DNase I Increases the Rate Constant of Depolymerization at the Pointed (-) End of Actin Filaments

The actin filament is quite dynamic in the cell. To determine the relationship between the structure and the dynamic properties of the actin filament, experiments using actin mutants are indispensable. We focused on Gln 137 to understand the relationships between two activities: the conformational changes relevant to the G-to F-actin transition and the activation of actin ATPase upon actin polymerization. To elucidate the function of Gln 137 in these activities, we characterized Gln 137 mutants of human cardiac muscle ␣-actin. Although all of the single mutants, Q137E, Q137K, Q137P, and Q137A, as well as the wild type were expressed by a baculovirus-based system, only Q137A and the wild type were purified to high homogeneity. The CD spectrum of Q137A was similar to that of the wild type, and Q137A showed the typical morphology of negatively stained Q137A F-actin images. However, Q137A had an extremely low critical concentration for polymerization. Furthermore, we found that Q137A polymerized 4-fold faster, cleaved the ␥-phosphate group of bound ATP 4-fold slower, and depolymerized 5-fold slower, as compared with the wild-type rates. These results suggest that Gln 137 plays dual roles in actin polymerization, in both the conformational transition of the actin molecule and the mechanism of ATP hydrolysis.

Section: Actinmentioning

confidence: 88%

“…Depolymerization Assay-Depolymerization was initiated by the addition of 7.5 M vitamin D-binding protein (Gc-globulin from Calbiochem) to 5 M F-actin at the early steady state of polymerization (25). The mixture was incubated on ice and centrifuged at 100,000 ϫ g for 30 min to pellet the F-actin.…”

Section: Methodsmentioning

confidence: 99%

Dual Roles of Gln137 of Actin Revealed by Recombinant Human Cardiac Muscle α-Actin Mutants

Iwasa

Maéda

Narita

et al. 2008

“…Micromolar concentrations of deoxyribonuclease I can also induce depolymerization of actin filaments (Fig. 4A), probably through the combined effects of monomer sequestration and destabilization of actin monomer binding to the filament pointed end (33,34). Although its effects on F-actin are complex, deoxyribonuclease I-driven depolymerization is advantageous (over the dilution method used above) in that it can be performed at constant actin and actin-binding protein concentrations under more physiological buffer conditions.…”

Section: Resultsmentioning

confidence: 99%

Dystrophin-Glycoprotein Complex Is Monomeric and Stabilizes Actin Filaments in Vitro through a Lateral Association

Rybakova

Ervasti

1997

The native molecular weight of the dystrophin-glycoprotein complex and its effect on actin depolymerization and polymerization were examined. First, we determined that the native molecular weight of purified dystrophin-glycoprotein complex is only large enough (M r 1,200,000) to contain one copy of each protein in the complex, including dystrophin. Using different approaches, we also demonstrated that dystrophin-glycoprotein complex significantly protected a fraction of actin filaments from disassembly, while individual recombinant actin binding fragments of dystrophin or calpain-digested dystrophin-glycoprotein complex had no effect on F-actin depolymerization. The protective effect of dystrophin-glycoprotein complex on F-actin depolymerization saturated at a dystrophin:actin molar ratio of 0.04, corresponding to 1 dystrophin/25 actin monomers, which is highly consistent with the 1:24 stoichiometry of dystrophin-glycoprotein complex binding to F-actin previously measured at equilibrium. However, dystrophin-glycoprotein complex did not bind Gactin or alter the kinetics or extent of actin polymerization. This excluded the possibility that dystrophinglycoprotein complex inhibited actin depolymerization by capping the ends of actin filaments. It therefore appears that actin binding domains separated on the dystrophin molecule from each other by almost 1,200 amino acids act in concert to protect F-actin from depolymerization. Our data suggest that dystrophin stabilizes Factin in vitro by binding alongside an actin filament and bridging actin monomers in a manner analogous to the actin side binding protein tropomyosin. It is noteworthy that we did not find any effect of skeletal muscle tropomyosin on dystrophin-glycoprotein complex binding to F-actin. This indicates that dystrophin-glycoprotein complex and tropomyosin may simultaneously bind the same actin filament and identifies another feature that distinguishes dystrophin from the other proteins in the actin-cross-linking superfamily.Skeletal muscle dystrophin can be purified as part of a large oligomeric complex also containing integral and peripheral sarcolemmal proteins, several of which are glycoproteins (1-3). The dystrophin-glycoprotein complex further interacts with the laminin family of extracellular matrix proteins by way of the 156-kDa dystrophin-associated glycoprotein (4, 5), now known as ␣-dystroglycan, as well as with F-actin via dystrophin (5-9). These studies suggest that the dystrophin-glycoprotein complex is an essential functional unit that links the actin-based membrane cytoskeleton with the extracellular matrix and may play a structural role in maintaining sarcolemmal membrane integrity during muscle contraction. In fact, numerous studies have demonstrated that the absence of, or abnormality in, the dystrophin-glycoprotein complex renders muscle more susceptible to necrosis (10, 11).Striated muscle dystrophin is predominantly expressed as a 427-kDa, four-domain protein with the first three domains exhibiting significant sequence homology with ...

“…In conclusion, association of actobindin or Cib with pointed ends prevents growth but not depolymerization. The behavior of Cib and actobindin has some similarity here with DNase I, which also prevents pointed end growth but does not prevent depolymerization (34). The failure of Cib and actobindin to prevent pointed end depolymerization may be in relation with the poor binding of these proteins to ADP-actin, which is exposed at depolymerizing pointed ends, while ATP-actin is at the end of growing filaments.…”

Section: Association Of Actobindin or Cib With Filament Pointed Ends mentioning

confidence: 99%

Control of Actin Dynamics by Proteins Made of β-Thymosin Repeats

Hertzog¹,

Yarmola²,

Didry³

et al. 2002

Actobindin is an actin-binding protein from amoeba, which consists of two ␤-thymosin repeats and has been shown to inhibit actin polymerization by sequestering G-actin and by stabilizing actin dimers. Here we show that actobindin has the same biochemical properties as the Drosophila or Caenorhabditis elegans homologous protein that consists of three ␤-thymosin repeats. These proteins define a new family of actin-binding proteins. They bind G-actin in a 1:1 complex with thermodynamic and kinetic parameters similar to ␤-thymosins. Like ␤-thymosins, they slow down nucleotide exchange on G-actin and make a ternary complex with G-actin and Latrunculin A. On the other hand, they behave as functional homologs of profilin because their complex with MgATP-G-actin, unlike ␤-thymosin-actin, participates in filament barbed end growth, like profilin-actin complex. Therefore these proteins play an active role in actinbased motility processes. In addition, proteins of the actobindin family interact with the pointed end of actin filaments and inhibit pointed end growth, maybe via the interaction of the ␤-thymosin repeats with two terminal subunits.