing different concentrations of actin. Supernatants and pellets were analyzed by SDS/PAGE, and actin was quantified by densitometry. The critical concentration of V159N was measured three times, and the values ranged from 0 to 20 nM. The critical concentration of wild-type yeast actin is 160 nM.

Figure 2

Figure 2

Depolymerization of gelsolin-capped actin filaments. (A) Latrunculin A (50 μM) was added to gelsolin-capped filaments, and depolymerization was monitored with pyrene fluorescence. The half time of depolymerization is 96 s for wild-type (wt) filaments and 230 s for V159N filaments. (B) To compare length distributions, actin filaments used for the depolymerization assay were stained with rhodamine phalloidin and observed by fluorescence microscopy. (Bar = 5 μm.)

Figure 3

Figure 3

Pi release during actin filament assembly. Polymerization of actin (25 μM) was monitored by light scattering at 400 nm, and Pi release measured with the EnzChek phosphate assay kit (Molecular Probes). The amount of total polymerized actin then was measured by pelleting the final polymerization mix and measuring the protein concentration of the pellet by using an SDS gel containing actin standards. (A) Wild-type yeast actin. (B) V159N actin. (C) V159N actin plus 25 μM cofilin. The solid lines represent actin assembly and the circles are free Pi.

Figure 4

Figure 4

Electron micrographs of yeast wild-type F-actin (a), ADP-BeF4− filaments (b), and V159N filaments (c). Tobacco mosaic virus particles with a helical pitch of 23 Å were used as a size standard (c). (Bar in a = 1,000 Å.)

Figure 5

Figure 5

Three-dimensional electron microscopy reconstructions and actin filament models. Reconstructions of yeast actin filaments (a_–_f) compared with models generated from β-actin (g_–_l). The wild-type yeast actin, shown as a surface view (d) and in a single cross section (a), has an open nucleotide-binding cleft (arrow, d). The open cleft results in a lack of density between subdomains 2 (S2) and 4 (S4), as seen in cross section (a). Subdomains 1 (S1′) and 3 (S3′) from a subunit on the opposite strand also are labeled in a, and four subdomains from a single actin subunit are labeled in e. When the wild-type F-ADP filaments are incubated with BeF4− (b and e), the nucleotide-binding cleft closes. The V159N mutant actin (c and f) also shows a closure of the nucleotide-binding cleft. The opening of the nucleotide-binding cleft also can be seen in a crystal structure of nonmuscle β-actin (27) (h and i). The G-actin subunit from this crystal structure (h) has been oriented to the Lorenz et al. model for F-actin (26), using a least-squares alignment of Cα atoms in subdomains 1, 3, and 4 (3.3-Å rms deviation). The filament axis after such an alignment is shown by the vertical line (h, front view; i, side view), and the open arrows in g indicate the corresponding views of the subunits in a low-resolution filament surface generated from the atomic model. The corresponding “closed” state of β-actin (26) after alignment to the Lorenz et al. model (3.1-Å rms deviation) is shown in k and l, with the low-resolution surface generated from this subunit in j. The main difference between these two forms is the rotation of subdomain 2 by about 15°, shown by the arrow in i. (The scale bar in a is 50 Å and applies to a_–_c.)

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