The human EMAP-like protein-70 (ELP70) is a microtubule destabilizer that localizes to the mitotic apparatus.
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 microm. 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.
Eichenmuller
Bernd
B
Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045, USA.
Everley
Patrick
P
Palange
Jean
J
Lepley
Denise
D
Suprenant
Kathy A
KA
D000595:Amino Acid Sequence; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D008869:Microtubule-Associated Proteins / Q000737:chemistry; D008870:Microtubules / Q000378:metabolism* / Q000502:physiology; D008969:Molecular Sequence Data; D017239:Paclitaxel / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D008941:Spindle Apparatus / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000972:Antineoplastic Agents, Phytogenic; C115941:EMAP protein, Strongylocentrotus purpuratus; C485471:EML2 protein, human; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D017239:Paclitaxel