Purification of a WD repeat protein, EMAP, that promotes microtubule dynamics through an inhibition of rescue.
The major microtubule-associated protein in echinoderms is a 77-kDa, WD repeat protein, called EMAP. EMAP-related proteins have been identified in sea urchins, starfish, sanddollars, and humans. We describe the purification of sea urchin EMAP and demonstrate that EMAP binding to microtubules is saturable at a molar ratio of 1 mol of EMAP to 3 mol of tubulin dimer. Unlike MAP-2, MAP-4, or tau proteins, EMAP binding to microtubules is not lost by cleavage of tubulin with subtilisin. In addition to binding to the microtubule polymer, EMAP binds to tubulin dimers in a 1:1 molar ratio. The abundance of EMAP in the egg suggests that it could function to regulate microtubule assembly. To test this hypothesis, we examined the effects of EMAP on the dynamic instability of microtubules nucleated from axoneme fragments as monitored by video-enhanced differential interference contrast microscopy. Addition of 2.2 microM EMAP to 21 microM tubulin results in a slight increase in the elongation and shortening velocities at the microtubule plus ends but not at the minus ends. Significantly, EMAP inhibits the frequency of rescue 8-fold without producing a change in the frequency of catastrophe. These results indicate that EMAP, unlike brain microtubule-associated proteins, promotes microtubule dynamics.
Hamill
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DR
Department of Biochemistry, Cell and Molecular Biology, University of Kansas, Lawrence, Kansas 66045, USA.
Howell
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B
Cassimeris
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Suprenant
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KA
D000818:Animals; D015153:Blotting, Western; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D019281:Dimerization; D004447:Echinodermata; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007700:Kinetics; D008297:Male; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008970:Molecular Weight; D010063:Ovum / Q000378:metabolism*; D012617:Sea Urchins / Q000378:metabolism*; D013082:Sperm Tail / Q000378:metabolism; D013215:Starfish; D013552:Swine; D014404:Tubulin / Q000302:isolation & purification / Q000378:metabolism*
C115941:EMAP protein, Strongylocentrotus purpuratus; C485471:EML2 protein, human; D008869:Microtubule-Associated Proteins; D014404:Tubulin