Striated microtubule-associated fibers: identification of assemblin, a novel 34-kD protein that forms paracrystals of 2-nm filaments in vitro.
Microtubule-associated fibers from the basal apparatus of the green flagellate alga Spermatozopsis similis exhibit a complex cross-striation pattern with 28-nm periodicity and consist of 2-nm filaments arranged in several layers. Fibers enriched by mechanical disintegration and high salt extraction (2 M NaCl) of isolated basal apparatuses are soluble in 2 M urea. Dialysis of solubilized fibers against 150 mM KCl yields paracrystals which closely resemble the native fibers in filament arrangement and striation pattern. Paracrystals purified through several cycles of disassembly and reassembly are greatly enriched (greater than 90%) in a single protein of 34 kD (assemblin) as shown by SDS-PAGE. A rabbit polyclonal antibody raised against assemblin labels the striated fibers as shown by indirect immunofluorescence of isolated cytoskeletons or methanol permeabilized cells and immunogold EM. Two-dimensional electrophoresis (isoelectric focusing and SDS-PAGE) resolves assemblin into at least four isoforms (a-d) with pI's of 5.45, 5.55, 5.75, and 5.85. The two more acidic isoforms are phosphoproteins as shown by in vivo 32PO4-labeling and autoradiography. Amino acid analysis of assemblin shows a high content of helix-forming residues (leucine) and a relatively low content of glycine. We conclude that assemblin may be representative of a class of proteins that form fine filaments alongside microtubules.
Universität zu Köln, Botanisches Institut, Germany.
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KF
Lechtreck
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Melkonian
D000596:Amino Acids / Q000032:analysis; D000460:Chlorophyta / Q000502:physiology / Q000648:ultrastructure*; D003460:Crystallization; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D016253:Microscopy, Immunoelectron; D008869:Microtubule-Associated Proteins / Q000302:isolation & purification / Q000502:physiology / Q000648:ultrastructure*; D008870:Microtubules / Q000502:physiology / Q000648:ultrastructure*; D008970:Molecular Weight; D010766:Phosphorylation
D000596:Amino Acids; D008869:Microtubule-Associated Proteins