Yeast actin: polymerization kinetic studies of wild type and a poorly polymerizing mutant.

Buzan, Jenny M.; Frieden, Carl

doi:10.1073/pnas.93.1.91

Cited by 64 publications

(49 citation statements)

References 30 publications

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“…Previous studies have shown that addition of a N-terminal His tag to yeast actin does not affect its polymerization or function in yeast (Buzan and Frieden, 1996;Buzan et al, 1999). Nevertheless, we tested whether the presence of the N-terminal tag affected polymerization of TgACT1 by mon-itoring the quenching of tryptophan fluorescence, which is an inherent property of actins.…”

Section: Polymerization Kinetics Of Tgact1mentioning

confidence: 99%

Unusual Kinetic and Structural Properties Control Rapid Assembly and Turnover of Actin in the ParasiteToxoplasma gondii

Sahoo

Beatty

Heuser

et al. 2006

MBoC

116

138

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Toxoplasma is a protozoan parasite in the phylum Apicomplexa, which contains a number of medically important parasites that rely on a highly unusual form of motility termed gliding to actively penetrate their host cells. Parasite actin filaments regulate gliding motility, yet paradoxically filamentous actin is rarely detected in these parasites. To investigate the kinetics of this unusual parasite actin, we expressed TgACT1 in baculovirus and purified it to homogeneity. Biochemical analysis showed that Toxoplasma actin (TgACT1) rapidly polymerized into filaments at a critical concentration that was 3-4-fold lower than conventional actins, yet it failed to copolymerize with mammalian actin. Electron microscopic analysis revealed that TgACT1 filaments were 10 times shorter and less stable than rabbit actin. Phylogenetic comparison of actins revealed a limited number of apicomplexan-specific residues that likely govern the unusual behavior of parasite actin. Molecular modeling identified several key alterations that affect interactions between monomers and that are predicted to destabilize filaments. Our findings suggest that conserved molecular differences in parasite actin favor rapid cycles of assembly and disassembly that govern the unusual form of gliding motility utilized by apicomplexans.

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Section: Polymerization Kinetics Of Tgact1mentioning

confidence: 99%

Unusual Kinetic and Structural Properties Control Rapid Assembly and Turnover of Actin in the ParasiteToxoplasma gondii

Sahoo

Beatty

Heuser

et al. 2006

MBoC

116

138

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“…Yeast, compared with muscle actin, polymerizes faster and exchanges its bound nucleotide faster. In contrast to muscle actin, the yeast actin filament releases its P i almost immediately after ATP hydrolysis and the filament fragments more readily (4). Yeast cells cannot survive with muscle actin as the sole actin, and with ␤-nonmuscle actin as the only actin, yeast cells are very sick (5).…”

mentioning

confidence: 99%

Actin Isoform-specific Conformational Differences Observed with Hydrogen/Deuterium Exchange and Mass Spectrometry

Stokasimov

Rubenstein

2009

Journal of Biological Chemistry

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Actin can exist in multiple conformations necessary for normal function. Actin isoforms, although highly conserved in sequence, exhibit different biochemical properties and cellular roles. We used amide proton hydrogen/deuterium (HD) exchange detected by mass spectrometry to analyze conformational differences between Saccharomyces cerevisiae and muscle actins in the G and F forms to gain insight into these differences. We also utilized HD exchange to study interdomain and allosteric communication in yeast-muscle hybrid actins to better understand the conformational dynamics of actin. Areas showing differences in HD exchange between G-and F-actins are areas of intermonomer contacts, consistent with the current filament models. Our results showed greater exchange for yeast G-actin compared with muscle actin in the barbed end pivot region and areas in subdomains 1 and 2 and for F-actin in monomer-monomer contact areas. These results suggest greater flexibility of the yeast actin monomer and filament compared with muscle actin. For hybrid G-actins, the muscle-like and yeastlike parts of the molecule generally showed exchange characteristics resembling their parent actins. A few exceptions were a peptide on top of subdomain 2 and the pivot region between subdomains 1 and 3 with muscle actin-like exchange characteristics although the areas were yeastlike. These results demonstrate that there is cross-talk between subdomains 1 and 2 and the large and small domains. Hybrid F-actin data showing greater exchange compared with both yeast and muscle actins are consistent with mismatched yeast-muscle interfaces resulting in decreased stability of the hybrid filament contacts.The ability of actin to engage in a wide range of physiological functions requires that it be subject to complex spatial and temporal control by a large array of actin-binding proteins (1-3). Such regulation demands that both the monomeric and filamentous forms of actin be able to exist in a number of different conformations. Some of these may be differentially recognized by different actin-binding proteins, and some might actually be induced by the interaction of actin with these proteins.Actin is highly conserved from yeast to humans. There is 87% sequence identity between yeast and muscle actin and 91% sequence identity between yeast and nonmuscle actins. Even with this high sequence similarity and minor differences in crystal structures, there are some major differences in yeast and muscle actin behavior. Yeast, compared with muscle actin, polymerizes faster and exchanges its bound nucleotide faster. In contrast to muscle actin, the yeast actin filament releases its P i almost immediately after ATP hydrolysis and the filament fragments more readily (4). Yeast cells cannot survive with muscle actin as the sole actin, and with ␤-nonmuscle actin as the only actin, yeast cells are very sick (5). A set of biochemical data indicates that the muscle filament is less flexible than yeast (6, 7). However, conformational and structural differences that could ex...

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“…It has long been appreciated that actin is a highly dynamic protein [Buzan and Frieden, 1996;Kim et al, 1996], and that allostery plays a major role in actin function based on the fact that alterations in actin structure can exert long-distance effects through the protein [McKane et al, 2005[McKane et al, , 2006. Supporting evidence lies in the fact that modification of the actin C-terminus by proteolysis, antibody binding and by mutagenesis can significantly impact actin polymerization properties [Johannes and Gallwitz, 1991;Crosbie et al, 1994;Orlova and Egelman, 1995;Phan et al 1997;Hegyi et al, 1998;Kim et al, 1998].…”

Section: Budding Yeast As An Expression System For Mutant Actinsmentioning

confidence: 89%

Insights into the effects of disease‐causing mutations in human actins

Rubenstein

Wen

2014

Cytoskeleton

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Mutations in all six actins in humans have now been shown to cause diseases. However, a number of factors have made it difficult to gain insight into how the changes in actin functions brought about by these pathogenic mutations result in the disease phenotype. These include the presence of multiple actins in the same cell, limited accessibility to pure mutant material, and complexities associated with the structures and their component cells that manifest the diseases. To try to circumvent these difficulties, investigators have turned to the use of model systems. This review describes these various approaches, the initial results obtained using them, and the insight they have provided into allosteric mechanisms that govern actin function. Although results so far have not explained a particular disease phenotype at the molecular level, they have provided valuable insight into actin function at the mechanistic level which can be utilized in the future to delineate the molecular bases of these different actinopathies. Key Words: actin; allostery; disease; mutation; isoforms Introduction A ctin mutations can cause human disease. However, little is known concerning the mechanisms by which these mutations lead to the disease phenotypes they produce. The goal of this review is to describe the general properties of actin, the diseases associated with mutations in actin, methodologies that might be employed to achieve a mechanistic understanding of actin based disease and efforts that have been made toward this goal. Properties of G-and F-ActinA discussion of the effects of pathogenic mutations on actin function requires a basic understanding of actin structure and its solution properties. Actin is an abundant 42 kD monomeric protein, found in virtually all eukaryotic cells, that can cycle between either a monomeric (G-actin) or polymeric (F-actin) state. It plays both contractile and structural roles in the cytoplasm, and recent studies have documented that actin in the nucleus acts as a promoter of transcription or as a member of a number of chromatin remodeling complexes Philimonenko et al., 2004;Miyamoto and Gurdon, 2011; Miyamoto et al., 2011a,b;Weston et al., 2012].G-actin is a clam-shaped protein with two domains, the inner and outer domains ( Fig. 1), connected by a hinge region, and separated by a deep cleft. Outer (subdomains 1 and 2) and inner (subdomains 3 and 4) refer to their position in the actin filament. The N and C-termini reside on opposite faces of the protein in subdomain 1. An adenine nucleotide binds deep within the inter-domain cleft, held in place by a divalent cation, and imparts stability to the protein and the filament. This latter role depends on its ability to cycle between the ATP and ADP state by virtue of a polymerization-dependent ATPase activity [Carlier et al., 1987;Korn et al., 1987]. F-actin is a polar structure with pointed (2) and barbed (1) ends referring to the arrowhead pattern formed when myosin S1 binds to F-actin in the absence of ATP [Craig et al., 1985]. The polarity of th...

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Yeast actin: polymerization kinetic studies of wild type and a poorly polymerizing mutant.

Cited by 64 publications

References 30 publications

Unusual Kinetic and Structural Properties Control Rapid Assembly and Turnover of Actin in the ParasiteToxoplasma gondii

Unusual Kinetic and Structural Properties Control Rapid Assembly and Turnover of Actin in the ParasiteToxoplasma gondii

Actin Isoform-specific Conformational Differences Observed with Hydrogen/Deuterium Exchange and Mass Spectrometry

Insights into the effects of disease‐causing mutations in human actins

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