Molecular cloning, over-expression, developmental regulation and immunolocalization of fragminP, a gelsolin-related actin-binding protein from Physarum polycephalum plasmodia.
FragminP is a Ca2+-dependent actin-binding and microfilament regulatory protein of the gelsolin family. We screened a Physarum polycephalum cDNA library with polyclonal fragminP antibodies and isolated a cDNA clone of 1,104 bp encoding 368 amino acids of fragminP, revealing two consensus phosphatidylinositol 4,5 bisphosphate-binding motifs in the central part of the protein. The first methionine is modified by an acetyl group, and three amino acids were missing from the protein coded for by the cDNA clone. Full-length recombinant fragminP was generated by PCR, purified after over-expression from Escherichia coli and displayed identical properties to native Physarum fragminP. Northern blot analysis against RNA, isolated from cultures at various stages of development, indicated that fragminP is absent from amoebae and that expression is initiated at an early stage during apogamic development, in a similar way to that observed for the profilin genes. In situ immunolocalization of fragminP in Physarum microplasmodia revealed that the protein is localized predominantly at the plasma membrane, suggesting a role in the regulation of the subcortical actin meshwork. Our data indicate that we have isolated the plasmodium-specific fragminP cDNA (frgP) and suggest that, in each of its two vegetative cell types, P. polycephalum uses a different fragmin isoform that performs different functions.
T'Jampens
D
D
Flanders Interuniversity Institute of Biotechnology, Department of Biochemistry, Universiteit Gent, Belgium.
Meerschaert
K
K
Constantin
B
B
Bailey
J
J
Cook
L J
LJ
De Corte
V
V
De Mol
H
H
Goethals
M
M
Van Damme
J
J
Vandekerckhove
J
J
Gettemans
J
J
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics; D018260:Gelsolin / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D008840:Microfilament Proteins / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D016839:Physarum polycephalum / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000199:Actins; D002352:Carrier Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D004271:DNA, Fungal; D016054:DNA, Protozoan; C034037:F-actin-binding proteins; D018260:Gelsolin; D008840:Microfilament Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins