Cloning and characterization of actin depolymerizing factor from Toxoplasma gondii1

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Abstract

We determined the predicted amino acid sequence of actin depolymerizing factor (ADF) from Toxoplasma gondii by sequencing the full-length cDNA. T. gondii ADF consists of 118 amino acids (calculated molecular weight 13 400) and shares a high degree of sequence similarity to other low molecular weight actin monomer sequestering proteins, especially Acanthamoeba actophorin, plant ADFs and yeast and vertebrate cofilin. ADF from T. gondii is smaller and does not contain a nuclear localization sequence like the related vertebrate proteins. Southern blot analysis indicates that T. gondii ADF is a single-copy gene. Homogeneous recombinant T. gondii ADF purified from E. coli is active in binding actin monomers and depolymerizing F-actin. Localization of ADF by immunofluorescence and immuno-electron microscopy indicates ADF is scattered throughout the cytoplasm and prominently localized beneath the plasma membrane in T. gondii.

Keywords

Toxoplasma gondii
Actin depolymerizing factor
Cytoskeleton

Abbreviations

ADF, actin depolymerizing factor (protein)
rADF, recombinant actin depolymerizing factor (protein)
G-actin, globular monomeric form of actin
F-actin, actin filaments
SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis
DTT, dithiothreitol
HEPES, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid
PCR, polymerase chain reaction
FBS, fetal bovine serum

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1

Note: The nucleic acid sequence in this paper has been submitted to GenBank™ under Accession Number U62146.

2

Present address: Fibrogen, Inc., 260 Littlefield Ave., South San Francisco, CA 94080, USA.

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