Characterization of the actin depolymerizing factor, AtADF1, from Arabidopsis thaliana
Abstract
The plant actin cytoskeleton participates in diverse intracellular processes including cytokinesis, cytoplasmic streaming, tip growth and pollen tube extension. The dynamic nature of the actin cytoskeleton is regulated by actin-binding proteins. Two Arabidopsis thaliana actin depolymerizing factor-like (AtADF1 and AtADF2) cDNA clones were identified from expressed sequence tag (EST) databases. Their theoretical translation products are 137 and 139 amino acids, respectively, with predicted molecular weights of $\sim$16 kDa. The deduced amino acid sequences were 85% identical to each other and 58-72% identical to ADF-like clones from Lilium, Brassica, and Zea mays. AtADF1 shared 48% identity with Acanthamoeba actophorin and 38% identity with yeast cofilin. Both AtADF1 and AtADF2 contained a stretch of 25 amino acids in their C-terminal region with 50-60% identity to the postulated actin-binding sites of pig cofilin. To confirm that the AtADF1 cDNA encoded a bona fide ADF family member, AtADF1 was overexpressed in E. coli, purified, and used in in vitro studies with rabbit skeletal muscle actin. Sedimentation assays demonstrated that recombinant AtADF1 sedimented with filamentous actin (F-actin). In the presence of AtADF1, the amount of F-actin that pelleted was reduced and there was a comparable increase of actin in the supernatant. Actin levels in the supernatants were greater at pH 8.0 than at pH 6.5. At both pH levels, the amount of actin in the supernatants increased until a 1:1 molar ratio of AtADF1-actin was present in the reaction. This result suggested that AtADF1 was not a simple monomer-sequestering protein. AtADF1 increased the rate of actin polymer elongation. The enhancement of polymer elongation rate was greater at pH 0.5 than at pH 8.0, which suggested that the interaction of AtADF1 with F-actin was pH-dependent. AtADF1 inhibited nucleotide exchange on the actin monomer (G-actin), independently of pH. The combined data are consistent with the hypothesis that AtADF1 interacts with both F- and G-actin and that AtADF1 likely plays a significant role in regulation of actin dynamics within plant cells.
Degree
Ph.D.
Advisors
Staiger, Purdue University.
Subject Area
Botany|Molecular biology
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