Fluorescence Probing of Yeast Actin Subdomain 3/4 Hydrophobic Loop 262–274

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The Arp2/3 complex creates filament branches leading to an enhancement in the rate of actin polymerization. Work with Arp complexes from different sources indicated that it was inactive by itself, required an activating factor such as the Wiskott-Aldrich syndrome protein (WASP), and might exhibit a preference for ATP or ADP-P i actin. However, with yeast actin, P i release is almost concurrent with polymerization, eliminating the presence of an ADP-P i cap. We thus investigated the ability of the yeast Arp2/3 complex (yArp2/3) to facilitate yeast actin polymerization in the presence and absence of the Arp2/3-activating factor Las17p WA. yArp2/3 significantly accelerates yeast actin but not muscle actin polymerization in the absence of Las17p WA. The addition of Las17p WA further enhances yeast actin polymerization by yArp2/3 and allows the complex to now assist muscle actin polymerization. This actin isoform difference is not observed with bovine Arp2/3 complex, because the neural WASP VCA fragment is required for polymerization of both actins. Observation of individual branching filaments showed that Las17p WA increased the persistence of filament branches. Compared with wild type actin, the V159N mutant actin, proposed to be more ATP-like in behavior, exhibited an enhanced rate of polymerization in the presence of the yArp2/3 complex. yArp2/3 caused a significant rate of P i release prior to observation of an increase in filament mass but while branched structures were present. Thus, yeast F-actin can serve as a primary yArp2/3-activating factor, indicating that a newly formed yeast actin filament has a topology, unlike that of muscle actin, that is recognized specifically by yArp2/3.

Section: Methodsmentioning

confidence: 99%

Acceleration of Yeast Actin Polymerization by Yeast Arp2/3 Complex Does Not Require an Arp2/3-activating Protein

Wen

Rubenstein

2005

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“…Furthermore, the conformational dynamics of the hydrophobic loop accompanying polymerization is also important. The hydrophobic loop detaches from the main body of actin and is inserted into the hydrophobic pocket, stabilizing the filaments (43,44). Thus, crosslinked actin, in which extension of the hydrophobic loop was inhibited, was unable to polymerize (45).…”

Section: R95c E226k G268dmentioning

confidence: 99%

Dominant Negative Mutant Actins Identified in Flightless Drosophila Can Be Classified into Three Classes

Noguchi

Gomibuchi

Murakami

et al. 2010

Strongly dominant negative mutant actins, identified by An and Mogami (An, H. S., and Mogami, K. (1996) J. Mol. Biol. 260, 492-505), in the indirect flight muscle of Drosophila impaired its flight, even when three copies of the wild-type gene were present. Understanding how these strongly dominant negative mutant actins disrupt the function of wild-type actin would provide useful information about the molecular mechanism by which actin functions in vivo. Here, we expressed and purified six of these strongly dominant negative mutant actins in Dictyostelium and classified them into three groups based on their biochemical phenotypes. The first group, G156D, G156S, and G268D actins, showed impaired polymerization and a tendency to aggregate under conditions favoring polymerization. G63D actin of the second group was also unable to polymerize but, unlike those in the first group, remained soluble under polymerizing conditions. Kinetic analyses using G63D actin or G63D actin⅐gelsolin complexes suggested that the pointed end surface is defective, which would alter the polymerization kinetics of wild-type actin when mixed and could affect formation of thin filament structures in indirect flight muscle. The third group, R95C and E226K actins, was normal in terms of polymerization, but their motility on heavy meromyosin surfaces in the presence of tropomyosin-troponin indicated altered sensitivity to Ca 2؉ . Cofilaments in which R95C or E226K actins were copolymerized with a 3-fold excess of wild-type actin also showed altered Ca 2؉ sensitivity in the presence of tropomyosin-troponin.Actin filaments are major components of the cytoskeletons of all eukaryotic cells and play key roles in a variety of cellular functions, including cell migration, organelle transport, and muscle contraction. These diverse processes depend on the dynamic nature of actin filaments and their interactions with various actin-binding proteins (1), which are thought to involve conformational changes in the subunits that make up actin filaments. For instance, the stability of actin filaments is affected by the conformational difference between actin subunits carrying ATP or ADP and those that do not (2, 3), whereas cofilin, a major actin filament severing protein, alters the twist of actin filaments (4). In addition, modification of actin filaments through chemical cross-linking causes motility defects with myosin, suggesting a possible link between actin-myosin interaction and the conformational dynamics of actin (5, 6). Moreover, the myosin-induced conformational changes appear to be cooperative (7-9), e.g. Ca 2ϩ -triggered conformational changes in the skeletal muscle actin filaments are enhanced by cooperative conformational changes induced by myosin (10). In many cases, however, details of the molecular relationship between conformational changes in actin and its physiological function remain unclear.Actin is a highly conserved protein, so that Dictyostelium actin 15 shares 91% amino acid identity with rabbit skeletal muscle actin. This implies ...

“…The actin concentration was determined by UV light absorbance at 290 nm using the extinction coefficient: ⑀ ϭ 25.6 cm Ϫ1 ⅐mM Ϫ1 . Dithiothreitol-free actin, 40 M, was labeled with 45 M N-(1-pyrenyl)maleimide as described previously (33). The pyrene-labeled F-actin was collected by centrifugation and depolymerized by dialysis against G buffer.…”

Section: Actin Purification and Labeling With Pyrene Maleimidementioning

confidence: 99%

A Mammalian Actin Substitution in Yeast Actin (H372R) Causes a Suppressible Mitochondria/Vacuole Phenotype

McKane

Wen

Boldogh

et al. 2005

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To determine the reason for the inviability of Saccharomyces cerevisiae with skeletal muscle actin, we introduced into yeast actin the first variant muscle residue from the C-terminal end, H372R. Arg is also found at this position in non-yeast nonmuscle actins. The substitution caused retarded growth on glucose and an inability to use glycerol as a sole carbon source. The mitochondria were clumped and had lost their DNA, the vacuole appeared hypervesiculated, and the actin cytoskeleton became somewhat depolarized. Introduction of the second muscle actin-specific substitution, S365A, rescued these defects. Suppression was also achieved by introducing the four acidic N-terminal residues of muscle actin in place of the two found in yeast actin. The H372R substitution results in an increase in polymerization-dependent fluorescence of Cys-374 pyrene-labeled actin. H372R actin polymerizes slightly faster than wild-type (WT) actin. Yeast actin-related proteins 2 and 3 (Arp2/3) accelerates the polymerization of H372R actin to a much greater extent than WT actin. The two suppressors did not affect the rate of H372R actin polymerization in the absence of an Arp2/3 complex. In contrast, the S365A substitution dampened the rate of Arp2/3 complex-stimulated H372R actin polymerization, and the addition of the four acidic N-terminal residues caused this rate to decrease below that observed with WT actin in the presence of Arp2/3. Structural analysis of the mutations suggests the presence of stringent steric and ionic requirements for the bottom of actin subdomain 1 and also suggests that there is allosteric communication through subdomain 1 within the actin monomer between the N and C termini.The actin C-terminal region, consisting of residues 364 -375, is located in subdomain 1 on the opposite side of the protein from that of the N terminus and is believed to play an important role in the interaction of actin with different actin-binding proteins such as profilin (1, 2), cofilin (3), and gelsolin (4) in the determination of actin filament stability and in the allosteric behavior of the actin filament. The crystal structure of the filament shows that a direct interaction of the N and C termini is virtually impossible because of the intervening mass of the core of subdomain 1 (Fig. 1).The sequence of the C-terminal peptide is highly conserved throughout the actin family. The sequences for mammalian and avian muscle actins are identical and there are only two differences between yeast and higher eukaryotic actins, a substitution of His in yeast at residue 372 for Arg and a substitution of Ser at residue 365 for Arg just outside of the helix in this peptide. In the first case the His and the Arg carry at least a partial positive charge.Removal of the C-terminal three residues in yeast actin causes cell inviability, whereas removal of either or both of the first two residues, beginning with the C terminus, shows milder phenotypes including temperature sensitivity and increased cell size (5). The actin C-terminal dipeptide or tripe...