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

Abstract

In this report, we show that the echinoderm microtubule (MT)-associated protein (EMAP) and related EMAP-like proteins (ELPs) share a similar domain organization with a highly conserved hydrophobic ELP (HELP) domain and a large tryptophan-aspartic acid (WD) repeat domain. To determine the function of mammalian ELPs, we generated antibodies against a 70-kDa human ELP and showed that ELP70 coassembled with MTs in HeLa cell extracts and colocalized with MTs in the mitotic apparatus. To determine whether ELP70 bound to MTs directly, human ELP70 was expressed and purified to homogeneity from baculovirus-infected Sf9 cells. Purified ELP70 bound to purified MTs with a stoichiometry of 0.40 ؎ 0.04 mol of ELP70/mol of tubulin dimer and with an intrinsic dissociation constant of 0.44 ؎ 0.13 M. Using a nucleated assembly assay and video-enhanced differential interference contrast microscopy, we demonstrated that ELP70 reduced seeded nucleation, reduced the growth rate, and promoted MT catastrophes in a concentration-dependent manner. As a result, ELP70-containing MTs were significantly shorter than MTs assembled from tubulin alone. These data indicate that ELP70 is a novel MT destabilizer. A lateral destabilization model is presented to describe ELP70's effects on microtubules.

Microtubule association of EML proteins and the EML4-ALK variant 3 oncoprotein require an N-terminal trimerization domain

“…We confirmed co-localization of YFP-tagged human EML1 to the interphase MT network of transiently transfected HeLa cells, as has been previously observed for endogenous EML proteins ( Figure 3) [5][6][7][8]. Overexpression of a tagged protein might affect its regulation and distribution on microtubules, and we shall not comment on these features of the data.…”

“…The archetypal member of this family was identified as the most abundant MAP in dividing echinoderm eggs and embryos [4]. Further members of the EML family associate with MTs and are essential for the proper formation of both the interphase MT network and the mitotic spindle [5][6][7][8]. EML proteins affect microtubule dynamics, and may act as scaffold proteins to localize mitotic kinases to MTs [5,9,10].…”

“…Further members of the EML family associate with MTs and are essential for the proper formation of both the interphase MT network and the mitotic spindle [5][6][7][8]. EML proteins affect microtubule dynamics, and may act as scaffold proteins to localize mitotic kinases to MTs [5,9,10]. Drosophila and C. elegans each have a single EML homologue, whereas six EML proteins are present in humans of which EML1-4 have molecular weights in the 70-120 kDa range while EML5 and 6 are in excess of 200 kDa.…”

“…Drosophila and C. elegans each have a single EML homologue, whereas six EML proteins are present in humans of which EML1-4 have molecular weights in the 70-120 kDa range while EML5 and 6 are in excess of 200 kDa. The EML proteins contain WD40 repeats, and in EML1-4 these fall within a ~70 kDa core region that begins with a conserved ~60 amino acid sequence that has been termed the 'hydrophobic EML protein' (HELP) motif [5]. We recently determined the crystal structure of the core HELP/WD region of human EML1, which forms a tandem atypical β-propeller (TAPE) domain that binds soluble tubulin [11].…”

EML proteins in microtubule regulation and human disease

“…Antibodies raised against EMAP detected a similar sized protein by Western blot in microtubule preparations from a variety of echinoderm species, while it decorated microtubules in sea urchin embryos and adult coelomocytes by immunofluorescence microscopy (Suprenant et al, 1993). Expression of recombinant proteins confirmed even association of mammalian EML1, 2, 3 and 4 with the microtubule lattice (Eichenmuller et al, 2002;Houtman et al, 2007;Pollmann et al, 2006;Richards et al, 2015;Tegha-Dunghu et al, 2008). However, analysis of endogenous EML localization has, at least until recently, been hampered by the lack of good, commercially available antibodies.…”