Saturable binding of the echinoderm microtubule-associated protein (EMAP) on microtubules, but not filamentous actin or vimentin filaments.
The echinoderm microtubule-associated protein (EMAP) is a 75-kDa, WD-repeat protein associated with the mitotic spindle apparatus. To understand EMAP's biological role, it is important to determine its affinity for microtubules (MTs) and other cytoskeletal components. To accomplish this goal, we utilized a low-cost, bubble-column bioreactor to express EMAP as a hexahistidine fusion (6his) protein in baculovirus-infected insect cells. After optimizing cell growth conditions, up to 30 mg of EMAP was obtained in the soluble cell lysate from a 1-liter culture. EMAP was purified to homogeneity in a two-step process that included immobilized metal-affinity chromatography (IMAC) and anion-exchange chromatography. In vitro binding studies on cytoskeletal components were performed with the 6his-EMAP. EMAP bound to MTs, but not actin or vimentin filaments, with an intrinsic dissociation constant of 0.18 microM and binding stoichiometry of 0.7 mol EMAP per mol tubulin heterodimer. In addition, we show that a strong MT binding domain resides in the 137 amino acid, NH(2)-terminus of EMAP and a weaker binding site in the WD-domain. Previous work has shown that the EMAP concentration in the sea urchin egg is over 4 microM. Together, these results show that there is sufficient EMAP in the egg to regulate the assembly of a large pool of maternally stored tubulin.
Eichenmüller
B
B
Department of Molecular Biosciences, University of Kansas, Lawrence 66045, USA.
Ahrens
D P
DP
Li
Q
Q
Suprenant
K A
KA
D008841:Actin Cytoskeleton / Q000378:metabolism*; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001665:Binding Sites; D019149:Bioreactors; D018929:Cell Culture Techniques; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics; D014746:Vimentin / Q000378:metabolism*
C115941:EMAP protein, Strongylocentrotus purpuratus; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D014746:Vimentin