Saturable binding of the echinoderm microtubule‐associated protein (EMAP) on microtubules, but not filamentous actin or vimentin filaments

Abstract

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.

Loss of dystrophin and the microtubule‐binding protein ELP‐1 causes progressive paralysis and death of adult C. elegans

“…All EMAP-like proteins are characterized as having a Hydrophobic ELP (HELP) domain/motif and a WD domain (Eichenmuller et al, 2002). The HELP domain is required for microtubule binding and it is assumed that the WD repeat domain is required for additional protein-protein interactions (Eichenmuller et al, 2001; Tegha-Dunghu et al, 2008). …”

Myosin-Va Binds to and Mechanochemically Couples Microtubules to Actin Filaments

“…Finally, MT-bound Myo5a retains its characteristic ATPdependent interactions with F-actin without dissociating from the MT as demonstrated by cosedimentation analysis (Figure 4) and the ATP-dependent dissociation of MTMyo5a-actin gels under low Ca 2ϩ conditions (Figures 3 and 4), and contraction of these gels into aster-like arrays in the presence of Ca 2ϩ (Figures 7-9) . The interaction of native Myo5a with MTs is saturable and occurs with high affinity, suggesting that Myo5a is a bona fide MAP and that MTs should be added to the ever-grow-ing list of cargos for this myosin (reviewed in Reck-Peterson et al, 2000;Karcher et al, 2002;Langford, 2002 Eichenmuller et al, 2001]). However, at saturation the ratio of Myo5a:tubulin is relatively low (1:22-24) compared with other MAPs such as (Pedrotti and Islam, 1994) MAP2 (1:12) (Burns and Islam, 1984), or XMAP215 (1:16) (Gard and Kirschner, 1987) that bind substoichiometrically to tubulin dimers in the MT.…”

The Human EMAP-like Protein-70 (ELP70) Is a Microtubule Destabilizer That Localizes to the Mitotic Apparatus

“…This high degree of conservation among ELP family members indicates that the HELP domain is likely to be critical to ELP function. For example, a preliminary study has shown that the NH 2 terminus of sea urchin EMAP, which contains the HELP domain, is sufficient for MT binding in vitro (43).…”