The cruciated microtubule-associated fibers of the green alga Dunaliella bioculata consist of a 31 kDa SF-assemblin.
Cytoskeletons of Dunaliella bioculata, the biflagellate wallless green alga, were isolated and analyzed using a monoclonal and a polyclonal antibody raised against SF-assemblin, the major protein of the two striated microtubule-associated fibers of the alga Spermatozopsis similis. Indirect immunofluorescence showed antigenic structures associated with the four microtubular flagellar roots. SDS-PAGE followed by immunoblot analysis revealed a cross-reacting polypeptide of 31 kDa. This protein of D. bioculata was isolated using gel filtration chromatography in 8 M urea and in vitro reassembly of striated fibers. Microsequencing of the purified protein yielded various peptides, which could be aligned along the sequence of SF-assemblin from S. similis. A complete sequence of the Dunaliella protein was obtained by cDNA cloning. It documents the non helical head domain followed by a helical rod domain with a 29 residue repeat pattern based on four heptads followed by a skip residue. Compared to SF-assemblin of S. similis the SF-assemblin of Dunaliella has a shorter head and a slightly longer rod domain. The two algal SF-assemblins share only 57% sequence identity. We conclude that SF-assemblin and related proteins in various protists are representatives of a new class of alpha-helical proteins characterized by the ability to form a special segmented coiled coil and to assemble into striated fibers of 2 nm protofilaments in vivo and in vitro.
Lechtreck
K F
KF
University of Minnesota, Department of Genetics and Cell Biology, St Paul, MN 55108-1095, USA.
Frins
S
S
Bilski
J
J
Teltenkötter
A
A
Weber
K
K
Melkonian
M
M
D000595:Amino Acid Sequence; D001483:Base Sequence; D000460:Chlorophyta / Q000737:chemistry* / Q000648:ultrastructure; D003001:Cloning, Molecular; D003599:Cytoskeleton / Q000737:chemistry / Q000648:ultrastructure; D018076:DNA, Complementary / Q000737:chemistry; D018744:DNA, Plant / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008869:Microtubule-Associated Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010940:Plant Proteins / Q000737:chemistry*
D018076:DNA, Complementary; D018744:DNA, Plant; D008869:Microtubule-Associated Proteins; D010940:Plant Proteins; C096668:striated fiber assemblin