A novel 49-kilodalton protein from Artemia cross-links microtubules in vitro.
A 49 kilodalton (kDa) protein, previously proposed to cross-link microtubules, was purified to apparent homogeneity from cell-free extracts of the brine shrimp Artemia. When incubated with tubulin under assembly conditions, the purified 49-kDa protein cross-linked the resulting microtubules. Preformed microtubules were also cross-linked when incubated with the 49-kDa protein. Upon centrifugation through sucrose cushions the 49-kDa protein cosedimented with microtubules, suggesting a stable association between the cross-linking protein and tubulin. Such microtubules were interconnected by particles which were circular, bilobed, or elongated in shape. Disruption of microtubule cross-linking and dissociation of the 49-kDa protein from microtubules occurred in the presence of ATP and 5'-adenylyl-imidodiphosphate (AMP-PNP), a nonhydrolyzable analogue of ATP. The 49-kDa protein was moderately resistant to heat, it did not stimulate tubulin assembly, and it did not react with antibodies to neural microtubule-associated proteins (MAPs) and kinesin. These observations indicate that the 49-kDa protein is different from many known MAPs, a conclusion strengthened by the inability of antibodies raised to the 49-kDa protein to recognize these proteins. The amino terminal 15 amino acid residues of the 49-kDa protein were determined by Edman digestion and an antibody raised to this peptide reacted with the 49-kDa protein on Western blots. Microtubule cross-linking was unaffected by the synthetic amino-terminal peptide, even when it was present at a fivefold molar excess over the 49-kDa protein. A search of three protein databanks revealed that the amino terminus of the 49-kDa protein is unique among published sequences.(ABSTRACT TRUNCATED AT 250 WORDS)
Zhang
J
J
Department of Biology, Dalhousie University, Halifax, N.S., Canada.
MacRae
T H
TH
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001156:Artemia / Q000737:chemistry*; D003429:Cross Reactions; D007120:Immunochemistry; D016547:Kinesins / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D014404:Tubulin / Q000378:metabolism
D008869:Microtubule-Associated Proteins; D014404:Tubulin; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D016547:Kinesins